Influence of polyethylene terephthalate (PET) microplastic on selected active substances in the intramural neurons of the porcine duodenum.

BACKGROUND: Currently, society and industry generate huge amounts of plastics worldwide. The ubiquity of microplastics is obvious, but its impact on the animal and human organism remains not fully understood. The digestive tract is one of the first barriers between pathogens and xenobiotics and a living organism. Its proper functioning is extremely important in order to maintain homeostasis. The aim of this study was to determine the effect of microplastic on enteric nervous system and histological structure of swine duodenum. The experiment was carried out on 15 sexually immature gilts, approximately 8 weeks old. The animals were randomly divided into 3 study groups (n = 5/group). The control group received empty gelatin capsules once a day for 28 days, the first research group received daily gelatin capsules with polyethylene terephthalate (PET) particles as a mixture of particles of various sizes (maximum particle size 300 µm) at a dose of 0.1 g/animal/day. The second study group received a dose ten times higher-1 g/animal/day. RESULTS: A dose of 1 g/day/animal causes more changes in the enteric nervous system and in the histological structure of duodenum. Statistically significant differences in the expression of cocaine and amphetamine regulated transcript, galanin, neuronal nitric oxide synthase, substance P, vesicular acetylcholine transporter and vasoactive intestinal peptide between control and high dose group was noted. The histopathological changes were more frequently observed in the pigs receiving higher dose of PET. CONCLUSION: Based on this study it may be assumed, that oral intake of microplastic might have potential negative influence on digestive tract, but it is dose-dependent.

A novel class of insecticidal alkylsulfones are potent inhibitors of vesicular acetylcholine transport.

Pyridine alkylsulfone derivatives typified by oxazosulfyl (Sumitomo Chemical Company Ltd.) and compound A2 (Syngenta) represent a new class of insecticides, with potent activity against several insect orders. Whilst the MOA of this class has been attributed to interaction with the voltage-gated sodium channel (VGSC), here we present strong evidence that their toxicity to insects is mediated primarily through inhibition of the vesicular acetylcholine transporter (VAChT). Alkylsulfone intoxication in insects is characterised by (i) a reduction in cholinergic synaptic transmission efficiency demonstrated by a depression of cercal afferent activity in giant-interneurone preparations of American cockroach (Periplaneta americana), (ii) selective block of cholinergic-transmission dependent post-synaptic potentials in the Drosophila giant-fibre pathway and (iii) abolition of miniature excitatory post-synaptic currents (mEPSCs) in an identified synapse in Drosophila larvae. Ligand-binding studies using a tritiated example compound ([3H]-A1) revealed a single saturable binding-site, with low nanomolar Kd value, in membrane fractions of green bottle fly (Lucilia sericata). Binding is inhibited by vesamicol and by several examples of a previously identified class of insecticidal compounds known to target VAChT, the spiroindolines. Displacement of this binding by analogues of the radioligand reveals a strong correlation with insecticidal potency. No specific binding was detected in untransformed PC12 cells but a PC12 line stably expressing Drosophila VAChT showed similar affinity for [3H]-A1 as that seen in fly head membrane preparations. Previously identified VAChT point mutations confer resistance to the spiroindoline class of insecticides in Drosophila by Gal-4/UAS directed expression in cholinergic neurones and by CRISPR gene-editing of VAChT, but none of these flies show detectable cross-resistance to this new chemical class. Oxazosulfyl was previously shown to stabilise voltage-gated sodium channels in their slow-inactivated conformation with an IC50 value of 12.3µM but inhibits binding of [3H]-A1 with approximately 5000 times greater potency. We believe this chemistry class represents a novel mode-of-action with high potential for invertebrate selectivity.

Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern.

