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1.
eNeuro ; 4(4)2017.
Article in English | MEDLINE | ID: mdl-28798955

ABSTRACT

The neuromuscular junction (NMJ) is a chemical synapse formed between motoneurons and skeletal muscle fibers. The vertebrate NMJ uses acetylcholine (ACh) as the neurotransmitter and features numerous invaginations of the postsynaptic muscle membrane termed junctional folds. ACh receptors (AChRs) are believed to be concentrated on the crest of junctional folds but their spatial organization remains to be fully understood. In this study, we utilized super-resolution microscopy to examine the nanoscale organization of AChRs at NMJ. Using Structured Illumination Microscopy, we found that AChRs appear as stripes within the pretzel-shaped mouse NMJs, which however, do not correlate with the size of the crests of junctional folds. By comparing the localization of AChRs with several pre- and postsynaptic markers of distinct compartments of NMJs, we found that AChRs are not distributed evenly across the crest of junctional folds as previously thought. Instead, AChR stripes are more closely aligned with the openings of junctional folds as well as with the presynaptic active zone. Using Stochastic Optical Reconstruction Microscopy (STORM) for increased resolution, we found that each AChR stripe contains an AChR-poor slit at the center that is equivalent to the size of the opening of junctional folds. Together, these findings indicate that AChRs are largely localized to the edges of crests surrounding the opening of folds to align with the presynaptic active zones. Such a nanoscale organization of AChRs potentially enables trans-synaptic alignment for effective synaptic transmission of NMJs.


Subject(s)
Neuromuscular Junction/physiology , Neuromuscular Junction/ultrastructure , Receptors, Cholinergic/metabolism , Receptors, Cholinergic/ultrastructure , Synaptic Transmission/physiology , Analysis of Variance , Animals , Animals, Newborn , Bungarotoxins/pharmacokinetics , Female , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Muscle, Skeletal/ultrastructure , Neuromuscular Junction/drug effects , Protein Binding/drug effects , Synaptic Transmission/drug effects
2.
J Neurosci ; 36(38): 9770-81, 2016 09 21.
Article in English | MEDLINE | ID: mdl-27656017

ABSTRACT

UNLABELLED: The neuromuscular junction (NMJ) is a tripartite synapse that is formed by motor nerve terminals, postjunctional muscle membranes, and terminal Schwann cells (TSCs) that cover the nerve-muscle contact. NMJ formation requires intimate communications among the three different components. Unlike nerve-muscle interaction, which has been well characterized, less is known about the role of SCs in NMJ formation and maintenance. We show that SCs in mice lead nerve terminals to prepatterned AChRs. Ablating SCs at E8.5 (i.e., prior nerve arrival at the clusters) had little effect on aneural AChR clusters at E13.5, suggesting that SCs may not be necessary for aneural clusters. SC ablation at E12.5, a time when phrenic nerves approach muscle fibers, resulted in smaller and fewer nerve-induced AChR clusters; however, SC ablation at E15.5 reduced AChR cluster size but had no effect on cluster density, suggesting that SCs are involved in AChR cluster maturation. Miniature endplate potential amplitude, but not frequency, was reduced when SCs were ablated at E15.5, suggesting that postsynaptic alterations may occur ahead of presynaptic deficits. Finally, ablation of SCs at P30, after NMJ maturation, led to NMJ fragmentation and neuromuscular transmission deficits. Miniature endplate potential amplitude was reduced 3 d after SC ablation, but both amplitude and frequency were reduced 6 d after. Together, these results indicate that SCs are not only required for NMJ formation, but also necessary for its maintenance; and postsynaptic function and structure appeared to be more sensitive to SC ablation. SIGNIFICANCE STATEMENT: Neuromuscular junctions (NMJs) are critical for survival and daily functioning. Defects in NMJ formation during development or maintenance in adulthood result in debilitating neuromuscular disorders. The role of Schwann cells (SCs) in NMJ formation and maintenance was not well understood. We genetically ablated SCs during development and after NMJ formation to investigate the consequences of the ablation. This study reveals a critical role of SCs in NMJ formation as well as maintenance.


