Search

Neonicotinoid insecticides are nicotine-derived molecules which exert acute neurotoxic effects over the insect central nervous system by activating nicotinic acetylcholine receptors (nAChRs). However, these receptors are also present in the mammalian central and peripheral nervous system, where the effects of neonicotinoids are faintly known. In mammals, cholinergic synapses are crucial for the control of vascular tone, blood pressure and skeletal muscle contraction. We therefore hypothesized that neonicotinoids could affect cholinergic networks in mammals and sought to highlight functional consequences of acute intoxication in rats with sub-lethal concentrations of the highly used acetamiprid (ACE) and clothianidin (CLO). In this view, we characterized their electrophysiological effects on rat α3β4 nAChRs, knowing that it is predominantly expressed in ganglia of the vegetative nervous system and the adrenal medulla, which initiates catecholamine secretion. Both molecules exhibited a weak agonist effect on α3β4 receptors. Accordingly, their influence on epinephrine secretion from rat adrenal glands was also weak at 100 μM, but it was stronger at 500 μM. Challenging ACE or CLO together with nicotine (NIC) ended up with paradoxical effects on secretion. In addition, we measured the rat arterial blood pressure (ABP) in vivo by arterial catheterization. As expected, NIC induced a significant increase in ABP. ACE and CLO did not affect the ABP in the same conditions. However, simultaneous exposure of rats to both NIC and ACE/CLO promoted an increase of ABP and induced a biphasic response. Modeling the interaction of ACE or CLO on α3β4 nAChR is consistent with a binding site located in the agonist pocket of the receptor. We present a transversal experimental approach of mammal intoxication with neonicotinoids at different scales, including in vitro, ex vivo, in vivo and in silico. It paves the way of the acute and chronic toxicity for this class of insecticides on mammalian organisms.

Parkinson's disease (PD) is associated with neuronal damage in the brain and gut. This work compares changes in the enteric nervous system (ENS) of commonly used mouse models of PD that exhibit central neuropathy and a gut phenotype. Enteric neuropathy was assessed in five mouse models: peripheral injection of MPTP; intracerebral injection of 6-OHDA; oral rotenone; and mice transgenic for A53T variant human α-synuclein with and without rotenone. Changes in the ENS of the colon were quantified using pan-neuronal marker, Hu, and neuronal nitric oxide synthase (nNOS) and were correlated with GI function. MPTP had no effect on the number of Hu+ neurons but was associated with an increase in Hu+ nuclear translocation (P < 0.04). 6-OHDA lesioned mice had significantly fewer Hu+ neurons/ganglion (P < 0.02) and a reduced proportion of nNOS+ neurons in colon (P < 0.001). A53T mice had significantly fewer Hu+ neurons/area (P < 0.001) and exhibited larger soma size (P < 0.03). Treatment with rotenone reduced the number of Hu+ cells/mm 2 in WT mice (P < 0.006) and increased the proportion of Hu+ translocated cells in both WT (P < 0.02) and A53T mice (P < 0.04). All PD models exhibited a degree of enteric neuropathy, the extent and type of damage to the ENS, however, was dependent on the model.

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 μM. This depolarization was markedly inhibited by the H 1 receptor antagonist triprolidine and mimicked by the H 1 receptor agonist 2-pyridylethylamine, thus implicating histamine H 1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H 1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca 2+ removal and attenuated by extracellular Na + replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H 1 receptors and G q/11 -protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either G q/11 signalling or intracellular Ca 2+ release and by the application of the TRPC blockers Pyr3, Gd 3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd 3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The use of online remote control for 24/7 behavioural monitoring can play a key role in estimating the environmental status of aquatic ecosystems. Recording the valve activity of bivalve molluscs is a relevant approach in this context. However, a clear understanding of the underlying disturbances associated with behaviour is a key step. In this work, we studied freshwater Asian clams after exposure to crude oil (measured concentration, 167 ± 28 μg·L -1 ) for three days in a semi-natural environment using outdoor artificial streams. Three complementary approaches to assess and explore disturbances were used: behaviour by high frequency non-invasive (HFNI) valvometry, tissue contamination with polycyclic aromatic hydrocarbons (PAH), and proteomic analysis. Two tissues were targeted: the pool adductor muscles - retractor pedal muscle - cerebral and visceral ganglia, which is the effector of any valve movement and the gills, which are on the frontline during contamination. The behavioural response was marked by an increase in valve closure-duration, a decrease in valve opening-amplitude and an increase in valve agitation index during opening periods. There was no significant PAH accumulation in the muscle plus nervous ganglia pool, contrary to the situation in the gills, although the latter remained in the low range of data available in literature. Major proteomic changes included (i) a slowdown in metabolic and/or cellular processes in muscles plus ganglia pool associated with minor toxicological effect and (ii) an increase of metabolic and/or cellular processes in gills associated with a greater toxicological effect. The nature of the proteomic changes is discussed in terms of unequal PAH distribution and allows to propose a set of explanatory mechanisms to associate behaviour to underlying physiological changes following oil exposure. First, the first tissues facing contaminated water are the inhalant siphon, the mantle edge and the gills. The routine nervous activity in the visceral ganglia should be modified by nervous information originating from these tissues. Second, the nervous activity in the visceral ganglia could be modified by its own specific contamination. Third, a decrease in nervous activity of the cerebral ganglia close to the mouth, including some kind of narcosis, could contribute to a decrease in visceral ganglia activity via a decrease or blockage of the downward neuromodulation by the cerebro-visceral connective. This whole set of events can explain the decrease of metabolic activity in the adductor muscles, contribute to initiate the catch mechanism and then deeply modify the valve behaviour., Competing Interests: Declaration of Competing Interest This work was partially supported by a grant from the TOTAL oil company but we certify that it did not inappropriately influence (bias) the present work by affecting our objectivity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diabetic cardiac autonomic neuropathy (DCAN) is a serious complication of diabetes mellitus, which often leads to cardiac dysfunction and even threatens patients' life. Osthole, a natural coumarin derivative, has anti-inflammatory, anti-oxidant and antihypertensive effects. The P2X3 receptor is related to DCAN. The objective of this study will investigate whether osthole relieves DCAN associated with the P2X3 receptor in the stellate ganglia of diabetic rats. A type 2 diabetes mellitus rat model was induced by a combination of diet and streptozotocin. Our results showed that osthole improved the abnormal changes of blood pressure, heart rate, and heart rate variability in diabetic rats and significantly reduced the up-regulated expression levels of the P2X3 receptor, tumor necrosis factor-α and interleukin-1β in stellate ganglia of diabetic rats. Meanwhile, osthole significantly decreased the elevated serum adrenaline concentration and phosphorylation level of extracellular regulated protein kinase 1/2. In addition, the molecular docking result indicated that osthole was a perfect fit for interacting with the P2X3 receptor. Overall, osthole alleviates the sympathetic relative excitation via inhibiting the expression of P2X3 receptors in the stellate ganglia, to achieve a balance between sympathetic and parasympathetic nerves, relieves the DCAN., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rotenone (ROT) was shown to affect cerebral ganglions (CGs) of Lumbricus terrestris as a pioneering observation in our earlier investigation. Though ROT is a well-known neurotoxin causing neurodegeneration (ND), the precipitation of movement dysfunction remains largely unknown. We have designed the current study to analyze motor abnormalities in worms by exposing them to different concentrations (0.0-0.4 ppm) of ROT for 7 days. GABA, cholinergic receptor, serotonin transporter (SERT), acetylcholine esterase (AchE), and dopamine-β-hydroxylase that are well known for their involvement in neuromuscular junctions were investigated by qRT-PCR. Further, neuronal mitochondrial genes (cytochrome C oxidase-2, NADH deydrogenase-1, cytochrome-b) and actin-1 that are essential for regeneration and calreticulin (phagocytosis) were investigated. The levels of neurotransmitters, lipids, ATPase, neuronal behavior analyses, and fluorescence analysis (lipid droplets) were performed in CGs which showed significant variations at 0.3 ppm. Ultrastructural changes in lipid droplet and neuromelatonin were prominent in 0.3 ppm. Dose-dependent effect of ROT on behavior alteration and expression of m-RNAs studied suggested that at 0.3 ppm, it could deteriorate motor and cognitive functions. We predict that perhaps, by virtue of its effect on cerebral ganglionic genes and their neurotransmitting potential, ROT may cause morbidities that resemble features characteristic of hemiparkinsonic degeneration.