How is the quantal size in neurotransmitter release adjusted for various firing levels? We explored the possible mechanisms that regulate acetylcholine (ACh) release from cholinergic interneurons using an ultra-mini superfusion system. After preloading [3 H]ACh in rat striatal cholinergic interneurons, the release was elicited by electrical stimulation under a condition in which presynaptic cholinergic and dopaminergic feedback was inhibited. [3 H]ACh release was reproducible at intervals of more than 10 min; shorter intervals resulted in reduced levels of ACh release. Upon persistent stimulation for 10 min, ACh release transiently increased, before gradually decreasing. Vesamicol, an inhibitor of the vesicular ACh transporter (VAChT), had no effect on the release induced by the first single pulse, but it reduced the release caused by subsequent pulses. Vesamicol also reduced the [3 H]ACh release evoked by multiple pulses, and the inhibition was enhanced by repetitive stimulation. The decreasing phase of [3 H]ACh release during persistent stimulation was accelerated by vesamicol treatment. Thus, it is likely that releasable ACh was slowly compensated for via VAChT during and after stimulation, changing the vesicular ACh content. In addition, ACh release per pulse decreased under high-frequency stimulation. The present results suggest that ACh release from striatal cholinergic interneurons may be adjusted by changes in the quantal size due to slow replenishment via VAChT, and by a reduction in release probability upon high-frequency stimulation. These two distinct processes likely enable the fine tuning of neurotransmission and neuroprotection/limitation against excessive output and have important physiological roles in the brain.

No Dopamine Agonist Modulation of Brain [18F]FEOBV Binding in Parkinson's Disease.

The [18F]fluoroethoxybenzovesamicol ([18F]FEOBV) positron emission tomography (PET) ligand targets the vesicular acetylcholine transporter. Recent [18F]FEOBV PET rodent studies suggest that regional brain [18F]FEOBV binding may be modulated by dopamine D2-like receptor agents. We examined associations of regional brain [18F]FEOBV PET binding in Parkinson's disease (PD) subjects without versus with dopamine D2-like receptor agonist drug treatment. PD subjects (n = 108; 84 males, 24 females; mean age 68.0 ± 7.6 [SD] years), mean disease duration of 6.0 ± 4.0 years, and mean Movement Disorder Society-revised Unified PD Rating Scale III 35.5 ± 14.2 completed [18F]FEOBV brain PET imaging. Thirty-eight subjects were taking dopamine D2-like agonists. Vesicular monoamine transporter type 2 [11C]dihydrotetrabenazine (DTBZ) PET was available in a subset of 54 patients. Subjects on dopamine D2-like agonists were younger, had a longer duration of disease, and were taking a higher levodopa equivalent dose (LED) compared to subjects not taking dopamine agonists. A group comparison between subjects with versus without dopamine D2-like agonist use did not yield significant differences in cortical, striatal, thalamic, or cerebellar gray matter [18F]FEOBV binding. Confounder analysis using age, duration of disease, LED, and striatal [11C]DTBZ binding also failed to show significant regional [18F]FEOBV binding differences between these two groups. Chronic D2-like dopamine agonist use in PD subjects is not associated with significant alterations of regional brain [18F]FEOBV binding.

Aberrant slow waves in the hippocampus during activation in mice with low cholinergic tone.

The effects of acetylcholine on cortical activation were studied in wild-type (WT) mice, compared to knockout (KO) mice depleted of the vesicular acetylcholine transporter (VAChT) gene in the basal forebrain, and knockdown (KD) mice with heterogeneous depletion of VAChT gene in the brain. Cortical activation was assessed by comparing power spectra of local field potentials (LFPs) during activated states of rapid-eye-movement sleep (REM) or walk (WLK), with those during non-activated states of slow-wave sleep (SWS) or awake-immobility (IMM). Activation-induced suppression of delta (1-4 Hz) and beta (13-30 Hz) power in the hippocampus, and delta power in frontal cortex, were reduced in KO and KD mice compared to WT mice. Mean theta frequency was higher in KD than KO mice during WLK and REM, but not different between WT and KO mice. Peak theta (4-12 Hz) and integrated gamma (30-150 Hz) power were not significantly different among mouse groups. However, theta-peak-frequency selected gamma2 (62-100 Hz) power was lower in KO than WT or KD mice during WLK, and theta-peak-frequency selected theta power during REM decreased faster with high theta frequency in KO than WT/ KD mice. Theta power increase during REM compared to WLK was lower in KO and KD mice compared to WT mice. Theta-gamma cross-frequency coherence, a measure of synchronization of gamma with theta phase, was not different among mouse groups. However, during REM, SWS, and IMM, delta-gamma coherence was significantly higher and proximal-distal delta coherence in CA1 was lower in KO than WT/KD mice. We conclude that a deficiency in basal forebrain acetylcholine release not only enhances slow waves and suppresses theta-associated gamma waves during activation, but also increases delta-gamma cross-frequency coherence during nonactivated states, with a possible effect of disrupting cognitive processing during any brain state.