Subject(s)
Neuromuscular Junction/cytology , Neuromuscular Junction/physiology , Schwann Cells/physiology , Action Potentials/physiology , Animals , Animals, Newborn , Bungarotoxins/pharmacokinetics , Electromyography , Embryo, Mammalian , Estrogen Antagonists , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Transgenic , Myelin Proteolipid Protein/genetics , Myelin Proteolipid Protein/metabolism , Neurofilament Proteins/metabolism , Neuromuscular Junction/drug effects , Neuromuscular Junction/ultrastructure , Proteins/genetics , Proteins/metabolism , Receptors, Cholinergic/genetics , Receptors, Cholinergic/metabolism , Rhodamines/pharmacokinetics , S100 Calcium Binding Protein beta Subunit/metabolism , Schwann Cells/drug effects , Synaptophysin/metabolism , Tamoxifen/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Exp Neurol ; 285(Pt A): 96-107, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27567739

ABSTRACT

In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400µM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p<0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the non-infused side (p<0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1µM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1-G93A mice, even at late stages of disease.


Subject(s)
Amyotrophic Lateral Sclerosis/complications , Enzyme Inhibitors/administration & dosage , Methylene Blue/administration & dosage , Neuromuscular Junction Diseases/drug therapy , Neuromuscular Junction Diseases/etiology , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Bungarotoxins/pharmacokinetics , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Delivery Systems , Enzyme Inhibitors/therapeutic use , Fluorescent Antibody Technique , Humans , Methylene Blue/therapeutic use , Mice , Mice, Transgenic , Motor Endplate/drug effects , Motor Endplate/physiology , Muscle Contraction/drug effects
4.
Brain Behav ; 6(6): e00468, 2016 06.
Article in English | MEDLINE | ID: mdl-27186443

ABSTRACT

INTRODUCTION: Motor endplates are composed of a motor neuron terminal and muscle fiber and are distributed in skeletal muscle, causing muscle contraction. However, traditional motor endplate staining methods are limited to the observation of partial skeletal muscle. The procedure was time-consuming due to strict incubation conditions, and usually provided unsatisfactory results. We explored a novel method to label motor endplate rapidly by in vivo injection of fluorescent α-bungarotoxin. METHODS: Fifty-two mice were randomly divided into two groups, an experiment group (n = 50), and a contrast group (n = 2). In experiment group, α-bungarotoxin was injected via the caudal vein. The injection dosages were designated as 0.1, 0.2, 0.3, 0.4, and 0.5 µg/g. The experimental mice were divided into five subgroups of ten mice per group. The contrast group was only injected with 200 µL normal saline solution. Bilateral gastrocnemius were acquired for microscope analysis and optical clearing to seek specific fluorescent signal. RESULTS: A dose of 0.3 µg/g of α-bungarotoxin with 1 h conjugation time could display the number and structure of motor endplate in plane view. Compared with the traditional procedure, this method was rapid, convenient, and time-saving. Combined with the optical clearing technique, spatial distribution could also be seen, helping to better understand the stereoscopic view of motor endplate position in skeletal muscle. CONCLUSIONS: In vivo injection of α-bungarotoxin proved effective for studying motor endplate in skeletal muscle.


Subject(s)
Bungarotoxins/pharmacokinetics , Fluorescent Dyes/pharmacokinetics , Motor Endplate/anatomy & histology , Staining and Labeling/methods , Animals , Bungarotoxins/administration & dosage , Fluorescent Dyes/administration & dosage , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Muscle, Skeletal
5.
J Neurosci ; 36(9): 2663-76, 2016 Mar 02.
Article in English | MEDLINE | ID: mdl-26937007

ABSTRACT

The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss- and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development. SIGNIFICANCE STATEMENT: In addition to the archetypal axon guidance cues, the extracellular matrix provides local information that guides motor axons from the spinal cord to their muscle targets. Many of the proteins involved are unknown. Using the zebrafish model, we identified an unexpected role of the extracellular matrix collagen XV in motor axon pathfinding. We show that the synthesis of collagen XV-B by slow muscle precursors and its deposition in the common motor path are dependent on a novel two-step mechanism that determines axon decisions at a choice point during motor axonogenesis. Zebrafish and humans use common molecular cues and regulatory mechanisms for the neuromuscular system development. And as such, our study reveals COL15A1 as a candidate gene for orphan neuromuscular disorders.