Aims: Cisplatin can damage spiral ganglion neurons (SGNs) and cause sensorineural hearing loss. Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea, but the role of Wnt signaling in protecting SGNs from cisplatin treatment has not yet been elucidated. This study was designed to investigate the neuroprotective effects of Wnt signaling against cisplatin-induced SGN damage., Results: First, we found that Wnt signaling was activated in SGNs after cisplatin treatment. Next, we discovered that overexpression (OE) of Wnt signaling in SGNs reduced cisplatin-induced SGN loss by inhibiting caspase-associated apoptosis, thus preventing the loss of SGN function after cisplatin treatment. In contrast, inhibition of Wnt signaling increased apoptosis, made SGNs more vulnerable to cisplatin treatment, and exacerbated hearing loss. TP53-induced glycolysis and apoptosis regulator (TIGAR), which scavenges intracellular reactive oxygen species (ROS), was upregulated in SGNs in response to cisplatin administration. Wnt/β-catenin activation increased TIGAR expression and reduced ROS level, while inhibition of Wnt/β-catenin in SGNs reduced TIGAR expression and increased the ROS level. Moreover, OE of TIGAR reduced ROS and decreased caspase 3 expression, as well as increased the survival of SGNs in Wnt-inhibited SGNs. Finally, antioxidant treatment rescued the more severe SGN loss induced by β-catenin deficiency after cisplatin treatment. Innovation and Conclusion: This study is the first to indicate that Wnt signaling activates TIGAR and protects SGNs against cisplatin-induced damage through the inhibition of oxidative stress and apoptosis in SGNs, and this might offer novel therapeutic targets for the prevention of SGN injury. Antioxid. Redox Signal. 00, 000-000.

Although it is well documented that exposure to aversive stimuli induces modulation of neural circuits and subsequent behavioral changes, the means by which an aversive stimulus concomitantly alters behaviors of different natures (e.g., defensive and appetitive) remains unclear. Here, we addressed this issue by using the learning-induced concurrent modulation of defensive and appetitive behaviors that occurs when the mollusk Aplysia is exposed to aversive stimuli. In Aplysia, aversive stimuli concomitantly enhance withdrawal reflexes (i.e., sensitization) and suppress feeding. Sensitization and feeding suppression, which are expressed in the short term and long term, depending on the training protocol, are accompanied by increased excitability of the tail sensory neurons (TSNs) controlling the withdrawal reflexes, and by decreased excitability of feeding decision-making neuron B51, respectively. Serotonin (5-HT) has been shown to mediate sensitization, but not feeding suppression. In this study, we examined which other neurotransmitter might be responsible for feeding suppression and its underlying cellular changes. Our results indicate that nitric oxide (NO) contributes to both short-term and long-term feeding suppression, as well as to the underlying decreased B51 excitability. NO was also necessary for the induction of long-term sensitization and for the expression of short-term increased TSN excitability in vitro, revealing a previously undocumented interaction between 5-HT and NO signaling cascades in sensitization. Overall, these results revealed a scenario in which multiple modulators contribute to the widespread changes induced by sensitizing stimuli in Aplysia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The non-proteinogenic amino acid beta-methyl-amino-l-alanine (BMAA) is a neurotoxin produced by cyanobacteria. BMAA accumulation in the brain of animals via biomagnification along the food web can contribute to the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), the latter being associated with a loss of dopaminergic neurons. Daphnia magna is an important microcrustacean zooplankton species that plays a key role in aquatic food webs, and BMAA-producing cyanobacteria often form part of their diet. Here, we tested the effects of BMAA on putative neurodegeneration of newly identified specific dopaminergic neurons in the optic ganglia/brain complex of D. magna using quantitative tyrosine-hydroxylase immunohistochemistry and fluorescence cytometry. The dopaminergic system was analysed in fed and starved isogenic D. magna adults incubated under different BMAA concentrations over 4 days. Increased BMAA concentration showed significant decrease in the stainability of dopaminergic neurons of D. magna , with fed animals showing a more extreme loss. Furthermore, higher BMAA concentrations tended to increase offspring mortality during incubation. These results are indicative of ingested BMAA causing neurodegeneration of dopaminergic neurons in D. magna and adversely affecting reproduction. This may imply similar effects of BMAA on known human neurodegenerative diseases involving dopaminergic neurons.

The Large Cell (LC) motor neurons of the crab cardiac ganglion have variable membrane conductance magnitudes even within the same individual, yet produce identical synchronized activity in the intact network. In a previous study we blocked a subset of K + conductances across LCs, resulting in loss of synchronous activity (Lane et al., 2016). In this study, we hypothesized that this same variability of conductances makes LCs vulnerable to desynchronization during neuromodulation. We exposed the LCs to serotonin (5HT) and dopamine (DA) while recording simultaneously from multiple LCs. Both amines had distinct excitatory effects on LC output, but only 5HT caused desynchronized output. We further determined that DA rapidly increased gap junctional conductance. Co-application of both amines induced 5HT-like output, but waveforms remained synchronized. Furthermore, DA prevented desynchronization induced by the K + channel blocker tetraethylammonium (TEA), suggesting that dopaminergic modulation of electrical coupling plays a protective role in maintaining network synchrony., Competing Interests: BL, DK, DW, SN, DS No competing interests declared, (© 2018, Lane et al.)