Vesicular Acetylcholine Transporter Alters Cholinergic Tone and Synaptic Plasticity in DYT1 Dystonia.

BACKGROUND: Acetylcholine-mediated transmission plays a central role in the impairment of corticostriatal synaptic activity and plasticity in multiple DYT1 mouse models. However, the nature of such alteration remains unclear. OBJECTIVE: The aim of the present work was to characterize the mechanistic basis of cholinergic dysfunction in DYT1 dystonia to identify potential targets for pharmacological intervention. METHODS: We utilized electrophysiology recordings, immunohistochemistry, enzymatic activity assays, and Western blotting techniques to analyze in detail the cholinergic machinery in the dorsal striatum of the Tor1a+/- mouse model of DYT1 dystonia. RESULTS: We found a significant increase in the vesicular acetylcholine transporter (VAChT) protein level, the protein responsible for loading acetylcholine (ACh) from the cytosol into synaptic vesicles, which indicates an altered cholinergic tone. Accordingly, in Tor1a+/- mice we measured a robust elevation in basal ACh content coupled to a compensatory enhancement of acetylcholinesterase (AChE) enzymatic activity. Moreover, pharmacological activation of dopamine D2 receptors, which is expected to reduce ACh levels, caused an abnormal elevation in its content, as compared to controls. Patch-clamp recordings revealed a reduced effect of AChE inhibitors on cholinergic interneuron excitability, whereas muscarinic autoreceptor function was preserved. Finally, we tested the hypothesis that blockade of VAChT could restore corticostriatal long-term synaptic plasticity deficits. Vesamicol, a selective VAChT inhibitor, rescued a normal expression of synaptic plasticity. CONCLUSIONS: Overall, our findings indicate that VAChT is a key player in the alterations of striatal plasticity and a novel target to normalize cholinergic dysfunction observed in DYT1 dystonia. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Decreased number of striatal cholinergic interneurons and motor deficits in dopamine receptor 2-expressing-cell-specific Dyt1 conditional knockout mice.

DYT1 early-onset generalized torsion dystonia is a hereditary movement disorder characterized by abnormal postures and repeated movements. It is caused mainly by a heterozygous trinucleotide deletion in DYT1/TOR1A, coding for torsinA. The mutation may lead to a partial loss of torsinA function. Functional alterations of the basal ganglia circuits have been implicated in this disease. Striatal dopamine receptor 2 (D2R) levels are significantly decreased in DYT1 dystonia patients and in the animal models of DYT1 dystonia. D2R-expressing cells, such as the medium spiny neurons in the indirect pathway, striatal cholinergic interneurons, and dopaminergic neurons in the basal ganglia circuits, contribute to motor performance. However, the function of torsinA in these neurons and its contribution to the motor symptoms is not clear. Here, D2R-expressing-cell-specific Dyt1 conditional knockout (d2KO) mice were generated and in vivo effects of torsinA loss in the corresponding cells were examined. The Dyt1 d2KO mice showed significant reductions of striatal torsinA, acetylcholine metabolic enzymes, Tropomyosin receptor kinase A (TrkA), and cholinergic interneurons. The Dyt1 d2KO mice also showed significant reductions of striatal D2R dimers and tyrosine hydroxylase without significant alteration in striatal monoamine contents or the number of dopaminergic neurons in the substantia nigra. The Dyt1 d2KO male mice showed motor deficits in the accelerated rotarod and beam-walking tests without overt dystonic symptoms. Moreover, the Dyt1 d2KO male mice showed significant correlations between striatal monoamines and locomotion. The results suggest that torsinA in the D2R-expressing cells play a critical role in the development or survival of the striatal cholinergic interneurons, expression of striatal D2R mature form, and motor performance. Medical interventions to compensate for the loss of torsinA function in these neurons may affect the onset and symptoms of this disease.