Subject(s)
Axons/physiology , Collagen/metabolism , Motor Neurons/physiology , Muscle, Skeletal/cytology , Age Factors , Animals , Animals, Genetically Modified , Axons/drug effects , Bungarotoxins/pharmacokinetics , Collagen/genetics , Cyclic AMP-Dependent Protein Kinases/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Humans , Morpholinos/pharmacology , Motor Neurons/drug effects , Mutation/genetics , RNA, Messenger/metabolism , Receptors, Cholinergic/metabolism , Signal Transduction/physiology , Touch , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , beta Catenin/metabolism
6.
Neuroscience ; 316: 167-77, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26711679

ABSTRACT

The objective of this study was to determine whether pre- to post-synaptic coupling of the neuromuscular junction (NMJ) could be maintained in the face of significant morphological remodeling brought about by exercise training, and whether aging altered this capacity. Eighteen young adult (8 mo) and eighteen aged (24 mo) Fischer 344 rats were randomly assigned to either endurance trained (treadmill running) or untrained control conditions resulting in four groups (N=9/group). After the 10-week intervention rats were euthanized and hindlimb muscles were surgically removed, quickly frozen at approximate resting length and stored at -85°C. The plantaris and EDL muscles were selected for study as they have different functions (ankle extensor and ankle flexor, respectively) but both are similarly and overwhelmingly comprised of fast-twitch myofibers. NMJs were stained with immunofluorescent procedures and images were collected with confocal microscopy. Each variable of interest was analyzed with a 2-way ANOVA with main effects of age and endurance training; in all cases significance was set at P⩽0.05. Results showed that no main effects of aging were detected in NMJs of either the plantaris or the EDL. Similarly, endurance training failed to alter any synaptic parameters of EDL muscles. The same exercise stimulus in the plantaris however, resulted in significant pre- and post-synaptic remodeling, but without altering pre- to post-synaptic coupling of the NMJs. Myofiber profiles of the same plantaris and EDL muscles were also analyzed. Unlike NMJs, myofibers displayed significant age-related atrophy in both the plantaris and EDL muscles. Overall, these results confirm that despite significant training-induced reconfiguration of NMJs, pre- to post-synaptic coupling remains intact underscoring the importance of maintaining proper apposition of neurotransmitter release and binding sites so that effective nerve to muscle communication is assured.


Subject(s)
Aging/physiology , Neuromuscular Junction/cytology , Neuromuscular Junction/physiology , Physical Conditioning, Animal , Presynaptic Terminals/physiology , Analysis of Variance , Animals , Body Mass Index , Bungarotoxins/pharmacokinetics , Male , Muscle, Skeletal/physiology , Myofibrils/metabolism , Rats , Rats, Inbred F344 , Receptors, Cholinergic/metabolism , Rhodamines/pharmacokinetics , Synaptic Vesicles/metabolism , Synaptophysin/metabolism
7.
Neuroscience ; 306: 91-9, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26283025

ABSTRACT

Bisabolol is a plant-derived monocyclic sesquiterpene alcohol with antinociceptive and antiinflammatory actions. However, molecular targets mediating these effects of bisabolol are poorly understood. In this study, using a two-electrode voltage-clamp and patch-clamp techniques and live cellular calcium imaging, we have investigated the effect of bisabolol on the function of human α7 subunit of nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes, interneurons of rat hippocampal slices. We have found that bisabolol reversibly and concentration dependently (IC50 = 3.1 µM) inhibits acetylcholine (ACh)-induced α7 receptor-mediated currents. The effect of bisabolol was not dependent on the membrane potential. Bisabolol inhibition was not changed by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free solution containing Ba(2+), suggesting that endogenous Ca(2+)-dependent Cl(-) channels are not involved in bisabolol actions. Increasing the concentrations of ACh did not reverse bisabolol inhibition. Furthermore, the specific binding of [(125)I] α-bungarotoxin was not attenuated by bisabolol. Choline-induced currents in CA1 interneurons of rat hippocampal slices were also inhibited with IC50 of 4.6 µM. Collectively, our results suggest that bisabolol directly inhibits α7-nAChRs via a binding site on the receptor channel.


Subject(s)
Hippocampus/physiology , Interneurons/physiology , Membrane Potentials/drug effects , Sesquiterpenes/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/physiology , Animals , Bungarotoxins/pharmacokinetics , Calcium/metabolism , Dose-Response Relationship, Drug , Female , Hippocampus/drug effects , Humans , Interneurons/drug effects , Male , Models, Molecular , Monocyclic Sesquiterpenes , Rats , Rats, Sprague-Dawley , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors
8.
Neurol Sci ; 36(7): 1167-71, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25964166