This study aimed to determine whether nerve regeneration by means of an artificial nerve conduit is promoted by ethanol-induced cervical sympathetic ganglion block (CSGB) in a canine model. This study involved two experiments-in part I, the authors examined the effect of CSGB by ethanol injection on long-term blood flow to the orofacial region; part II involved evaluation of the effect of CSGB by ethanol injection on inferior alveolar nerve (IAN) repair using polyglycolic acid-collagen tubes. In part I, seven Beagles were administered left CSGB by injection of 99.5% ethanol under direct visualization by means of thoracotomy, and changes in oral mucosal blood flow in the mental region and nasal skin temperature were evaluated. The increase in blood flow on the left side lasted for 7 weeks, while the increase in average skin temperature lasted 10 weeks on the left side and 3 weeks on the right. In part II, fourteen Beagles were each implanted with a polyglycolic acid-collagen tube across a 10-mm gap in the left IAN. A week after surgery, seven of these dogs were administered CSGB by injection of ethanol. Electrophysiological findings at 3 months after surgery revealed significantly higher sensory nerve conduction velocity and recovery index (ratio of left and right IAN peak amplitudes) after nerve regeneration in the reconstruction+CSGB group than in the reconstruction-only group. Myelinated axons in the reconstruction+CSGB group were greater in diameter than those in the reconstruction-only group. Administration of CSGB with ethanol resulted in improved nerve regeneration in some IAN defects. However, CSGB has several physiological effects, one of which could possibly be the long-term increase in adjacent blood flow.

Objectives: To evaluate neuronal nitric oxide (NO) synthase (nNOS) phosphorylation, nNOS uncoupling, and oxidative stress in the penis and major pelvic ganglia (MPG), before and after the administration of the cAMP-dependent protein kinase A (PKA) agonist colforsin in a rat model of bilateral cavernous nerve injury (BCNI),which mimics nerve injury after prostatectomy., Materials and Methods: Adult male Sprague-Dawley rats were divided into BCNI and sham-operated groups. Each group included two subgroups: vehicle and colforsin (0.1 mg/kg/day i.p.). After 3 days, erectile function (intracavernosal pressure) was measured and penis and MPG were collected for molecular analyses of phospho (P)-nNOS (Ser-1412 and Ser-847), total nNOS, nNOS uncoupling, binding of protein inhibitor of nNOS (PIN) to nNOS, gp91 phox subunit of NADPH oxidase, active caspase 3, PKA catalytic subunit α (PKA-Cα; by Western blot) and oxidative stress (hydrogen peroxide [H 2 O 2 ] and superoxide by Western blot and microdialysis method)., Results: Erectile function was decreased 3 days after BCNI and normalized by colforsin. nNOS phosphorylation on both positive (Ser-1412) and negative (Ser-847) regulatory sites, and nNOS uncoupling, were increased after BCNI in the penis and MPG, and normalized by colforsin. H 2 O 2 and total reactive oxygen species production were increased in the penis after BCNI and normalized by colforsin. Protein expression of gp91 phox was increased in the MPG after BCNI and was normalized by colforsin treatment. Binding of PIN to nNOS was increased in the penis after BCNI and was normalized by colforsin treatment. Protein expression of active Caspase 3 was increased in the MPG after BCNI and was normalized by colforsin treatment. Protein expression of PKA-Cα was decreased in the penis after BCNI and normalized by colforsin., Conclusion: Collectively, BCNI impairs nNOS function in the penis and MPG by mechanisms involving its phosphorylation and uncoupling in association with increased oxidative stress, resulting in erectile dysfunction. PKA activation by colforsin reverses these molecular changes and preserves penile erection in the face of BCNI., (© 2017 The Authors BJU International © 2017 BJU International Published by John Wiley & Sons Ltd.)

Neurons of the Grueneberg ganglion respond to cool temperatures as well as to distinct odorants and extend axonal processes to the olfactory bulb of the brain. Analyses of transgenic mice, in which Grueneberg ganglion neurons and their axons are labeled, revealed that these axons innervated nine distinct glomeruli distributed in a characteristic topographical pattern in dorsal, lateral, ventral, and medial regions of rather posterior areas in the bulb. To assess activation of these glomeruli (hereinafter designated as Grueneberg glomeruli) upon stimulation of Grueneberg ganglion neurons, mice were exposed to the odorant 2,3-dimethylpyrazine (2,3-DMP) and the expression of the activity-dependent marker c-Fos in juxtaglomerular cells of the relevant glomeruli was monitored. It was found that all of these glomeruli were activated, irrespective of their localization in the bulb. To verify that the activation of juxtaglomerular cells in Grueneberg glomeruli was indeed based on stimulation of Grueneberg ganglion neurons, the 2,3-DMP-induced responses in these glomeruli were investigated in mice lacking the cyclic nucleotide-gated channel CNGA3 which is critical for chemo- and thermosensory signal transduction in Grueneberg ganglion neurons. This approach revealed that elimination of CNGA3 led to a reduction of the odorant-induced activity in Grueneberg glomeruli, indicating that the activation of these glomeruli is based on a preceding stimulation of the Grueneberg ganglion. Analyzing whether Grueneberg glomeruli in the bulb might also process thermosensory information, it was found that upon exposure to coolness, Grueneberg glomeruli were activated. Investigating mice lacking CNGA3, the activation of these glomeruli by cool temperatures was attenuated.

Cocaine- and amphetamine-regulated transcript (CART) peptides are widely expressed not only in the brain but also in numerous endocrine/neuroendocrine cells as well as in neurons of the peripheral nervous system. The present study investigated the distribution patterns of CART-like immunoreactivity in the pelvic plexus (PP) of the female pig. The co-expression of CART with principal neurotransmitter markers: choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5-HT) or biologically active neuropeptides: pituitary adenylate cyclase-activating polypeptide (PACAP), substance P (SP), calbindin was analyzed using double immunohistochemical stainings. Amongst neurons immunopositive to Hu C/D panneuronal marker as many as 4.1 ± 1.2% in right and 4.4 ± 1.6% in left pelvic ganglia were found to express CART. The vast majority of CART-IR ganglionic neurons were predominantly small in size and were evenly scattered throughout particular ganglia. Immunoreactivity to CART was also detected in numerous nerve terminals (which frequently formed pericellular formations around CART-negative perikarya) as well as in numerous nerve fibres within nerve branches interconnecting the unilateral pelvic ganglia. Immunohistochemistry revealed that virtually all CART-IR neurons were cholinergic in nature and CART-IR basket-like formations frequently encircled TH-positive/CART-negative perikarya. None of CART-IR ganglionic neurons showed immunoreactivity to SP, PACAP, 5-HT or calbindin. CART-IR nerve fibres ran in a close vicinity to serotonin-containing cells or faintly labelled SP-expressing neurons. On the other hand, PACAP-IR, SP-IR (but not 5-HT-positive) nerve terminals were found to run in close proximity to CART-IR neurons. Our results indicate that: 1) CART present in PP may influence the activity of pelvic ganglionic neurons/SIF cells, 2) PP should be considered as a potential source of CART-like supply to pelvic viscera and 3) functional interactions between CART and SP or PACAP are possible at the periphery.

