43 results on '"Gould TW"'
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2. Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.
- Author
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Hannigan KI, Ni Bhraonain EP, Gould TW, Keef KD, and Cobine CA
- Subjects
- Animals, Mice, Myocytes, Smooth Muscle metabolism, Calcium Signaling physiology, Nitric Oxide metabolism, Electric Stimulation, Interstitial Cells of Cajal metabolism, Interstitial Cells of Cajal physiology, Anal Canal innervation, Anal Canal metabolism, Calcium metabolism
- Abstract
The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca
2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG -nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1 R) and small-conductance Ca2+ -activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium. NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).- Published
- 2024
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3. Perisynaptic Schwann Cells: Guardians of Neuromuscular Junction Integrity and Function in Health and Disease.
- Author
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Gould TW, Ko CP, Willison H, and Robitaille R
- Abstract
The neuromuscular junction (NMJ) is a highly reliable synapse to carry the control of the motor commands of the nervous system over the muscles. Its development, organization, and synaptic properties are highly structured and regulated to support such reliability and efficacy. Yet, the NMJ is also highly plastic, able to react to injury, and able to adapt to changes. This balance between structural stability and synaptic efficacy on one hand and structural plasticity and repair on another hand is made possible by perisynaptic Schwann cells (PSCs), glial cells at this synapse. They regulate synaptic efficacy and structural plasticity of the NMJ in a dynamic, bidirectional manner owing to their ability to decode synaptic transmission and by their interactions with trophic-related factors. Alteration of these fundamental roles of PSCs is also important in the maladapted response of NMJs in various diseases and in aging., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)
- Published
- 2024
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4. Postsynaptic Calcium Extrusion at the Mouse Neuromuscular Junction Alkalinizes the Synaptic Cleft.
- Author
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Durbin RJ, Heredia DJ, Gould TW, and Renden RB
- Subjects
- Female, Male, Animals, Mice, Neuromuscular Junction metabolism, Synaptic Transmission physiology, Cholinergic Agents, Mammals, Calcium metabolism, Synapses physiology
- Abstract
Neurotransmission is shaped by extracellular pH. Alkalization enhances pH-sensitive transmitter release and receptor activation, whereas acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. Previous work has shown that the synaptic cleft can either acidify because of synaptic vesicular release and/or alkalize because of Ca
2+ extrusion by the plasma membrane ATPase (PMCA). The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain ambiguous. We set out to elucidate the extracellular pH transients that occur at this cholinergic synapse under near-physiological conditions and identify their sources. We monitored pH-dependent changes in the synaptic cleft of the mouse levator auris longus using viral expression of the pseudoratiometric probe pHusion-Ex in the muscle. Using mice from both sexes, a significant and prolonged alkalization occurred when stimulating the connected nerve for 5 s at 50 Hz, which was dependent on postsynaptic intracellular Ca2+ release. Sustained stimulation for a longer duration (20 s at 50 Hz) caused additional prolonged net acidification at the cleft. To investigate the mechanism underlying cleft alkalization, we used muscle-expressed GCaMP3 to monitor the contribution of postsynaptic Ca2+ Activity-induced liberation of intracellular Ca2+ in muscle positively correlated with alkalization of the synaptic cleft, whereas inhibiting PMCA significantly decreased the extent of cleft alkalization. Thus, cholinergic synapses of the mouse NMJ typically alkalize because of cytosolic Ca2+ liberated in muscle during activity, unless under highly strenuous conditions where acidification predominates. SIGNIFICANCE STATEMENT Changes in synaptic cleft pH alter neurotransmission, acting on receptors and channels on both sides of the synapse. Synaptic acidification has been associated with a myriad of diseases in the central and peripheral nervous system. Here, we report that in near-physiological recording conditions the cholinergic neuromuscular junction shows use-dependent bidirectional changes in synaptic cleft pH-immediate alkalinization and a long-lasting acidification under prolonged stimulation. These results provide further insight into physiologically relevant changes at cholinergic synapses that have not been defined previously. Understanding and identifying synaptic pH transients during and after neuronal activity provides insight into short-term synaptic plasticity synapses and may identify therapeutic targets for diseases., (Copyright © 2023 Durbin et al.)- Published
- 2023
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5. Kir4.1 is specifically expressed and active in non-myelinating Schwann cells.