ABSTRACT

Myasthenia gravis (MG) is an autoimmune disease associated with antibodies directed to the postsynaptic muscle components of the neuromuscular junction. The heterogeneous nature of the acetylcholine receptor (AChR) antibody response had led to the categorization of AChR antibodies into 3 types: binding, blocking, and modulating antibodies. The purpose of this study is to compare the AChR antibodies' type with the clinical severity of MG patients. The patients enrolled in the study had been tested for both binding and blocking antibodies and had disease duration exceeding 2 years since diagnosis. The patients were divided into five main classes by the Myasthenia Gravis Foundation of America clinical classification. Again, the enrolled patients were divided into ocular and generalized group. We compared the type and titer of antibodies and the thymus status between the ocular and generalized group. Thirty-five patients met the inclusion criteria. Of these, 16 patients (47 %) had both blocking and binding AChR antibodies, 11 patients (31 %) had only binding antibodies, and 8 patients (22 %) had only blocking antibodies. By defined clinical classification, the ocular and generalized groups included 10 and 25 patients, respectively. Sixteen patients in the generalized group possessed both AChR antibodies, with the remaining patients displaying only the binding antibody. All the patients with only blocking antibody were classified into ocular group. Use of binding and blocking antibodies' tests may, therefore, be more helpful in predicting the prognosis and diagnoses of MG patient.


Subject(s)
Antibodies, Blocking/blood , Autoantibodies/blood , Myasthenia Gravis/immunology , Receptors, Cholinergic/immunology , Adult , Binding Sites, Antibody/physiology , Bungarotoxins/pharmacokinetics , Female , Humans , Longitudinal Studies , Male , Middle Aged , Myasthenia Gravis/classification , Retrospective Studies , Statistics, Nonparametric , Thymus Gland/pathology
9.
Int J Dev Neurosci ; 34: 9-18, 2014 May.
Article in English | MEDLINE | ID: mdl-24412779

ABSTRACT

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni-quantal end-plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.


Subject(s)
Neuromuscular Junction/growth & development , Neuromuscular Junction/metabolism , Neurotransmitter Agents/metabolism , Age Factors , Animals , Animals, Newborn , Bungarotoxins/pharmacokinetics , Electric Stimulation , Female , Gallic Acid/analogs & derivatives , Gallic Acid/pharmacokinetics , Male , Neuromuscular Junction/drug effects , Rats , Reaction Time/physiology , Receptors, Nicotinic/metabolism , Ryanodine/pharmacokinetics , Synaptic Potentials/physiology , Synaptophysin/metabolism
10.
J Alzheimers Dis ; 38(1): 49-62, 2014.
Article in English | MEDLINE | ID: mdl-23948929

ABSTRACT

Amyloid-ß (Aß) peptides are constitutively produced in the brain throughout life via mechanisms that can be regulated by synaptic activity. Although Aß has been extensively studied as the pathological plaque-forming protein species in Alzheimer's disease (AD), little is known about the normal physiological function(s) and signaling pathway(s). We previously discovered that physiologically-relevant, low picomolar amounts of Aß can enhance synaptic plasticity and hippocampal-dependent cognition in mice. In this study, we demonstrated that astrocytes are cellular candidates for participating in this type of Aß signaling. Using calcium imaging of primary astrocyte cultures, we observed that picomolar amounts of Aß peptides can enhance spontaneous intracellular calcium transient signaling. After application of 200 pM Aß42 peptides, the frequency and amplitude averages of spontaneous cytosolic calcium transients were significantly increased. These effects were dependent on α7 nicotinic acetylcholine receptors (α7-nAChRs), as the enhancement effects were blocked by a pharmacological α7-nAChR inhibitor and in astrocytes from an α7 deficient mouse strain. We additionally examined evoked intercellular calcium wave signaling but did not detect significant picomolar Aß-induced alterations in propagation parameters. Overall, these results indicate that at a physiologically-relevant low picomolar concentration, Aß peptides can enhance spontaneous astrocyte calcium transient signaling via α7-nAChRs. Since astrocyte-mediated gliotransmission has been previously found to have neuromodulatory roles, Aß peptides may have a normal physiological function in regulating neuron-glia signaling. Dysfunction of this signaling process may underlie glia-based aspects of AD pathogenesis.