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm 2 ) at 60 mM. In the case of cumulative dose-response of bethanecol, the maximum response (37.42 mN/mm 2 ) was reached at 3.2 × 10 -4  M. The response to ATP was clearly smaller (3.8 mN/mm 2 ). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans.

Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders distributing in the hilly areas of southern China. In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive to ω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of ∼51.3 mg/L. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons., (Copyright © 2016. Published by Elsevier Inc.)

Rotenone is well-documented to cause neurodegenerative condition such as Parkinson's, in the exposed systems. However, its detrimental effect on particular sites of neuronal pathway is still under investigation. We aimed at elucidating the impact of rotenone on cerebral ganglions (CG) of Lumbricus terrestris which control movement and behaviour of the worms. Worms were exposed to 0-0.4 ppm/mL of rotenone. Mitochondrial and lysosomal integrities were found to be affected beyond 0.2 ppm/mL of rotenone. Activities of cholinergic enzymes and the expression of tyrosine hydroxylase showed an impaired neuronal transmission in CGs at the dose of 0.2 ppm/mL of rotenone. Histopathological and immunoflourescent analyses showed neuronal apoptosis, reduced nucleic acid content and inhibited of neurosecretion at 0.3 ppm/mL. Electron microscopy showed that the neurons and neuromuscular junctions were affected at 0.2 ppm/mL. Dose-dependent changes were also observed in the motor function such as burrowing behaviours and locomotion. Conduction velocity (CV) and locomotion assessment showed that the CV of lateral giant fiber (LGF) was reduced while that of MGF remains unaffected at 0.2 ppm, the dose at which the burrowing behaviour was also not affected. LGF, cholinergic enzymes and tyrosine hydroxylase are primarily targeted by rotenone affecting locomotion at 0.2 ppm/mL while MGF, neuropile and the burrowing behaviour were affected at 0.3 ppm/mL. We demonstrate, in addition to dose-dependent effects, that the bioaccumulation factors range 0.28-0.32 ppm/μg of rotenone cause degenerative impact on giant fibers affecting neuronal behaviors/locomotion of worms. We also propose worms for studying mechanisms of neuronal pathology caused by chemicals prevailing in earth's atmosphere., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Erectile dysfunction (ED) is a serious medical condition in which current treatments are ineffective in prostatectomy and diabetic patients, due to injury to the cavernous nerve (CN), which causes irreversible remodeling of the penis (decreased smooth muscle and increased collagen), through a largely undefined mechanism. We propose that sonic hedgehog (SHH) and neural innervation, are indispensable regulators of collagen in the penis, with decreased SHH protein being an integral component of the fibrotic response to loss of innervation. We examined collagen abundance and morphology in control (Peyronie's), prostatectomy and diabetic patients, and in rat models of penile development, CN injury, SHH inhibition and under regenerative conditions, utilizing self-assembling peptide amphiphile (PA) nanofiber hydrogels for SHH delivery. Collagen abundance increased in penis of ED patients. In rats, collagen increased with CN injury in a defined time frame independent of injury severity. An inverse relationship between SHH and collagen abundance was identified; SHH inhibition increased and SHH treatment decreased penile collagen. SHH signaling in the pelvic ganglia (PG)/CN is important to maintain CN integrity and when inhibited, downstream collagen induction occurs. Collagen increased throughout penile development and with age, which is important when considering how to treat fibrosis clinically. These studies show that SHH PA treatment reduces collagen under regenerative post-prostatectomy conditions, indicating broad application for ED prevention in prostatectomy, diabetic and aging patients and in other peripheral nerve injuries. The PA nanofiber protein vehicle may be widely applicable as an in vivo delivery tool., Statement of Significance: We use self-assembling peptide amphiphiles (PA) as biological delivery vehicles to prevent cavernous nerve (CN) injury induced erectile dysfunction (ED). These versatile hydrogels were molecularly pre-programmed for sonic hedgehog (SHH) protein delivery, either from an injectable solution with fast, in situ assembly into a soft hydrogel, or by highly aligned monodomain nanofiber bundles. We used PAs to examine a novel neuronal component to collagen regulation and the role of SHH in the fibrotic response to CN injury. SHH perturbation in the penis or the CN, selectively impacts collagen, with SHH inhibition increasing and SHH treatment suppressing collagen. These results suggest that SHH treatment by PA has translational potential to suppress collagen induction and remodelling, an irreversible component of ED development., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChR or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntington's disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntington's disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntington's disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells., (© 2015. Published by The Company of Biologists Ltd.)

This study was aimed to analyze the alteration of membrane protein profiles in Aplysia juliana Quoy & Gaimard (A. juliana) pleural–pedal ganglia under MP exposure. Both the results of GC–MS analysis and the activity assay of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT) reveal that MP toxicological effects on Aplysia left and right pleural–pedal ganglia are different under 7 and 14 days of exposure. Therefore, Aplysia were subjected for exposure at two concentrations (1 and 2 mg/l) of MP for 7 and 14 days for membrane proteomic study. As a result, 19 and 14 protein spots were differentially expressed in A. juliana left pleural–pedal ganglia under 7 and 14 days treatment, and 20 and 14 protein spots found with differential expressions in their right ganglia under the same treatment, respectively. Several proteins with expression variations were detected from both the left and right pleural–pedal ganglia; however, most proteins have distinctive expressions, indicating different mechanisms might be involved in initiating MP toxicology in left and right ganglia. Among the total differential protein spots obtained, 29 proteins were classed as membrane proteins. These proteins are mainly involved in the metabolism process, cell redox homeostasis, signal transduction, immunology, intracellular transport and catalysis, indicating MP toxicity in mollusks seems to be complex and diverse. Some differentially expressed proteins were further confirmed by Western blotting and quantitative real-time PCR. These results might provide renovated insights to reveal the mechanism of MP-induced neurotoxicity, and the novel candidate biomarkers might have potential application for environmental evaluation of MP pollution level.

Background: Taste disorders are frequently observed in cancer patients undergoing chemotherapy and are serious adverse events which impair the quality of life (QoL) of the cancer patient. Nevertheless, taste disorder mechanisms in cancer patients undergoing chemotherapy have not yet been fully elucidated. The aim of this study was to reveal taste disorder-related peripheral mechanisms using the two-bottle preference test (TBPT) and histological examination of tongues by hematoxylin-eosin staining and immunohistochemistry with protein-gene product 9.5., Methods: In the TBPT, one bottle was filled with the 0.01 mM quinine hydrochloride (quinine), as a bitter compound, and the other was filled with water. Doses of 50 and 100 mg kg(-1) day(-1) S-1 (tegafur/gimeracil/oteracil potassium) are lethal to Wistar rats. Therefore, doses ranging from 2-20 mg kg(-1) day(-1) were administered to the rats for 3 weeks. The S-1 dose of 2 mg kg(-1) day(-1) corresponds to the clinical dose administered to cancer patients. The part of the tongue containing the circumvallate papillae was excised the following TBPT., Results: The rate of increase in terms of the average preference rate for the quinine vs. all intake (quinine plus water) was significant from the initial S-1 period to the final one, compared with that in control rats, suggesting the possibility of a worsening sensation for the bitter taste. In S-1 rats, the area of taste nerve fibers were significantly decreased and the rate of degeneration of intra-tongue ganglionic nerve cells was significantly increased. These changes were significantly correlated with the rate of increase in average preference rate of the quinine., Conclusion: Neuropathy of the gustatory nerve at the periphery may be involved in taste disorders induced by an anticancer drug.