- Author
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Procacci NM, Hastings RL, Aziz AA, Christiansen NM, Zhao J, DeAngeli C, LeBlanc N, Notterpek L, Valdez G, and Gould TW
- Subjects
- Mice, Animals, Mice, Transgenic, Sciatic Nerve metabolism, Tamoxifen pharmacology, Schwann Cells metabolism, Myelin Sheath metabolism
- Abstract
Non-myelinating Schwann cells (NMSC) play important roles in peripheral nervous system formation and function. However, the molecular identity of these cells remains poorly defined. We provide evidence that Kir4.1, an inward-rectifying K+ channel encoded by the KCNJ10 gene, is specifically expressed and active in NMSC. Immunostaining revealed that Kir4.1 is present in terminal/perisynaptic SCs (TPSC), synaptic glia at neuromuscular junctions (NMJ), but not in myelinating SCs (MSC) of adult mice. To further examine the expression pattern of Kir4.1, we generated BAC transgenic Kir4.1-CreER
T2 mice and crossed them to the tdTomato reporter line. Activation of CreERT2 with tamoxifen after the completion of myelination onset led to robust expression of tdTomato in NMSC, including Remak Schwann cells (RSC) along peripheral nerves and TPSC, but not in MSC. In contrast, activating CreERT2 before and during the onset of myelination led to tdTomato expression in NMSC and MSC. These observations suggest that immature SC express Kir4.1, and its expression is then downregulated selectively in myelin-forming SC. In support, we found that while activating CreERT2 induces tdTomato expression in immature SC, it fails to induce tdTomato in MSC associated with sensory axons in culture. NMSC derived from neonatal sciatic nerve were shown to express Kir4.1 and exhibit barium-sensitive inwardly rectifying macroscopic K+ currents. Thus, this study identified Kir4.1 as a potential modulator of immature SC and NMSC function. Additionally, it established a novel transgenic mouse line to introduce or delete genes in NMSC., (© 2022 Wiley Periodicals LLC.)- Published
- 2023
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6. High signal-to-noise imaging of spontaneous and 5 ns electric pulse-evoked Ca2+ signals in GCaMP6f-expressing adrenal chromaffin cells isolated from transgenic mice.
- Author
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Viola C, Gould TW, Procacci N, Leblanc N, Zaklit J, and Craviso GL
- Subjects
- Animals, Cattle, Mice, Mice, Transgenic, Adrenal Glands metabolism, Electricity, Cells, Cultured, Calcium metabolism, Chromaffin Cells physiology
- Abstract
In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca2+]i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca2+indicator GCaMP6f. Initial experiments confirmed that Ca2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca2+ responses as well as spontaneous Ca2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca2+ channels but not tetrodotoxin-sensitive voltage-gated Na+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Viola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2023
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7. Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels.
- Author
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Yang L, Pierce S, Gould TW, Craviso GL, and Leblanc N
- Subjects
- Animals, Cations metabolism, Cattle, Membrane Potentials, RNA, Messenger metabolism, TRPC Cation Channels metabolism, Wortmannin metabolism, Wortmannin pharmacology, Chromaffin Cells metabolism, TRPM Cation Channels metabolism
- Abstract
In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of -70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na
+ that displayed inward rectification and a reversal potential near -3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1-6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2 ) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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8. Propulsive colonic contractions are mediated by inhibition-driven poststimulus responses that originate in interstitial cells of Cajal.