Subject(s)
Amyloid beta-Peptides/pharmacology , Astrocytes/drug effects , Calcium Signaling/drug effects , Calcium/metabolism , Animals , Animals, Newborn , Bungarotoxins/pharmacokinetics , Calcium Signaling/genetics , Cells, Cultured , Dose-Response Relationship, Drug , Glial Fibrillary Acidic Protein/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/pharmacology , Prosencephalon/cytology , Protein Binding/drug effects , Time Factors , alpha7 Nicotinic Acetylcholine Receptor/deficiency , alpha7 Nicotinic Acetylcholine Receptor/metabolism
11.
Neuroscience ; 230: 121-31, 2013 Jan 29.
Article in English | MEDLINE | ID: mdl-23159316

ABSTRACT

Nicotinic receptors have been linked to a wide range of cognitive and behavioral functions, but surprisingly little is known about their involvement in cost benefit decision making. The goal of these experiments was to determine how nicotinic acetylcholine receptor (nAChR) expression is related to two forms of cost benefit decision making. Male Long Evans rats were tested in probability- and delay-discounting tasks, which required discrete trial choices between a small reward and a large reward associated with varying probabilities of omission and varying delays to reward delivery, respectively. Following testing, radioligand binding to α4ß2 and α7 nAChR subtypes in brain regions implicated in cost benefit decision making was examined. Significant linear relationships were observed between choice of the large delayed reward in the delay discounting task and α4ß2 receptor binding in both the dorsal and ventral hippocampus. Additionally, trends were found suggesting that choice of the large costly reward in both discounting tasks was inversely related to α4ß2 receptor binding in the medial prefrontal cortex and nucleus accumbens shell. Similar trends suggested that choice of the large delayed reward in the delay discounting task was inversely related to α4ß2 receptor binding in the orbitofrontal cortex, nucleus accumbens core, and basolateral amygdala, as well as to α7 receptor binding in the basolateral amygdala. These data suggest that nAChRs (particularly α4ß2) play both unique and common roles in decisions that require consideration of different types of reward costs.


Subject(s)
Brain Mapping , Decision Making/physiology , Receptors, Nicotinic/metabolism , Analysis of Variance , Animals , Brain/diagnostic imaging , Brain/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Bungarotoxins/pharmacokinetics , Iodine Isotopes/pharmacokinetics , Male , Nicotinic Agonists/pharmacokinetics , Probability , Protein Binding/drug effects , Protein Binding/physiology , Pyridines/pharmacokinetics , Radionuclide Imaging , Rats , Rats, Long-Evans , Reaction Time/drug effects , Reward , Statistics as Topic , Time Factors , alpha7 Nicotinic Acetylcholine Receptor
12.
Brain Res ; 1483: 96-104, 2012 Nov 05.
Article in English | MEDLINE | ID: mdl-22995368

ABSTRACT

Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, ß2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4ß2) and [¹²5I]-α-bungarotoxin ([¹²5I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²5I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²5I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, ß2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients.


Subject(s)
Dystrophin/deficiency , Hippocampus/metabolism , Receptors, Nicotinic/metabolism , Age Factors , Alkaloids/pharmacokinetics , Analysis of Variance , Animals , Azocines/pharmacokinetics , Bungarotoxins/metabolism , Bungarotoxins/pharmacokinetics , Dose-Response Relationship, Drug , Dystrophin/genetics , Hippocampus/drug effects , Isotopes/pharmacokinetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Nicotinic Antagonists/pharmacokinetics , Protein Binding/drug effects , Protein Binding/genetics , Quinolizines/pharmacokinetics , RNA, Messenger/metabolism , Receptors, Nicotinic/genetics
13.
J Neurochem ; 123(4): 504-14, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22913551

ABSTRACT

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and ß2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or ß4 subunits reduced to about half, surface α-bungarotoxin binding sites and acetylcholine-gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only ß4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, ß4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3ß4(α5) receptors.


Subject(s)
Gene Expression Regulation/physiology , Protein Subunits/metabolism , Receptors, Nicotinic/metabolism , Acetylcholine/pharmacology , Analysis of Variance , Animals , Biophysics , Bungarotoxins/pharmacokinetics , Cattle , Cells, Cultured , Choline/pharmacology , Cholinergic Agents/pharmacology , Chromaffin Cells , Electric Stimulation , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Humans , Iodine Isotopes/pharmacokinetics , Larva , Lipotropic Agents/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/genetics , Microinjections , Oocytes , Patch-Clamp Techniques , Protein Binding/drug effects , Protein Binding/physiology , Protein Subunits/genetics , RNA, Messenger/metabolism , Receptors, Nicotinic/genetics , Xenopus , alpha7 Nicotinic Acetylcholine Receptor
14.
Neurol Res ; 34(9): 908-14, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22909999