Evidence has shown gender differences regarding the critical roles of histamine in the prevalence of asthma, anaphylaxis, and angina pectoris. Histamine depolarizes unmyelinated C-type neurons without any effects on myelinated A-type vagal ganglion neurons (VGNs) in male rats. However, little is known if VGNs from females react to histamine in a similar manner. Membrane depolarization and inward currents were tested in VGNs isolated from adult rats using a whole-cell patch technique. Results from males were consistent with the literature. Surprisingly, histamine-induced depolarization and inward currents were observed in both unmyelinated C-type and myelinated A- and Ah-type VGNs from female rats. In Ah-type neurons, responses to 1.0 μM histamine were stronger in intact females than in males and significantly reduced in ovariectomized (OVX) females. In C-type neurons, histamine-induced events were significantly smaller (pA/pF) in intact females compared with males and this histamine-induced activity was dramatically increased by OVX. Female A-types responded to histamine, which was further increased following ovariectomy. Histamine at 300 nM depolarized Ah-types in females, but not Ah-types in OVX females. In contrast, the sensitivity of A- and C-types to histamine was upregulated by OVX. These data demonstrate gender differences in VGN chemosensitivity to histamine for the first time. Myelinated Ah-types showed the highest sensitivity to histamine across female populations, which was changed by OVX. These novel findings improve the understanding of gender differences in the prevalence of asthma, anaphylaxis, and pain. Changes in sensitivity to histamine by OVX may explain alterations in the prevalence of certain pathophysiological conditions when women reach a postmenopausal age.

The motor cortex represents muscle and joint control and projects to spinal cord interneurons and-in many primates, including humans-motoneurons, via the corticospinal tract (CST). To examine these spinal CST anatomical mechanisms, we determined if motor cortex sites controlling individual forelimb joints project differentially to distinct cervical spinal cord territories, defined regionally and by the locations of putative last-order interneurons that were transneuronally labeled by intramuscular injection of pseudorabies virus. Motor cortex joint-specific sites were identified using intracortical-microstimulation. CST segmental termination fields from joint-specific sites, determined using anterograde tracers, comprised a high density core of terminations that was consistent between animals and a surrounding lower density projection that was more variable. Core terminations from shoulder, elbow, and wrist control sites overlapped in the medial dorsal horn and intermediate zone at C5/C6 but were separated at C7/C8. Shoulder sites preferentially terminated dorsally, in the dorsal horn; wrist/digit sites, more ventrally in the intermediate zone; and elbow sites, medially in the dorsal horn and intermediate zone. Pseudorabies virus injected in shoulder, elbow, or wrist muscles labeled overlapping populations of predominantly muscle-specific putative premotor interneurons, at a survival time for disynaptic transfer from muscle. At C5/C6, CST core projections from all joint zones were located medial to regions of densely labeled last-order interneurons, irrespective of injected muscle. At C7/C8 wrist CST core projections overlapped the densest interneuron territory, which was located in the lateral intermediate zone. In contrast, elbow CST core projections were located medial to the densest interneuron territories, and shoulder CST core projections were located dorsally and only partially overlapped the densest interneuron territory. Our findings show a surprising fractionation of CST terminations in the caudal cervical enlargement that may be organized to engage different spinal premotor circuits for distal and proximal joint control.

We have devised a new caged GABA based on ruthenium bipyridyl coordination chemistry. This phototrigger delivers GABA upon irradiation with wavelengths up to 532 nm undergoing heterolytic photocleavage, in a clean and very fast (a few nanoseconds) photoreaction. With an absorptivity coefficient ε(MAX) = 5300 M(-1) cm(-1) at 447 nm and a quantum efficiency φ ~ 0.09, RuBiGABA-2 is among the most active caged-GABAs, especially at long wavelengths. This highly hydrophilic caged GABA can be synthesized in a simple one-pot reaction. The synthesis, chemical characterization and photochemical properties are presented. Finally, the usefulness of this caged compound is demonstrated by photodelivering free GABA on leech motoneurons.

Contractile activity of damaged neuronal axons of Lymnaea stagnalis and Planorbis corneus vulgaris mollusks and the possibility of inhibiting their retraction by cytochalasin B were studied. In experimental series I (control), the neuronal axons contracted in Ringer's fluid in 90% cases. In series II and III (cytochalasin B in concentrations of 0.02 and 0.2 mM), the percentage of non-contracting neurons was 50 and 70%, respectively. Presumably, the fiber retraction mechanism was involved in the formation of diastasis after nerve cutting and damage to conduction tracts. The nerve diastasis formed at the expense of not only elastic characteristics of the nerve sheath and glia, but also due to nerve fiber retraction. Experiments with cytochalasin B demonstrated that F-actin filaments were involved in the retraction of myelin-free nerve fibers.

The structural and functional plasticity of Aplysia mechanosensory presynaptic neurons has been studied in relation with the mechanism underlying learning and memory. Long-term facilitation (LTF), which is a well-known cellular model for long-term memory in Aplysia, is accompanied by new synaptic structural growth or change. We developed a combined atomic force microscope and confocal laser scanning microscope (AFM-CLSM) system integrated with a MATLAB routine for image processing to concurrently obtain high-resolution 3-dimensional (3D) outer-surface morphological images and 3D interior fluorescence images. With our combined AFM-CLSM system, volumetric changes in the presynaptic structures (varicosities) of Aplysia live sensory-motor neuron cocultures were observed. The spatial distribution of synaptic vesicle molecules in the preexisting varicosities was monitored together with a volumetric change in the varicosities. Our combined AFM-CLSM system is successfully adapted for measuring learning-related structural changes and the movement of synaptic molecules in the single live neuron through interaction force and fluorescence imaging.