- Author
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Koh SD, Drumm BT, Lu H, Kim HJ, Ryoo SB, Kim HU, Lee JY, Rhee PL, Wang Q, Gould TW, Heredia D, Perrino BA, Hwang SJ, Ward SM, and Sanders KM
- Subjects
- Colon physiology, Gastrointestinal Motility physiology, Myocytes, Smooth Muscle, Peristalsis, Interstitial Cells of Cajal
- Abstract
The peristaltic reflex is a fundamental behavior of the gastrointestinal (GI) tract in which mucosal stimulation activates propulsive contractions. The reflex occurs by stimulation of intrinsic primary afferent neurons with cell bodies in the myenteric plexus and projections to the lamina propria, distribution of information by interneurons, and activation of muscle motor neurons. The current concept is that excitatory cholinergic motor neurons are activated proximal to and inhibitory neurons are activated distal to the stimulus site. We found that atropine reduced, but did not block, colonic migrating motor complexes (CMMCs) in mouse, monkey, and human colons, suggesting a mechanism other than one activated by cholinergic neurons is involved in the generation/propagation of CMMCs. CMMCs were activated after a period of nerve stimulation in colons of each species, suggesting that the propulsive contractions of CMMCs may be due to the poststimulus excitation that follows inhibitory neural responses. Blocking nitrergic neurotransmission inhibited poststimulus excitation in muscle strips and blocked CMMCs in intact colons. Our data demonstrate that poststimulus excitation is due to increased Ca2+ transients in colonic interstitial cells of Cajal (ICC) following cessation of nitrergic, cyclic guanosine monophosphate (cGMP)-dependent inhibitory responses. The increase in Ca2+ transients after nitrergic responses activates a Ca2+-activated Cl− conductance, encoded by Ano1, in ICC. Antagonists of ANO1 channels inhibit poststimulus depolarizations in colonic muscles and CMMCs in intact colons. The poststimulus excitatory responses in ICC are linked to cGMP-inhibited cyclic adenosine monophosphate (cAMP) phosphodiesterase 3a and cAMP-dependent effects. These data suggest alternative mechanisms for generation and propagation of CMMCs in the colon.
- Published
- 2022
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9. Colonic Motility Is Improved by the Activation of 5-HT 2B Receptors on Interstitial Cells of Cajal in Diabetic Mice.
- Author
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Jin B, Ha SE, Wei L, Singh R, Zogg H, Clemmensen B, Heredia DJ, Gould TW, Sanders KM, and Ro S
- Subjects
- Animals, Calcium Signaling, Colon metabolism, Colon physiopathology, Constipation etiology, Constipation metabolism, Constipation physiopathology, Diabetes Complications metabolism, Diabetes Complications physiopathology, Disease Models, Animal, Female, Genes, Reporter, Interstitial Cells of Cajal metabolism, Male, Mice, Inbred C57BL, Mice, Transgenic, Ovariectomy, Proto-Oncogene Proteins c-kit genetics, Proto-Oncogene Proteins c-kit metabolism, Receptor, Serotonin, 5-HT2B metabolism, Serotonin metabolism, Tryptophan Hydroxylase genetics, Tryptophan Hydroxylase metabolism, Mice, Colon drug effects, Constipation prevention & control, Diabetes Complications prevention & control, Gastrointestinal Motility drug effects, Indoles pharmacology, Interstitial Cells of Cajal drug effects, Myoelectric Complex, Migrating drug effects, Receptor, Serotonin, 5-HT2B drug effects, Serotonin 5-HT2 Receptor Agonists pharmacology, Thiophenes pharmacology
- Abstract
Background & Aims: Constipation is commonly associated with diabetes. Serotonin (5-HT), produced predominantly by enterochromaffin (EC) cells via tryptophan hydroxylase 1 (TPH1), is a key modulator of gastrointestinal (GI) motility. However, the role of serotonergic signaling in constipation associated with diabetes is unknown., Methods: We generated EC cell reporter Tph1-tdTom, EC cell-depleted Tph1-DTA, combined Tph1-tdTom-DTA, and interstitial cell of Cajal (ICC)-specific Kit-GCaMP6 mice. Male mice and surgically ovariectomized female mice were fed a high-fat high-sucrose diet to induce diabetes. The effect of serotonergic signaling on GI motility was studied by examining 5-HT receptor expression in the colon and in vivo GI transit, colonic migrating motor complexes (CMMCs), and calcium imaging in mice treated with either a 5-HT
