BACKGROUND/AIMS: Gastric peristalsis begins in the orad corpus and propagates to the pylorus. Directionality of peristalsis depends upon orderly generation and propagation of electrical slow waves and a frequency gradient between proximal and distal pacemakers. We sought to understand how chronotropic agonists affect coupling between corpus and antrum. METHODS: Electrophysiological and imaging techniques were used to investigate regulation of gastric slow wave frequency by muscarinic agonists in mice. We also investigated the expression and role of cholinesterases in regulating slow wave frequency and motor patterns in the stomach. RESULTS: Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to varicose processes of motor neurons. Inhibition of AChE in the absence of stimulation increased slow wave frequency in corpus and throughout muscle strips containing corpus and antrum. CCh caused depolarization and increased slow wave frequency. Stimulation of cholinergic neurons increased slow wave frequency but did not cause depolarization. Neostigmine (1 muM) increased slow wave frequency, but uncoupling between corpus and antrum was not detected. Motility mapping of contractile activity in gastric muscles showed similar effects of enteric nerve stimulation on the frequency and propagation of slow waves, but neostigmine (> 1 muM) caused aberrant contractile frequency and propagation and ectopic pacemaking. CONCLUSIONS: Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and suggest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns.
Animals ; Cholinergic Neurons ; Cholinesterases* ; Metabolism ; Mice* ; Motor Neurons ; Muscarinic Agonists ; Muscle, Smooth ; Muscles ; Neostigmine ; Peristalsis ; Pylorus ; Stomach

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. At present, no definite ALS biomarkers are available. In this study, exosomes from the plasma of patients with ALS and healthy controls were extracted, and differentially expressed exosomal proteins were compared. Among them, the expression of exosomal coronin-1a (CORO1A) was 5.3-fold higher than that in the controls. CORO1A increased with disease progression at a certain proportion in the plasma of patients with ALS and in the spinal cord of ALS mice. CORO1A was also overexpressed in NSC-34 motor neuron-like cells, and apoptosis, oxidative stress, and autophagic protein expression were evaluated. CORO1A overexpression resulted in increased apoptosis and oxidative stress, overactivated autophagy, and hindered the formation of autolysosomes. Moreover, CORO1A activated Ca²⁺-dependent phosphatase calcineurin, thereby blocking the fusion of autophagosomes and lysosomes. The inhibition of calcineurin activation by cyclosporin A reversed the damaged autolysosomes. In conclusion, the role of CORO1A in ALS pathogenesis was discovered, potentially affecting the disease onset and progression by blocking autophagic flux. Therefore, CORO1A might be a potential biomarker and therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/pathology* ; Calcineurin/metabolism* ; Motor Neurons/pathology* ; Microfilament Proteins/metabolism* ; Cytoskeletal Proteins/metabolism*

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.
Animals ; Animals, Genetically Modified ; Green Fluorescent Proteins/pharmacology* ; Microtubules/metabolism* ; Motor Neurons ; Zebrafish/genetics* ; Zebrafish Proteins/genetics*

OBJECTIVETo explore the mechanism of cytotoxic effect of 2, 5-hexanedione (2, 5-HD) on motor neuron.

METHODSVsc4.1 (a cell line from motor neuron) was incubated with a series concentration of 2, 5-HD. The cell viability, Ca(2+) Mg(2+) ATPase and Na(+)K(+) ATPase were detected. Laser scanning confocal microscope (LSCM) was used for detecting intracellular calcium level. The average calcium level in VSC4.1 was measured by flow cytometry.

RESULTSThe cell viability was decreased when Vsc4.1 cells were treated with 2, 5-HD at the dosage of 2.5, 5.0, 7.5, 10, 15 and 20 mmol/L for 24 hours. Compared with the control group the activity of Ca(2+) Mg(2+) ATPase was decreased to 70.02%, 77.44% and 47.47% respectively; the activity of Na(+)K(+) ATPase was decreased to 82.07%, 72.45% and 50.71%. The difference was significant. Intracellular free calcium of VSC4.1 cell was increased rapidly within 10 s and then recovered within 40 seconds when it was exposed to 33.5 mmol/L 2, 5-HD. An increase in intracellular calcium was observed when the VSC4.1 was treated with 33.5 mmol/L 2, 5-HD. The peak of intracellular calcium level occurred ten minutes later.

CONCLUSIONThe disturbance of calcium homeostasis may be involved in the mechanisms of neurotoxicity of 2, 5-HD.

Animals ; Ca(2+) Mg(2+)-ATPase ; metabolism ; Calcium ; metabolism ; Cell Line ; Dose-Response Relationship, Drug ; Hexanones ; toxicity ; Motor Neurons ; drug effects ; metabolism ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism

AIMTo investigate the changes of neurotransmitter concentration in spinal cord after exercise-induced central fatigue and study the mechanism of central fatigue at spinal level.

METHODSEstablish exercise-induced central fatigue model according to Bedford incremental loading training. The rats were divided into three groups, control group (C), immediately after training group (IT), rest 3 hours after training group (RT). Then using high performance liquid chromatography (HPLC) to detect the concentration of neurotransmitter in spinal cord.