Neurodegeneration is one of the most important complications of diabetes mellitus (DM). The exact mechanisms underlying neurodegeneration related to diabetic complications such as cognitive deficits and peripheral neuropathy are not clarified yet. Due to the fact that CCAAT/enhancer binding proteins (C/EBPs) have roles in cognitive functions, memory, synaptic plasticity, inflammation, lipid storage, and response to neurotrophic factors, it is possible to suggest that these transcription factors could have roles in neurodegeneration. Hence, in this study, the effects of experimental diabetes on C/EBPs in the hippocampus, sciatic nerve, and ganglia tissues were examined. After experimentally induced diabetes, immunoreactivity of related proteins was measured by western blotting. C/EBPα immunoreactivity in the hippocampus was not altered at 4-weeks but significantly decreased at 12-weeks of diabetes. C/EBPβ immunoreactivity was not altered at 4-weeks whereas significantly increased at 12-weeks of diabetes. In the ganglion, C/EBPα immunoreactivity was significantly decreased in diabetes, but C/EBPβ immunoreactivity was not affected. In the sciatic nerve, C/EBPα and β immunoreactivities were significantly decreased in diabetic rats. Furthermore, insulin therapy prevented diabetes-induced alterations in C/EBPα and β immunoreactivities. This study indicated, for the first time, that DM altered the immunoreactivity of C/EBPs in the nervous system. C/EBPs might be one of the important molecular targets which are responsible for neurodegeneration seen in diabetes.

We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1-2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 μM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.

To understand the relationship among cholesterolemia, hyperglycemic stage in non obese type 2 diabetes mellitus, and histological perturbations on liver, retina, hippocampus, and Harderian gland, we maintained rat on a diet high in cholesterol for fourteen weeks, then analyzed blood lipid profiles, blood glucose, hepatic enzymes, and microscopic lesion of those tissues. We observed that high cholesterol-treated rat elevated in cholesterol and low density lipoprotein with not correlated to hyperglycemia. Histopathological changing in Goto-Kakizaki rat on liver (microvesicular steatosis) and Harderain gland (tubular lesions) were related to hyperglycemic effect rather than cholesterolemic effect. These may be related to hypoinsulinemic characteristic of this diabetic model. However increasing pyknotic nuclei on hippocampus and reducing of retinal ganglionic cell were related to the high level of cholesterol loaded with synergized effect due to diabetic stage.

The aim of this study was to determine the effects of VEGF treatment on focal cerebral ischemia in rats. Rats were administered PBS or VEGF at concentrations of 10, 20 or 30 µg/ml. The effects of VEGF on the rat infarct volume and neurological deficits were investigated. Transmission electron microscopy was used to observe the ultrastructure of the cerebral cortex. Treatments with VEGF reduced the infarct volume and improved neurological functions. VEGF increased microvessel generation and also inhibited apoptosis in the cerebral cortex and basal ganglia. For the rats in the 30 µg/ml VEGF group, an even higher number of proliferative endothelial cells were observed by electron microscopy. In conclusion, VEGF treatment has protective effects on focal cerebral ischemia in rats.

Histamine is an endogenous biogenic amine that is synthesized from the basic amino acid histidine. Ability to mimic anaphylaxis is one of the first described functions of histamine and it has been demonstrated that histamine plays a significant role in the regulation of immune system and neuronal function, influences neuronal morphology and is involved in mast cells (MCs) chemotaxis. MCs as histamine releasers, may thus also interact with neuronal function. In the present study, we aimed to evaluate the role of histamine on mast cell density and neuronal morphology in the gastrointestinal tract of the mouse. Ten mice were daily injected intraperitoneally for 7 days with 20 mg/kg of histamine diluted in 0.5 ml physiological serum. After 7 days, mice were euthanised and samples from stomach, small bowel, colon and appendix were processed for histological examination. Immunohistochemistry was performed employing primary antibodies directed against triptase for mast cells and PGP 9.5 antigen for neuronal structures. The density of triptase and PGP 9.5 positive cells and the morphology of the ganglia were quantitatively evaluated by digital image analysis. The number of ganglia was higher in stomach, small bowel, colon and appendices of the histamine group when compared with the control group. Only in appendices and colon, the number of Schwann cells was significantly higher than that of the control group. The PGP 9.5 expression and the mean area of ganglia showed a significant increase only in appendices. In histamine group the MCs were clustered especially in the lamina propria. Mast cell density (MCD) was significantly higher than the control group in the small bowel, colon and appendices tissues. The intraperitoneally injection histamine increases the MCD and induces the neuronal hypertrophy and after the comparison of the organs in the gastrointestinal tract the results indicated the most effected organ as the appendices., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3'-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.

The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and ageing. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF), are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuroprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent the death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H(2)O(2)). H(2)O(2) treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, bis-benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H(2)O(2)-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The aim of the present study was to find out the mechanism by which the inflammatory mediator, bradykinin, induces an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in enteric neurons. For this purpose, ganglia in the isolated submucosa from rat colon were loaded with the Ca(2+)-sensitive dye, fura-2, and were exposed to bradykinin (2·10(-8)mol/l). Under control conditions, the kinin evoked a transient increase in [Ca(2+)](i). Preincubation with quinacrine or arachidonyltrifluoromethylketone (AACOCF(3)), i.e. blockers of cytosolic phospholipase A(2), prevented the raise of [Ca(2+)](i). This inhibition was mimicked by 5,8,11,14-eicosatetrayonic acid (ETYA), an inhibitor of cyclooxygenases as well as lipoxygenases, and by BWA4C, a selective inhibitor of lipoxygenases, whereas indomethacin was ineffective, suggesting the mediation of the kinin response by a lipoxygenase metabolite. Indeed, a leukotriene, leukotriene D(4) (LTD(4)), mimicked the effect of bradykinin. The LTD(4) receptor blocker, MK-571, inhibited the increase in [Ca(2+)](i) evoked by LTD(4) and by bradykinin. Consequently, bradykinin receptors in submucosal ganglia from rat colon are coupled to a stimulation of phospholipase A(2), the release of arachidonic acid and the production of LTD(4), which seems to be finally responsible for the change in the cytosolic Ca(2+) concentration., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: Acrylamide is a neurotoxic, genotoxic substance present in many commonly consumed food products and has been shown to have carcinogenic effects in rodents. The protective effects (if any) of potato fiber preparations, composed of cell wall material from potatoes, against the toxic influence of dietary acrylamide on the small intestinal wall were investigated., Methods: Male mice of the BALB/c strain were used in the study. Acrylamide was administered to the mice in their drinking water (0.5 mg/kg of body weight per day) and one of two types of potato fiber preparations (heated or raw potato fiber preparation) was added to their feed (2% addition to their feed). Histomorphometry of the small intestinal wall, hemoglobin adducts of acrylamide, animal weight, and feed and water consumption analyses were performed., Results: Acrylamide altered the morphology and histology of the small intestinal wall, decreasing proliferation, myenteron and submucosal thicknesses, villus length, fractal dimension, crypt depth, crypt number, and the small intestinal absorptive surface. Conversely, apoptosis, hemoglobin adduct levels, intensity of epithelium staining, enterocyte number, villus epithelial thickness, and crypt width and parameters associated with nerve ganglia were increased. The two potato fiber preparations that were used abolished the negative influences of acrylamide on the small intestinal wall and had no influence on the hemoglobin adduct levels of acrylamide., Conclusion: The negative impact of acrylamide on the histologic structure, regeneration, and innervation of the small intestinal wall and the absorptive function of the small intestinal mucosa can be abolished by dietary potato fiber preparations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 μM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 μM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.