Effect of Dopamine D2 Receptor Antagonists on [18F]-FEOBV Binding.

The interaction of dopaminergic and cholinergic neurotransmission in, e.g., Parkinson's disease has been well established. Here, D2 receptor antagonists were used to assess changes in [18F]-FEOBV binding to the vesicular acetylcholine transporter (VAChT) in rodents using positron emission tomography (PET). After pretreatment with either 10 mg/kg haloperidol, 1 mg/kg raclopride, or vehicle, 90 min dynamic PET scans were performed with arterial blood sampling. The net influx rate (Ki) was obtained from Patlak graphical analysis, using a metabolite-corrected plasma input function and dynamic PET data. [18F]-FEOBV concentration in whole-blood or plasma and the metabolite-corrected plasma input function were not significantly changed by the pretreatments (adjusted p > 0.07, Cohen's d 0.28-1.89) while the area-under-the-curve (AUC) of the parent fraction of [18F]-FEOBV was significantly higher after haloperidol treatment (adjusted p = 0.022, Cohen's d = 2.51) than in controls. Compared to controls, the AUC of [18F]-FEOBV, normalized for injected dose and body weight, was nonsignificantly increased in the striatum after haloperidol (adjusted p = 0.4, Cohen's d = 1.77) and raclopride (adjusted p = 0.052, Cohen's d = 1.49) treatment, respectively. No changes in the AUC of [18F]-FEOBV were found in the cerebellum (Cohen's d 0.63-0.74). Raclopride treatment nonsignificantly increased Ki in the striatum 1.3-fold compared to control rats (adjusted p = 0.1, Cohen's d = 1.1) while it reduced Ki in the cerebellum by 28% (adjusted p = 0.0004, Cohen's d = 2.2) compared to control rats. Pretreatment with haloperidol led to a nonsignificant reduction in Ki in the striatum (10%, adjusted p = 1, Cohen's d = 0.44) and a 40-50% lower Ki than controls in all other brain regions (adjusted p < 0.0005, Cohen's d = 3.3-4.7). The changes in Ki induced by the selective D2 receptor antagonist raclopride can in part be quantified using [18F]-FEOBV PET imaging. Haloperidol, a nonselective D2/σ receptor antagonist, either paradoxically decreased cholinergic activity or blocked off-target [18F]-FEOBV binding to σ receptors. Hence, further studies evaluating the binding of [18F]-FEOBV to σ receptors using selective σ receptor ligands are necessary.

Remodeling of cholinergic input to the hippocampus after noise exposure and tinnitus induction in Guinea pigs.

Here, we investigate remodeling of hippocampal cholinergic inputs after noise exposure and determine the relevance of these changes to tinnitus. To assess the effects of noise exposure on the hippocampus, guinea pigs were exposed to unilateral noise for 2 hr and 2 weeks later, immunohistochemistry was performed on hippocampal sections to examine vesicular acetylcholine transporter (VAChT) expression. To evaluate whether the changes in VAChT were relevant to tinnitus, another group of animals was exposed to the same noise band twice to induce tinnitus, which was assessed using gap-prepulse Inhibition of the acoustic startle (GPIAS) 12 weeks after the first noise exposure, followed by immunohistochemistry. Acoustic Brainstem Response (ABR) thresholds were elevated immediately after noise exposure for all experimental animals but returned to baseline levels several days after noise exposure. ABR wave I amplitude-intensity functions did not show any changes after 2 or 12 weeks of recovery compared to baseline levels. In animals assessed 2-weeks following noise-exposure, hippocampal VAChT puncta density decreased on both sides of the brain by 20-60% in exposed animals. By 12 weeks following the initial noise exposure, changes in VAChT puncta density largely recovered to baseline levels in exposed animals that did not develop tinnitus, but remained diminished in animals that developed tinnitus. These tinnitus-specific changes were particularly prominent in hippocampal synapse-rich layers of the dentate gyrus and areas CA3 and CA1, and VAChT density in these regions negatively correlated with tinnitus severity. The robust changes in VAChT labeling in the hippocampus 2 weeks after noise exposure suggest involvement of this circuitry in auditory processing. After chronic tinnitus induction, tinnitus-specific changes occurred in synapse-rich layers of the hippocampus, suggesting that synaptic processing in the hippocampus may play an important role in the pathophysiology of tinnitus.