ABSTRACT

OBJECTIVES: The present study was conducted to examine whether repeated crush injuries have significant effects on motor functional recovery of peripheral nerves. METHODS: Repeated crush injuries of the sciatic nerve were inflicted on adult rats at 1-week intervals, and functionality of the sciatic nerve was assessed by the static sciatic index each week for 8 weeks after the final injury. To determine the effects of repeated crush injuries on motor functional recovery of the sciatic nerve, tibialis anterior muscle fibers from single and triple crush injuries were examined, and fiber size and fiber reinnervation during the 2- to 4-week period after the final injury were measured. RESULTS: Compared to single crush injuries, which completely recovered by post-injury week 4, double crush injuries resulted in retarded, but complete recovery by post-injury week 6, whereas triple crush injuries resulted in marked retardation in the regenerative process with incomplete recovery during week 8 of the experimental period. Muscle fiber size for rats with triple crush did not recover to normal range at post-injury week 4, despite its normal size for rats with single crush. The rate of reinnervation increased prominently between post-injury weeks 2 and 3 in both injuries, but the rate with triple crush was lower than that with single crush at post-injury week 3. DISCUSSION: These results, which contradict those of a previous study that reported early functional recovery, indicate that repeated crush injuries inhibit motor functional recovery of the damaged sciatic nerve, as evidenced by delayed and incomplete regeneration, atrophied muscle fibers, and delayed reinnervation.


Subject(s)
Movement Disorders/etiology , Nerve Crush/methods , Recovery of Function/physiology , Sciatic Neuropathy/complications , Analysis of Variance , Animals , Bungarotoxins/pharmacokinetics , Disease Models, Animal , Female , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Neuromuscular Junction/pathology , Rats , Rats, Wistar , Sciatic Neuropathy/pathology , Synaptophysin/metabolism , Time Factors
15.
J Neurochem ; 122(5): 1065-80, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22747514

ABSTRACT

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.


Subject(s)
Acetylcholinesterase/deficiency , Adaptation, Physiological/genetics , Brain/enzymology , Gene Expression Regulation/genetics , Membrane Proteins/deficiency , Nerve Tissue Proteins/deficiency , Acetylcholine/metabolism , Acetylcholinesterase/metabolism , Adaptation, Physiological/drug effects , Animals , Animals, Newborn , Body Temperature/drug effects , Body Temperature/genetics , Brain/anatomy & histology , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Bungarotoxins/pharmacokinetics , Choline/metabolism , Cholinergic Agents/pharmacology , Cholinesterase Inhibitors/pharmacology , Collagen/deficiency , Dihydro-beta-Erythroidine/pharmacology , Dose-Response Relationship, Drug , Exploratory Behavior/drug effects , Exploratory Behavior/physiology , Gait/drug effects , Gait/genetics , Gene Expression Regulation/drug effects , In Vitro Techniques , Maze Learning/drug effects , Maze Learning/physiology , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Mice, Knockout , Microdialysis , Motor Activity/drug effects , Motor Activity/genetics , Muscarinic Antagonists/pharmacokinetics , Muscle Proteins/deficiency , Nails, Ingrown , Neostigmine/pharmacology , Neurons/drug effects , Neurons/physiology , Pirenzepine/analogs & derivatives , Pirenzepine/pharmacokinetics , Protein Binding/drug effects , Pyridines/pharmacokinetics , Radioisotopes/pharmacokinetics , Receptors, Muscarinic/metabolism , Rotarod Performance Test , Scopolamine/pharmacology , Spinal Cord/cytology , Statistics, Nonparametric , Tritium/pharmacokinetics
16.
Brain Res ; 1470: 24-34, 2012 Aug 27.
Article in English | MEDLINE | ID: mdl-22765913