RESULTSAmino acid neurotransmitters in spinal cord increased in IT group: Glu, GABA increased significantly (P < 0.05), Gly also increased but have no statistic difference; while in RT group, amino acid neurotransmitters got back to normal. However monoamine neurotransmitters NE, 5-HT tended to decrease in IT group. In RT group 5-HT decreased dramatically (P < 0.05).

CONCLUSIONExercise-induced fatigue changed the concentration of neurotransmitter in spinal cord.The results suggested that neurotransmitter in spinal cord might have relationship with exercise-induced fatigue, especially 5-HT might have more important effect on recovery of fatigue.

Animals ; Fatigue ; metabolism ; Male ; Motor Neurons ; metabolism ; Neurotransmitter Agents ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Wistar ; Spinal Cord ; metabolism ; Synaptic Transmission

BACKGROUND: Free radical reactions are a part of normal human metabolism. When produced in excess, radicals can cause tissue injury. The present study was aimed to investigate neurotoxic effect of oxygen free radicals and neuroprotective effect of antioxidant(glutathione). METHODS: Neurotixic effect of oxygen radicals was evaluated by MTT[3-(4,5-dimethylthiazol-2,5- diphenyltetrazolium bromide] assay and neurofilament enzyme-immunoassay after culturing of spinal motor neuron cell line of mouse(NSC-34). Then these cells were exposed to various concentrations of xanthine oxidase(XO) and hypoxanthine(HX). In addition, neuroprotective effect of antioxidant against oxidant-induced neurototoxicity on these cultures was examined. RESULTS: Exposure of neurons to 25mU/ml XO and 0.2mM HX for 3 hours resulted in a significant cell death and also glutathione(GSH) blocked the neurotoxicity induced by oxygen radicals in cultured mouse spinal motor neurons. CONCLUSION: From the above results, it is suggested that oxygen radicals are toxic in NSC-34. Selective antioxidants such as GSH are effective in blocking oxidant-induced neurotoxicity on these cultures.
Animals ; Antioxidants ; Cell Death ; Cell Line ; Free Radicals ; Glutathione* ; Humans ; Metabolism ; Mice ; Motor Neurons* ; Neurons ; Neuroprotective Agents ; Oxygen ; Reactive Oxygen Species ; Xanthine

Spinal muscular atrophy(SMA) is a genetic disorder of the motor neurons that cause muscular weakness and muscular atrophy due to anterior horn cell degeneration. Classic spinal muscular atrophy patient is caused by mutation in the chromosome 5(q11.2-q13.3), and the majority of the patient shows homozygous deletion of the telomeric survival motor neuron(SMN) gene in the chromosome 5. Deletion of exon 7 and 8 of the SMN gene and deletion of exon 4 and 5 of the neuronal apoptosis inhibitory protein(NAIP) are typically observed in SMA patients. The SMN protein plays a role in an essential cell metabolism process, the splicing of pre mRNA in the spliceosomes. We report a 7 month old male with SMA. He showed rapidly aggrdvatial muscular weakness and died at 7 months. His DNA analysis proved deletion of exon 7 and 8 of the telomeric copy of the SMN gene.
Anterior Horn Cells ; Apoptosis ; Chromosomes, Human, Pair 5 ; DNA ; Exons* ; Humans ; Infant ; Male ; Metabolism ; Motor Neurons* ; Muscle Weakness ; Muscular Atrophy ; Muscular Atrophy, Spinal* ; Neurons ; RNA Precursors ; Spliceosomes

OBJECTIVETo explore the effect of 2,5-hexanedione (2,5-HD) on the levels of nerve growth factor (NGF) in sciatic nerve of rats and motor-neurons.

METHODA total of 50 Wistar rats were randomly designed into five groups and intoxicated with 400 mgxkg(-1)xd(-1) 2,5-HD for 0, 7, 14, 21, 28 d. Immunohistochemistry and real-time PCR were used to detect the levels of NGF and NGF mRNA. Motor neuron VSC4.1 cells were administrated with 0, 2.5, 5.0, 10.0, 20.0 mmol/L 2,5-HD for 24 h and 10.0 mmol/L 2,5-HD was chosen to intoxicated VSC4.1 cells for 0, 1, 3, 6, 12, 24, 48 h respectively. Immunofluorescence technique was selected to detect the levels of NGF.