SLC18A3 variants lead to fetal akinesia deformation sequence early in pregnancy.

Fetal akinesia deformation sequence (FADS) and lethal multiple pterygium syndrome (LMPS) are clinically overlapping syndromes manifesting with reduced or absent fetal movement, arthrogryposis, and several anomalies during fetal life. The etiology of these syndromes is heterogeneous, and in many cases it remains unknown. In order to determine the genetic etiology of FADS in two fetuses with fetal akinesia, arthrogryposis, edema, and partial cleft palate, we utilized exome sequencing. Our investigations revealed a homozygous nonsense variant [c.1116C>A, p.(Cys372Ter)] in the SLC18A3 gene, which encodes for the vesicular acetylcholine transporter (VAChT) responsible for active transport of acetylcholine in the neuromuscular junction. This is the first description of a nonsense variant in the SLC18A3 gene, as only missense variants and whole gene deletions have been previously identified in patients. The previously detected SLC18A3 defects have been associated with congenital myasthenic syndromes, and therefore our findings extend the clinical spectrum of SLC18A3 defects to severe prenatal phenotypes. Our findings suggest that nonsense variants in SLC18A3 cause a more severe phenotype than missense variants and are in line with previous studies showing a lethal phenotype in VAChT knockout mice. Our results underline the importance of including SLC18A3 sequencing in the differential diagnostics of fetuses with arthrogryposis, FADS, or LMPS of unknown etiology.

Kinetic modeling of [18 F]VAT, a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non-human primate brain.

Molecular imaging of vesicular acetylcholine transporter (VAChT) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VAChT-specific positron emission tomography radiotracer, ((-)-(1-(-8-(2-fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone) ([18 F]VAT) in macaques. Total volume distribution (VT ) estimates were compared for one-tissue compartment model (1TCM), two-tissue compartment model (2TCM), Logan graphic analysis (LoganAIF) and multiple linear analysis (MA1) with arterial blood input function using data from three macaques. Using the cerebellum-hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM), simplified RTM (SRTM), and Logan graphic analysis (LoganREF). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [18 F]VAT; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r > 0.93, p < 0.05). Test-retest studies demonstrated that [18 F]VAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate [18 F]VAT is a promising VAChT positron emission tomography tracer for quantitative assessment of VAChT levels in the brain of living subjects.

Decreased vesicular acetylcholine transporter related to memory deficits in epilepsy: A [18 F] VAT positron emission tomography brain imaging study.

OBJECTIVE: Vesicular acetylcholine transporter (VAChT) is a rate-limiting factor for synaptic acetylcholine transport. Our study focused on whether [18 F] VAT, a novel positron emission tomography (PET) tracer, could be used in detecting cognitive deficits in epilepsy. METHODS: Morris water maze test was used to evaluate learning and memory deficits in pilocarpine-induced chronic epilepsy rats 12 weeks after status epilepticus. Interictal [18 F] VAT PET was performed 13 weeks after status epilepticus to evaluate the level of VAChT in cholinergic pathways compared with [18 F] fluorodeoxyglucose PET. The association between VAChT levels and memory measures was analyzed. Neuropathological tests were performed. RESULTS: Epileptic rats exhibited significant memory deficits in Morris water maze test. [18 F] VAT uptake decreased in septum, hippocampus, thalamus, and basal forebrain, and correlated to memory function. Of note, the level of VAChT in basal forebrain significantly decreased, yet no glucose hypometabolism was detected. Immunofluorescence and Western blot demonstrated decreased expression of VAChT in hippocampus and basal forebrain in the epilepsy group, but no change of expression of acetyltransferase or activity of acetylcholinesterase was detected. SIGNIFICANCE: [18 F] VAT PET is a promising method to test the level of VAChT as a valuable biomarker for memory deficits in pilocarpine-induced chronic epileptic rats.