Oxytocin (OT) has been reported to have a potential protective effect on stress-induced functional gastrointestinal disorders. This study determined whether colonic contraction in adults was affected by antenatal maternal hypoxia, and whether OT is involved in antenatal maternal hypoxia induced colonic contraction disorder. Isometric spontaneous contractions were recorded in colonic longitudinal muscle strips in order to investigate colonic contractions and the effects of exogenous OT on the contraction in antenatal maternal hypoxia and control mice. Both high potassium and carbachol-induced contractions of proximal colon but not distal colon were reduced in antenatal maternal hypoxia mice. Exogenous OT decreased the contractions of proximal colonic smooth muscle strips in control mice, while it increased contractions in antenatal maternal hypoxia mice. OT increased the contractions of distal colonic smooth muscle strips in both antenatal maternal hypoxia and control mice. Hexamethonium blocked the OT-induced potentiation of proximal colon but not distal colon in antenatal maternal hypoxia mice. These results suggest that exogenous oxytocin reverses the decrease of proximal colonic smooth muscle contraction in antenatal maternal hypoxia mice via ganglia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: To value the possibility and the future feasibility of the use of autograft cells transplantation in disorders of the enteric neural system, we postulate that isolated neonatal nongenetically modified neural crest progenitors could survive and differentiate into neurons and glia in homogenic denervated rats and, therefore, restore partial intestinal function after transplantation., Methods: Neural crest progenitors were isolated from neonatal rats. After passages, the cells were labeled with CM-DiI. The labeled cells were then delivered into the muscular distal denervated colon of rats whose neural plexuses were eliminated using benzalkonium chloride. The treated colons of recipients were harvested at 1, 4, and 8 wk, and identified by immunofluorescent staining. The physiologic and functional improvements on treated colons were well examined after transplantation 8 wk., Results: Progenitors could generate neurospheres and differentiate into neurons and glia in vitro. After transplantation, red fluorescent cells were observed in the injected tissue for up to 8 wk, and they differentiated into neurons and glia in the host colon. Functional examinations indicated that symptoms and intestinal dysfunction of the denervated model were reversed., Conclusions: We provide herein further evidence that autologous cell transplantation is a feasible therapy for enteric nervous system disorders., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

Bardet-Biedl Syndrome (BBS) is a heterogeneous syndromic form of retinal degeneration. We have identified a novel transcript of a known BBS gene, BBS3 (ARL6), which includes an additional exon. This transcript, BBS3L, is evolutionally conserved and is expressed predominantly in the eye, suggesting a specialized role in vision. Using antisense oligonucleotide knockdown in zebrafish, we previously demonstrated that bbs3 knockdown results in the cardinal features of BBS in zebrafish, including defects to the ciliated Kupffer's Vesicle and delayed retrograde melanosome transport. Unlike bbs3, knockdown of bbs3L does not result in Kupffer's Vesicle or melanosome transport defects, rather its knockdown leads to impaired visual function and mislocalization of the photopigment green cone opsin. Moreover, BBS3L RNA, but not BBS3 RNA, is sufficient to rescue both the vision defect as well as green opsin localization in the zebrafish retina. In order to demonstrate a role for Bbs3L function in the mammalian eye, we generated a Bbs3L-null mouse that presents with disruption of the normal photoreceptor architecture. Bbs3L-null mice lack key features of previously published Bbs-null mice, including obesity. These data demonstrate that the BBS3L transcript is required for proper retinal function and organization., Competing Interests: The authors have declared that no competing interests exist.

Bradykinin acts as an inflammatory mediator in the gut. In the present study we characterized bradykinin-induced changes in the intracellular calcium concentration ([Ca(2+)](i)) in whole-mount submucosal preparations from rat distal colon and examined the bradykinin receptors and subsequent signalling cascades involved. Bradykinin (2.10(-10)-2.10(-7)mol/l) evoked a concentration-dependent increase in [Ca(2+)](i) in about 90% of the investigated neurones. This Ca(2+) response was abolished by the bradykinin B(2) receptor antagonist HOE 140. The B(2) receptor agonist [Hyp(3)]-bradykinin mimicked the kinin response. In contrast, the B(1) receptor antagonist [des-Arg(10)]-HOE 140 and the B(1) receptor agonist bradykinin fragment 1-8 were ineffective. Immunohistochemical experiments confirmed the presence of bradykinin B(2) receptors in submucosal neurones. The effect of bradykinin on [Ca(2+)](i) was not mediated by a release of prostaglandins, as it was resistant against the cyclooxygenase inhibitor indomethacin. Blocking of G(q/11) proteins with YM-254890 suppressed the action of bradykinin, revealing that neuronal bradykinin B(2) receptors are coupled to this G protein. However, the subsequent signalling cascade differed from the classical phospholipase C signalling pathway, as the bradykinin response was resistant against the phospholipase C inhibitor U-73221, the ryanodine receptor antagonist dehydroryanodine, and only marginally sensitive against the blocker of IP(3)-receptors xestospongin C. Vice versa, the effect of bradykinin was nearly completely dependent on the presence of external Ca(2+) and could be reduced by lanthanum, a blocker of voltage-operated Ca(2+) channels, suggesting that the bradykinin-induced Ca(2+) response is achieved by an influx from the extracellular space via voltage-operated Ca(2+) channels., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10(-6)M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies <0.7Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.

Introduction: Immunophilin ligands function by binding to receptor proteins such as FK506 binding proteins (FKBPs). FKBPs are studied for their roles in neuroprotection., Aim: Compare the effect of FK506 (FK) and rapamycin (RAP) on erectile function (EF) recovery and FKBP expressions in penis and major pelvic ganglion (MPG) after cavernous nerve (CN) injury., Methods: Adult male rats were divided into four groups: sham surgery (CN exposure only) + vehicle; bilateral CN injury (BCNI; bilateral crush, 3 minutes with hemostat clamp) + vehicle; BCNI + FK (5 mg/kg/day, 5 days, sc); and BCNI + RAP (2 mg/kg/day, 5 days, sc). At both 24 hours (Day 1) or 1 week (Day 7) after BCNI, EF was assessed by intracavernosal pressure measurement and FKBPs 12, 38, 52, and 65 expressions were evaluated by Western blot analysis in collected penises and MPGs., Main Outcome Measures: EF and change in protein expressions of FKBPs in the rat penis and MPG after BCNI with and without immunophilin ligand treatment., Results: Both FK- and RAP-treated rats had preserved EF compared with vehicle-treated rats after BCNI. FKBPs changed variably following injury and treatment. In particular, in the penis at Day 1, FKBP 38 expression was decreased after BCNI and both FK and RAP attenuated this decrease. In MPG at Day 1, FKBP 38 expression was also decreased after BCNI and FK attenuated the decrease, while at Day 7, FKBP 38 expression was still decreased and RAP attenuated the decrease. Also, in the penis at Day 1, FKBP 65 expression decreased after BCNI and FK attenuated the decrease. In the MPG, FKBP 65 expression increased at both Days 1 and 7 with FK treatment., Conclusions: Improved EF after BCNI, as shown with RAP, further suggests a role of immunophilin ligands as a protective therapy of CN injury associated erectile dysfunction. Our findings also suggest that select FKBPs, such as FKBP 38 and FKBP 65, may mediate these effects.