2B receptor (HTR2B) antagonist or agonist., Results: Colonic transit was delayed in males with diabetes, although colonic Tph1+ cell density and 5-HT levels were increased. Colonic transit was not further reduced in diabetic mice by EC cell depletion. The HTR2B protein, predominantly expressed by colonic ICCs, was markedly decreased in the colonic muscles of males and ovariectomized females with diabetes. Ca2+ activity in colonic ICCs was decreased in diabetic males. Treatment with an HTR2B antagonist impaired CMMCs and colonic motility in healthy males, whereas treatment with an HTR2B agonist improved CMMCs and colonic motility in males with diabetes. Colonic transit in ovariectomized females with diabetes was also improved significantly by the HTR2B agonist treatment., Conclusions: Impaired colonic motility in mice with diabetes was improved by enhancing HTR2B signaling. The HTR2B agonist may provide therapeutic benefits for constipation associated with diabetes., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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10. Oculomotor nerve guidance and terminal branching requires interactions with differentiating extraocular muscles.
- Author
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Bjorke B, Weller KG, Jones LE, Robinson GE, Vesser M, Chen L, Gage PJ, Gould TW, and Mastick GS
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- Animals, Axons metabolism, Female, Gene Expression genetics, Gene Expression Regulation genetics, Homeodomain Proteins metabolism, Mice, Muscle Development, Myogenic Regulatory Factor 5 metabolism, Oculomotor Muscles growth & development, Oculomotor Muscles metabolism, Oculomotor Nerve metabolism, Pregnancy, Transcription Factors metabolism, Homeobox Protein PITX2, Oculomotor Muscles innervation, Oculomotor Nerve embryology
- Abstract
Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the transcription factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2021. Published by Elsevier Inc.)
- Published
- 2021
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11. Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins.
- Author
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Brawley J, Etter E, Heredia D, Intasiri A, Nennecker K, Smith J, Welcome BM, Brizendine RK, Gould TW, Bell TW, and Cremo C
- Subjects
- 4-Hydroxycoumarins chemical synthesis, Adenosine Triphosphatases antagonists & inhibitors, Molecular Docking Simulation, Muscle, Skeletal drug effects, Muscle, Skeletal enzymology, Muscle, Skeletal metabolism, Structure-Activity Relationship, 4-Hydroxycoumarins chemistry, 4-Hydroxycoumarins pharmacology, Imines chemistry, Myosins antagonists & inhibitors
- Abstract
Inhibitors of muscle myosin ATPases are needed to treat conditions that could be improved by promoting muscle relaxation. The lead compound for this study ((3-( N -butylethanimidoyl)ethyl)-4-hydroxy-2 H -chromen-2-one; BHC) was previously discovered to inhibit skeletal myosin II. BHC and 34 analogues were synthesized to explore structure-activity relationships. The properties of analogues, including solubility, stability, and toxicity, suggest that the BHC scaffold may be useful for developing therapeutics. Inhibition of actin-activated ATPase activity of fast skeletal and cardiac muscle myosin II, inhibition of skeletal muscle contractility ex vivo , and slowing of in vitro actin-sliding velocity were measured. Several analogues with aromatic side arms showed improved potency (half-maximal inhibitory concentration (IC
50 ) <1 μM) and selectivity (≥12-fold) for skeletal myosin versus cardiac myosin compared to BHC. Several analogues blocked neurotransmission, suggesting that they are selective for nonmuscle myosin II over skeletal myosin. Competition and molecular docking studies suggest that BHC and blebbistatin bind to the same site on myosin.- Published
- 2020
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12. Calcium Signaling in Schwann cells.