Radiosynthesis and evaluation of a fluorine-18 labeled radioligand targeting vesicular acetylcholine transporter.

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing the loss of cholinergic neurons in the brain that is associated with cognitive impairment of patients. 5-Hydrotetralin compound (±)-5-OH-VAT is potent (Ki = 4.64 ±â€¯0.32 nM) and selective for VAChT (>1800-fold and 398-fold for σ1 and σ2 receptor, respectively) with favorable hydrophilicity (LogD = 1.78), while (-)-5-OH-VAT originally serves as the radiolabeling precursor of (-)-[18F]VAT, a promising VAChT radiotracer with a logD value of 2.56. To evaluate (-)-5-OH-[18F]VAT as a radiotracer for VAChT, we performed in vitro binding assay to determine the potency of the minus enantiomer (-)-5-OH-VAT and plus enantiomer (+)-5-OH-VAT, indicating that (-)-5-OH-VAT is a more potent VAChT enantiomer. Radiosynthesis of (-)-5-OH-[18F]VAT was explored using three strategies. (-)-5-OH-[18F]VAT was achieved with a good yield (24 ±â€¯6%) and high molar activity (∼37 GBq/µmol, at the end of synthesis) using a microwave assisted two-step one-pot procedure that started with di-MOM protected nitro-containing precursor (-)-6. MicroPET studies in the brain of nonhuman primate (NHP) suggest that (-)-5-OH-[18F]VAT readily penetrated the blood brain barrier and specifically accumulated in the VAChT-enriched striatum with improved washout kinetics from striatum compared to [18F]VAT. Nevertheless, the lower target to non-target ratio may limit its use for in vivo measurement of the VAChT level in the brain.

Regulation of Cognitive Processing by Hippocampal Cholinergic Tone.

Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric diseases. Here we tested how information processing is regulated by cholinergic tone in genetically modified mice targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine release. We measured long-term potentiation of Schaffer collateral-CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of paired associates learning task (PAL). Acquisition of information in the mouse PAL task correlated to levels of hippocampal VAChT, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signaling. Disrupted forebrain cholinergic signaling also affected working memory, a result reproduced by selectively decreasing VAChT in the hippocampus. In contrast, spatial memory was relatively preserved, whereas reversal spatial memory was sensitive to decreased hippocampal cholinergic signaling. This work provides a refined roadmap of how synaptically secreted acetylcholine influences distinct behaviors and suggests that distinct forms of cognitive processing may be regulated in different ways by cholinergic activity.

Cholinergic/glutamatergic co-transmission in striatal cholinergic interneurons: new mechanisms regulating striatal computation.

It is well established that neurons secrete neuropeptides and ATP with classical neurotransmitters; however, certain neuronal populations are also capable of releasing two classical neurotransmitters by a process named co-transmission. Although there has been progress in our understanding of the molecular mechanism underlying co-transmission, the individual regulation of neurotransmitter secretion and the functional significance of this neuronal 'bilingualism' is still unknown. Striatal cholinergic interneurons (CINs) have been shown to secrete glutamate (Glu) in addition to acetylcholine (ACh) and are recognized for their role in the regulation of striatal circuits and behavior. Our review highlights the recent research into identifying mechanisms that regulate the secretion and function of Glu and ACh released by CINs and the roles these neurons play in regulating dopamine secretion and striatal activity. In particular, we focus on how the transporters for ACh (VAChT) and Glu (VGLUT3) influence the storage of neurotransmitters in CINs. We further discuss how these individual neurotransmitters regulate striatal computation and distinct aspects of behavior that are regulated by the striatum. We suggest that understanding the distinct and complementary functional roles of these two neurotransmitters may prove beneficial in the development of therapies for Parkinson's disease and addiction. Overall, understanding how Glu and ACh secreted by CINs impacts striatal activity may provide insight into how different populations of 'bilingual' neurons are able to develop sophisticated regulation of their targets by interacting with multiple receptors but also by regulating each other's vesicular storage. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

Newly raised anti-VAChT and anti-ChAT antibodies detect cholinergic cells in chicken embryos.