The coupling between Ca2+ extrusion and H+ uptake by the ubiquitous plasma membrane calcium ATPase (PMCA) has not been measured in any neurone. I have investigated this with Ca2+- and pH-sensitive microelectrodes in large voltage-clamped snail neurones, which have no Na+-Ca2+ exchangers. The recovery of [Ca2+]i and surface pH after a brief depolarization or Ca2+ injection was not slowed by hyperpolarization to -90 mV from a holding potential of -50 mV, consistent with a 1 Ca2+ : 2 H+ coupling ratio. Since Ca2+ injections proved difficult to quantify, and Ca2+ currents through channels were obscured by K+ currents, Ba2+ was used as a substitute. When the cell was bathed in Ca2+-free Ba2+ Ringer solution, the K+ currents were blocked and large inward currents were revealed on depolarization. The Ca2+-sensitive microelectrodes were sensitive to intracellular Ba2+ as well as Ca2+. With equal depolarizations Ba2+ entry appeared larger than Ca2+ entry and generated similar but slower pH changes. Ba2+ extrusion was insensitive to hyperpolarization, blocked by eosin or high pH, and about 5 times slower than Ca2+ extrusion. The ratio of the pH change caused by the extrusion of unit charge of Ba2+ influx to that caused by unit charge of H+ injection was 0.85 +/- 0.08 (s.e.m., n = 8), corresponding to a Ba2+ : H+ ratio of 1 : 1.7. Both this ratio and the electroneutrality of the PMCA suggest that the Ca2+ : H+ ratio is 1 : 2, ensuring that after a Ca2+ influx [Ca2+]i recovery is not influenced by the membrane potential and maximizes the conversion of Ca2+ influxes into possible pH signals.

Vagal pulmonary myelinated afferents are normally not activated by capsaicin, a selective agonist of transient receptor potential vanilloid type 1 (TRPV1) receptors. This study was carried out to investigate whether the expression of TRPV1 in these afferents is altered when chronic airway inflammation is induced by ovalbumin (Ova) sensitization. Two groups of Brown-Norway rats (sensitized and control) were exposed to aerosolized Ova and vehicle, respectively, 3 days per week for 3 weeks. After the C-fibre conduction in both vagus nerves was blocked, right-atrial injection of capsaicin elicited augmented breaths in sensitized rats breathing spontaneously, but not in control rats, indicating a stimulation of rapidly adapting receptors (RARs) by capsaicin. Single-unit fibre activities of RARs and slow adapting receptors (SARs), identified by their firing behaviour and adaptation indexes in response to lung inflation, were recorded in anaesthetized, vagotomized and artificially ventilated rats. Capsaicin injection evoked either negligible or no response in both RARs and SARs of control rats. However, in striking contrast, the same dose of capsaicin evoked an immediate stimulatory effect on these myelinated afferents in sensitized rats. Furthermore, the immunohistochemistry experiments showed that there was a significant increase in the proportion of TRPV1-expressing pulmonary neurones in nodose ganglia of sensitized rats; this increase in TRPV1 expression was found mainly in neurofilament-positive (myelinated) neurones. In conclusion, allergen-induced airway inflammation clearly elevated capsaicin sensitivity in myelinated pulmonary afferents, which probably resulted from an increased expression of TRPV1 in these sensory nerves.

Objective: To investigate whether copolymer-1 (Cop-1), a synthesized analogue of myelin basic protein, can protect the retina ganglion cells (RGCs) and possible mechanism thereof., Methods: Vortex veins of rats were ligated to induce an increase of intraocular pressure (IOP) so as to establish glaucoma models. 106 rat models were randomly divided into 2 equal groups: Cop-1 Group undergoing subcutaneous injection of 200 microg Cop-1, and phosphate buffered solution (PBS) Group undergoing PBS injection as controls. Fluorogold was used to retrogradely label the RGCs. 10, 17, 24, and 31 days after the immunization some rats were killed with their eyeballs taken out. The fluorogold positive spots were calculated. Anti-rat T cell receptor (TCR) monoclonal antibody was used to mark T cells in the retina., Results: 17, 24, and 31 days after the immunization, the RGC density of the Cop-1 Group were (2617 +/- 17)/mm(2), (2588 +/- 206)/mm(2), and (2394 +/- 15)/mm(2) respectively, all significantly higher than those of the PBS Group [(2357 +/- 37)/mm(2), (2277 +/- 340)/mm(2), and (2129 +/- 17)/mm(2) respectively, all P < 0.05]. 10, 17, 24, and 31 days after the immunization the numbers of T cells in the retina of Cop-1 Group were (11.3 +/- 2.8)/mm(2), (36.7 +/- 5.2)/mm(2), (33.9 +/- 3.0)/mm(2), and (21.4 +/- 5.9)/mm(2), all significantly higher than those of PBS Group (4.7 +/- 3.6)/mm(2), (19.7 +/- 2.4)/mm(2), (15.3 +/- 4.0)/mm(2), and (13.3 +/- 4.4)/mm(2) respectively, all P < 0.01)., Conclusion: Cop-1 protects RGCs. The injury of RGCs induced by increased IOP results in congregation of T cells. Cop-1 increases the number of T-lymphocyte cells congregating in the retina, which may be related to its neuroprotection.

Many central pattern generating networks are influenced by synaptic input from modulatory projection neurons. The network response to a projection neuron is sometimes mimicked by bath applying the neuronally-released modulator, despite the absence of network interactions with the projection neuron. One interesting example occurs in the crab stomatogastric ganglion (STG), where bath applying the neuropeptide pyrokinin (PK) elicits a gastric mill rhythm which is similar to that elicited by the projection neuron modulatory commissural neuron 1 (MCN1), despite the absence of PK in MCN1 and the fact that MCN1 is not active during the PK-elicited rhythm. MCN1 terminals have fast and slow synaptic actions on the gastric mill network and are presynaptically inhibited by this network in the STG. These local connections are inactive in the PK-elicited rhythm, and the mechanism underlying this rhythm is unknown. We use mathematical and biophysically-realistic modeling to propose potential mechanisms by which PK can elicit a gastric mill rhythm that is similar to the MCN1-elicited rhythm. We analyze slow-wave network oscillations using simplified mathematical models and, in parallel, develop biophysically-realistic models that account for fast, action potential-driven oscillations and some spatial structure of the network neurons. Our results illustrate how the actions of bath-applied neuromodulators can mimic those of descending projection neurons through mathematically similar but physiologically distinct mechanisms.