RESULTSThe NGF level in sciatic nerve of rats administrated with 400 mgxkg(-1)xd(-1) 2,5-HD showed increase tendency at begin and then decrease after exposure. The NGF mRNA level in 14 d (2(-DeltaDeltaCt)= 3.46), 21 d (2(-DeltaDeltaCt)= 5.28) and 28 d (2(-DeltaDeltaCt)= 3.10) were higher than those in 0 d (2(-DeltaDeltaCt)= 1) and 7 d (2(-DeltaDeltaCt)= 0.78). In vitro tests of VSC4.1 cells showed that NGF levels in 5.0 mmol/L (43.24 +/- 7.52), 10.0 mmol/L (43.48 +/- 10.86) and 20.0 mmol/L (63.13 +/- 10.68) were higher than those in 0 mmol/L (16.32 +/- 4.20)(q values were 19.92, 19.72, 32.78, respectively, P < 0.01) and 2.5 mmol/L (19.78 +/- 2.66) (q values were 17.50, 17.42, 30.63, respectively, P < 0.01) in 24 h and the NGF level in 20.0 mmol/L was higher than those in 5.0 mmol/L (q = 13.04, P < 0.01) and 10.0 mmol/L (q = 11.71, P < 0.01). The NGF levels of VSC4.1 cells with 10.0 mmol/L 2,5-HD in 6 h (18.66 +/- 2.89), 12 h (23.14 +/- 6.08), 24 h (27.66 +/- 6.11) and 48 h (17.25 +/- 3.05) were increased compared with that in 0 h (10.18 +/- 1.81) (q values were 9.64, 15.74, 21.76, 8.50, respectively, P < 0.01), 1 h (9.31 +/- 1.28) (q values were 10.28, 16.17, 21.95, 9.20, respectively, P < 0.01) and 3 h (10.44 +/- 2.13) (q values were 9.25, 15.24, 21.17, 8.10, respectively, P < 0.01), and NGF levels in 12 h and 24 h increased compared with those in 6 h (q values were 5.24, 10.77, respectively, P < 0.01) and 48 h (q values were 7.31, 13.26, respectively, P < 0.01).

CONCLUSION2,5-HD could increase NGF levels in sciatic nerve of rats and motor-neurons, and the dose or time dependent effects were observed in this study.

Animals ; Cell Line ; Hexanones ; toxicity ; Male ; Motor Neurons ; drug effects ; metabolism ; Nerve Growth Factor ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; metabolism

Spinal α-motoneurons directly innervate skeletal muscles and function as the final common path for movement and behavior. The processes that determine the excitability of motoneurons are critical for the execution of motor behavior. In fact, it has been noted that spinal motoneurons receive various neuromodulatory inputs, especially monoaminergic one. However, the roles of histamine and hypothalamic histaminergic innervation on spinal motoneurons and the underlying ionic mechanisms are still largely unknown. In the present study, by using the method of intracellular recording on rat spinal slices, we found that activation of either H or H receptor potentiated repetitive firing behavior and increased the excitability of spinal α-motoneurons. Both of blockage of K channels and activation of Na-Ca exchangers were involved in the H receptor-mediated excitation on spinal motoneurons, whereas the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were responsible for the H receptor-mediated excitation. The results suggest that, through switching functional status of ion channels and exchangers coupled to histamine receptors, histamine effectively biases the excitability of the spinal α-motoneurons. In this way, the hypothalamospinal histaminergic innervation may directly modulate final motor outputs and actively regulate spinal motor reflexes and motor execution.
Animals ; Histamine ; pharmacology ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; metabolism ; Motor Neurons ; drug effects ; physiology ; Rats ; Receptors, Histamine H2 ; metabolism ; Sodium-Calcium Exchanger ; metabolism

The experiments were carried out on adult Sprague-Dawley rats. We investigated the discharge response of respiratory neurons (RNs) in the pre-Bözinger complex (PBC) to electrical stimulation of the facial nucleus in which the motor neurons were retrogradely degenerated and the antagonistic effects of microiontophoresis of CNQX, bicuculline (BIC), strychnine (Stry) and atropine on the discharge responses of the neurons. In 12 rats with retrograde degeneration of the facial motor neurons, 116 RNs in the PBC ipsilateral to the facial nerve sectioned were extracellularly recorded. The response of pre-inspiratory (Pre-I) (24 / 26) and inspiratory (I) (30 / 35) neurons to the electrical stimulation of the facial nucleus was mainly excitatory, and the response of expiratory (E) (20 / 22) and inspiratory-expiratory phase-spanning (I-E) (28 / 33) neurons was mainly inhibitory. CNQX partially or completely block the excitatory effect of the stimulation on Pre-I (18 / 24) and I (23 / 27) neurons. Stry could partially or completely block the immediate transient inhibition on Pre-I (12 / 18) and I (14 / 23) neurons and the inhibitory effect on I-E (20 / 28) and E (9 / 16) neurons induced by the stimulation. BIC partially or completely blocked the inhibitory effect on I-E (22 / 25) and E (9 / 9) neurons induced by the stimulation. Atropine did not have obvious influence on the response of RNs to the stimulation. These results suggest that non-motoneurons in the facial nucleus may participate in the modulation of respiration by affecting the activities of RNs in the PBC and that Glu, GABA and Gly serve as neurotransmitters or modulators to regulate the activities of the RNs in the PBC and hence the rhythmic respiratory movement.
Animals ; Electric Stimulation ; Facial Nerve ; physiology ; Female ; Glutamic Acid ; metabolism ; Glycine ; metabolism ; Male ; Medulla Oblongata ; physiology ; Motor Neurons ; physiology ; Neurons ; physiology ; Neurotransmitter Agents ; metabolism ; Rats ; Rats, Sprague-Dawley ; Respiration ; Respiratory Center ; physiology ; gamma-Aminobutyric Acid ; metabolism