ABSTRACT

Clinical reports suggest a positive association between fat consumption and the incidence of hyperactivity, impulsivity and cognitive abnormalities. To investigate possible mechanisms underlying these disturbances under short-term conditions, we examined in Sprague-Dawley rats the influence of 7-day consumption of a high-fat diet (HFD) compared to chow on anxiety, novelty-seeking and exploratory behaviors and also on acetylcholine (ACh) neurotransmission that may mediate these behaviors. The HFD consumption, which elevated circulating fatty acids but produced no change in caloric intake or body weight, stimulated novelty-seeking and exploration in an open field, while reducing anxiety in an elevated plus maze. Using the Ellman assay to measure ACh esterase (AChE) activity that breaks down ACh, the second experiment showed HFD consumption to significantly reduce AChE activity in the frontal cortex, hypothalamus and midbrain. With measurements of [¹²5I]-epibatidine or [¹²5I]-bungarotoxin binding to nicotinic ACh receptors (nAChRs) containing ß2 or α7 subunits, respectively, the results also showed HFD consumption to increase both ß2-nAChR binding in the medial prefrontal cortex and substantia nigra and α7-nAChR binding in the lateral and ventromedial hypothalamus. When treated with an acute dose of the nicotinic antagonist, mecamylamine (0.5 mg/kg, sc), the HFD animals responded with significantly reduced exploratory and novelty-seeking behaviors, whereas the chow-consuming rats exhibited no response. These findings suggest that the exploratory and novelty-seeking behaviors induced by dietary fat may be mediated by enhanced nicotinic cholinergic activity, which is accompanied by increased density of ß2-nAChRs in cortical and midbrain regions associated with impulsivity and locomotor activity and of α7-nAChRs in hypothalamic regions associated with arousal and energy balance.


Subject(s)
Behavior, Animal/drug effects , Brain/drug effects , Brain/metabolism , Dietary Fats/pharmacology , Acetylcholinesterase/metabolism , Analysis of Variance , Animals , Anxiety/chemically induced , Autoradiography , Body Weight/drug effects , Brain Mapping , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Bungarotoxins/pharmacokinetics , Energy Intake/drug effects , Exploratory Behavior/drug effects , Fatty Acids/metabolism , Iodine Isotopes/pharmacokinetics , Male , Maze Learning/drug effects , Nicotinic Agonists/pharmacokinetics , Protein Binding/drug effects , Pyridines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Receptors, Nicotinic/metabolism
17.
BMC Neurosci ; 13: 73, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22727315

ABSTRACT

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dß1-Dß3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. RESULTS: A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric 'triplet' nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments. CONCLUSION: Data are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.


Subject(s)
Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Ion Channels/metabolism , Protein Subunits/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Acetylcholine/pharmacology , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Bungarotoxins/pharmacokinetics , Cell Line , Cloning, Molecular , Dose-Response Relationship, Drug , Drosophila , Drosophila Proteins/genetics , Female , Gene Expression/genetics , Humans , Ion Channels/genetics , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Molecular Sequence Data , Nicotinic Agonists/pharmacokinetics , Patch-Clamp Techniques , Protein Binding/drug effects , Protein Subunits/genetics , Pyridines/pharmacokinetics , Radiopharmaceuticals/pharmacokinetics , Receptors, Nicotinic/genetics , Receptors, Serotonin, 5-HT3/genetics , Receptors, Serotonin, 5-HT3/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection , Xenopus laevis , ras Proteins/genetics , ras Proteins/metabolism
18.
Neurology ; 79(5): 449-54, 2012 Jul 31.
Article in English | MEDLINE | ID: mdl-22592360

ABSTRACT

OBJECTIVE: To characterize the molecular basis of a novel fast-channel congenital myasthenic syndrome. METHODS: We used the candidate gene approach to identify the pathogenic mutation in the acetylcholine receptor (AChR) ε subunit, genetically engineered the mutant AChR into HEK cells, and evaluated the level of expression and kinetic properties of the mutant receptor. RESULTS: An 8-year-old boy born to consanguineous parents had severe myasthenic symptoms since birth. He is wheelchair bound and pyridostigmine therapy enables him to take only a few steps. Three similarly affected siblings died in infancy. He carries a homozygous p.W55R mutation at the α/ε subunit interface of the AChR agonist binding site. The mutant protein expresses well in HEK cells. Patch-clamp analysis of the mutant receptor expressed in HEK cells reveals 30-fold reduced apparent agonist affinity, 75-fold reduced apparent gating efficiency, and strikingly attenuated channel opening probability (P(open)) over a range agonist concentrations. CONCLUSION: Introduction of a cationic Arg into the anionic environment of α/ε AChR binding site hinders stabilization of cationic ACh by aromatic residues and accounts for the markedly perturbed kinetic properties of the receptor. The very low P(open) explains the poor response to pyridostigmine and the high fatality of the disease.