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.

Cholinergic PET imaging in infections and inflammation using 11C-donepezil and 18F-FEOBV.

INTRODUCTION: Immune cells utilize acetylcholine as a paracrine-signaling molecule. Many white blood cells express components of the cholinergic signaling pathway, and these are up-regulated when immune cells are activated. However, in vivo molecular imaging of cholinergic signaling in the context of inflammation has not previously been investigated. METHODS: We performed positron emission tomography (PET) using the glucose analogue 18F-FDG, and 11C-donepezil and 18F-FEOBV, markers of acetylcholinesterase and the vesicular acetylcholine transporter, respectively. Mice were inoculated subcutaneously with Staphylococcus aureus, and PET scanned at 24, 72, 120, and 144 h post-inoculation. Four pigs with post-operative abscesses were also imaged. Finally, we present initial data from human patients with infections, inflammation, and renal and lung cancer. RESULTS: In mice, the FDG uptake in abscesses peaked at 24 h and remained stable. The 11C-donepezil and 18F-FEOBV uptake displayed progressive increase, and at 120-144 h was nearly at the FDG level. Moderate 11C-donepezil and slightly lower 18F-FEOBV uptake were seen in pig abscesses. PCR analyses suggested that the 11C-donepezil signal in inflammatory cells is derived from both acetylcholinesterase and sigma-1 receptors. In humans, very high 11C-donepezil uptake was seen in a lobar pneumonia and in peri-tumoral inflammation surrounding a non-small cell lung carcinoma, markedly superseding the 18F-FDG uptake in the inflammation. In a renal clear cell carcinoma no 11C-donepezil uptake was seen. DISCUSSION: The time course of cholinergic tracer accumulation in murine abscesses was considerably different from 18F-FDG, demonstrating in the 11C-donepezil and 18F-FEOBV image distinct aspects of immune modulation. Preliminary data in humans strongly suggest that 11C-donepezil can exhibit more intense accumulation than 18F-FDG at sites of chronic inflammation. Cholinergic PET imaging may therefore have potential applications for basic research into cholinergic mechanisms of immune modulation, but also clinical applications for diagnosing infections, inflammatory disorders, and cancer inflammation.

Comparison of spinal cord contusion and transection: functional and histological changes in the rat urinary bladder.

OBJECTIVE: To compare the effect of complete transection (tSCI) and contusion spinal cord injury (cSCI) on bladder function and bladder wall structure in rats. MATERIALS AND METHODS: A total of 30 female Sprague-Dawley rats were randomly divided into three equal groups: an uninjured control, a cSCI and a tSCI group. The cSCI group underwent spinal cord contusion, while the tSCI group underwent complete spinal cord transection. At 6 weeks post-injury, 24-h metabolic cage measurement and conscious cystometry were performed. RESULTS: Conscious cystometry analysis showed that the cSCI and tSCI groups had significantly larger bladder capacities than the control group. The cSCI group had significantly more non-voiding detrusor contractions than the tSCI group. Both injury groups had more non-voiding contractions compared with the control group. The mean threshold pressure was significantly higher in the tSCI group than in the control and cSCI groups. The number of voids in the tSCI group was lower compared with the control group. Metabolic cage analysis showed that the tSCI group had larger maximum voiding volume as compared with the control and cSCI groups. Vesicular acetylcholine transporter/smooth muscle immunoreactivity was higher in the control than in the cSCI or tSCI rats. The area of calcitonin gene-related peptide staining was smaller in the tSCI group than in the control or cSCI groups. CONCLUSIONS: Spinal cord transection and contusion produce different bladder phenotypes in rat models of SCI. Functional data suggest that the tSCI group has an obstructive high-pressure voiding pattern, while the cSCI group has more uninhibited detrusor contractions.