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Heredia DJ, De Angeli C, Fedi C, and Gould TW
- Subjects
- Animals, Humans, Neuroglia metabolism, Neurons metabolism, Calcium Signaling physiology, Cell Communication physiology, Neuromuscular Junction physiology, Schwann Cells metabolism
- Abstract
In addition to providing structural, metabolic and trophic support to neurons, glial cells of the central, peripheral and enteric nervous systems (CNS, PNS, ENS) respond to and regulate neural activity. One of the most well characterized features of this response is an increase of intracellular calcium. Astrocytes at synapses of the CNS, oligodendrocytes along axons of the CNS, enteric glia associated with the cell bodies and axonal varicosities of the ENS, and Schwann cells at the neuromuscular junction (NMJ) and along peripheral nerves of the PNS, all exhibit this response. Recent technical advances have facilitated the imaging of neural activity-dependent calcium responses in large populations of glial cells and thus provided a new tool to evaluate the physiological significance of these responses. This mini-review summarizes the mechanisms and functional role of activity-induced calcium signaling within Schwann cells, including terminal/perisynaptic Schwann cells (TPSCs) at the NMJ and axonal Schwann cells (ASCs) within peripheral nerves., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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13. Activity within specific enteric neurochemical subtypes is correlated with distinct patterns of gastrointestinal motility in the murine colon.
- Author
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Gould TW, Swope WA, Heredia DJ, Corrigan RD, and Smith TK
- Subjects
- Animals, Animals, Genetically Modified, Enteric Nervous System drug effects, Enteric Nervous System physiology, Enzyme Inhibitors pharmacology, Mice, Muscle Contraction drug effects, Muscle Contraction physiology, Myoelectric Complex, Migrating drug effects, Myoelectric Complex, Migrating physiology, Nitric Oxide Synthase antagonists & inhibitors, Optogenetics, Cholinergic Neurons drug effects, Cholinergic Neurons physiology, Colon innervation, Colon physiology, Nitrergic Neurons drug effects, Nitrergic Neurons physiology, Nitroarginine pharmacology, Peristalsis drug effects, Peristalsis physiology
- Abstract
The enteric nervous system in the large intestine generates two important patterns relating to motility: 1 ) propagating rhythmic peristaltic smooth muscle contractions referred to as colonic migrating motor complexes (CMMCs) and 2 ) tonic inhibition, during which colonic smooth muscle contractions are suppressed. The precise neurobiological substrates underlying each of these patterns are unclear. Using transgenic animals expressing the genetically encoded calcium indicator GCaMP3 to monitor activity or the optogenetic actuator channelrhodopsin (ChR2) to drive activity in defined enteric neuronal subpopulations, we provide evidence that cholinergic and nitrergic neurons play significant roles in mediating CMMCs and tonic inhibition, respectively. Nitrergic neurons [neuronal nitric oxide synthase (nNOS)-positive neurons] expressing GCaMP3 exhibited higher levels of activity during periods of tonic inhibition than during CMMCs. Consistent with these findings, optogenetic activation of ChR2 in nitrergic neurons depressed ongoing CMMCs. Conversely, cholinergic neurons [choline acetyltransferase (ChAT)-positive neurons] expressing GCaMP3 markedly increased their activity during the CMMC. Treatment with the NO synthesis inhibitor N
ω -nitro-l-arginine also augmented the activity of ChAT-GCaMP3 neurons, suggesting that the reciprocal patterns of activity exhibited by nitrergic and cholinergic enteric neurons during distinct phases of colonic motility may be related. NEW & NOTEWORTHY Correlating the activity of neuronal populations in the myenteric plexus to distinct periods of gastrointestinal motility is complicated by the difficulty of measuring the activity of specific neuronal subtypes. Here, using mice expressing genetically encoded calcium indicators or the optical actuator channelrhodopsin-2, we provide compelling evidence that cholinergic and nitrergic neurons play important roles in mediating coordinated propagating peristaltic contractions or tonic inhibition, respectively, in the murine colon.- Published
- 2019
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14. Glial cells maintain synapses by inhibiting an activity-dependent retrograde protease signal.