Subject(s)
Mutation/genetics , Myasthenic Syndromes, Congenital/genetics , Receptors, Nicotinic/genetics , Acetylcholine/pharmacology , Binding Sites/drug effects , Binding Sites/genetics , Biophysical Phenomena/drug effects , Biophysical Phenomena/genetics , Bungarotoxins/pharmacokinetics , Cell Line, Transformed , Child , Cholinergic Agonists/pharmacology , DNA Mutational Analysis , Dose-Response Relationship, Drug , Electric Stimulation , Humans , Iodine Isotopes/pharmacokinetics , Ion Channel Gating/drug effects , Ion Channel Gating/genetics , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Patch-Clamp Techniques , Protein Binding/drug effects , Transfection
19.
Curr Alzheimer Res ; 9(10): 1210-20, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22515492

ABSTRACT

It is already known that progressive degeneration of cholinergic neurons in brain areas such as the hippocampus and the cortex leads to memory deficits, as observed in Alzheimer's disease. This work verified the effects of the infusion of amyloid-ß (Aß) peptide associated to an attentional rehearsal on the density of α7 nicotinic cholinergic receptor (nAChR) in the brain of male Wistar rats. Animals received intracerebroventricular infusion of Aß or vehicle (control - C) and their attention was stimulated weekly (Stimulated Aß group: S-Aß and Stimulated Control group: SC) or not (Non- Stimulated Aß group: N-SAß and Non-Stimulated Control group: N-SC), using an active avoidance apparatus. Conditioned avoidance responses (CAR) were registered. Chronic infusion of Aß caused a 37% reduction in CAR for N-SAß. In S-Aß, this reduction was not observed. At the end, brains were extracted and autoradiography for α7 nAChR was conducted using [125I]-α-bungarotoxin. There was an increase in α7 density in hippocampus, cortex and amygdala of SAß animals, together with the memory preservation. In recent findings from our lab using mice infused with Aß and the α7 antagonist methyllycaconitine, and stimulated weekly in the same apparatus, it was observed that memory maintenance was abolished. So, the increase in α7 density in brain areas related to memory might be related to a participation of this receptor in the long-lasting change in synaptic plasticity, which is important to improve and maintain memory consolidation.


Subject(s)
Amyloid beta-Peptides/pharmacology , Attention/drug effects , Brain/drug effects , Brain/metabolism , Peptide Fragments/pharmacology , Receptors, Nicotinic/metabolism , Analysis of Variance , Animals , Autoradiography , Avoidance Learning/drug effects , Bungarotoxins/pharmacokinetics , Humans , Iodine Isotopes/pharmacokinetics , Male , Protein Binding/drug effects , Rats , Rats, Wistar , Reaction Time/drug effects , Time Factors , alpha7 Nicotinic Acetylcholine Receptor
20.
J Neurosci Res ; 90(4): 732-42, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22420030

ABSTRACT

Motor neurons (MNs) are designated as alpha/gamma and fast/slow based on their target sites and the types of muscle fibers innervated; however, few molecular markers that distinguish between these subtypes are available. Here we report that osteopontin (OPN) is a selective marker of alpha MNs in the mouse spinal cord. OPN was detected in approximately 70% of postnatal choline acetyltransferase (ChAT)-positive MNs with relatively large somas, but not in those with smaller somas. OPN+/ChAT+ MNs were also positive for NeuN, an alpha MN marker, but were negative for Err3, a gamma MN marker. The size distribution of OPN+/ChAT+ cells was nearly identical to that of NeuN+/ChAT+ alpha MNs. Group Ia proprioceptive terminals immunoreactive for vesicular glutamate transporter-1 were selectively detected on the OPN+/ChAT+ cells. OPN staining was also detected at motor axon terminals at neuromuscular junctions, where the OPN+ terminals were positive or negative for SV2A, a marker distinguishing fast/slow motor endplates. Finally, retrograde labeling following intramuscular injection of fast blue indicated that OPN is expressed in both fast and slow MNs. Collectively, our findings show that OPN is an alpha MN marker present in both the soma and the endplates of alpha MNs in the postnatal mouse spinal cord.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Motor Neurons/metabolism , Osteopontin/metabolism , Spinal Cord/cytology , Amidines/metabolism , Animals , Animals, Newborn , Bungarotoxins/pharmacokinetics , Cell Count , Choline O-Acetyltransferase/metabolism , Gene Expression Regulation, Developmental/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscles/innervation , Muscles/metabolism , Neuromuscular Junction/drug effects , Neuromuscular Junction/metabolism , Osteopontin/deficiency , Phosphopyruvate Hydratase/metabolism , Posterior Horn Cells/metabolism , Protein Binding/drug effects , Protein Binding/genetics , Receptors, Estrogen/metabolism , Vesicular Glutamate Transport Protein 1/metabolism
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