- Author
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Gould TW, Dominguez B, de Winter F, Yeo GW, Liu P, Sundararaman B, Stark T, Vu A, Degen JL, Lin W, and Lee KF
- Subjects
- Animals, Gene Expression Profiling, Mice, Motor Neurons metabolism, Motor Neurons pathology, Muscle, Skeletal metabolism, Nerve Degeneration genetics, Neuroglia, Neuromuscular Junction growth & development, Schwann Cells metabolism, Thrombin genetics, Antithrombin III genetics, Heparin Cofactor II genetics, Neuromuscular Junction genetics, Prothrombin genetics, Synapses genetics
- Abstract
Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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15. Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm.
- Author
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Heredia DJ, Hennig GW, and Gould TW
- Subjects
- Animals, Calcium metabolism, Calcium Signaling genetics, Fluorescent Dyes metabolism, Mice, Mice, Transgenic, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Calcium Signaling physiology, Diaphragm innervation, Neuromuscular Junction metabolism, Optical Imaging methods
- Abstract
The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca
2+ ) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes. However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+ , thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.- Published
- 2018
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16. Postnatal Restriction of Activity-Induced Ca 2+ Responses to Schwann Cells at the Neuromuscular Junction Are Caused by the Proximo-Distal Loss of Axonal Synaptic Vesicles during Development.
- Author
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Heredia DJ, Feng CY, Agarwal A, Nennecker K, Hennig GW, and Gould TW
- Subjects
- Animals, Female, Male, Mice, Inbred C57BL, Mice, Transgenic, Neuromuscular Junction metabolism, Neuromuscular Junction ultrastructure, Phrenic Nerve physiology, Presynaptic Terminals ultrastructure, Schwann Cells ultrastructure, Synaptic Vesicles ultrastructure, Calcium Signaling, Neuromuscular Junction embryology, Presynaptic Terminals metabolism, Schwann Cells metabolism, Synaptic Vesicles metabolism
- Abstract
Terminal or perisynaptic Schwann cells (TPSCs) are nonmyelinating, perisynaptic glial cells at the neuromuscular junction (NMJ) that respond to neural activity by increasing intracellular calcium (Ca
2+ ) and regulate synaptic function. The onset of activity-induced TPSC Ca2+ responses, as well as whether axonal Schwann cells (ASCs) along the nerve respond to nerve stimulation during development, is unknown. Here, we show that phrenic nerve stimulation in developing male and female mice elicited Ca2+ responses in both ASCs and TPSCs at embryonic day 14. ASC responses were lost in a proximo-distal gradient over time, but could continue to be elicited by bath application of neurotransmitter, suggesting that a loss of release rather than a change in ASC competence accounted for this response gradient. Similar to those of early postnatal TPSCs, developing ASC/TPSC responses were mediated by purinergic P2Y1 receptors. The loss of ASC Ca2+ responses was correlated to the proximo-distal disappearance of synaptophysin immunoreactivity and synaptic vesicles in phrenic axons. Accordingly, developing ASC Ca2+ responses were blocked by botulinum toxin. Interestingly, the loss of ASC Ca2+ responses was also correlated to the proximo-distal development of myelination. Finally, compared with postnatal TPSCs, neonatal TPSCs and ASCs displayed Ca2+ signals in response to lower frequencies and shorter durations of nerve stimulation. Together, these results with GCaMP3-expressing Schwann cells provide ex vivo evidence that both axons and presynaptic terminals initially exhibit activity-induced vesicular release of neurotransmitter, but that the subsequent loss of axonal synaptic vesicles accounts for the postnatal restriction of vesicular release to the NMJ. SIGNIFICANCE STATEMENT Neural activity regulates multiple aspects of development, including myelination. Whether the excitation of developing neurons in vivo results in the release of neurotransmitter from both axons and presynaptic terminals is unclear. Here, using mice expressing the genetically encoded calcium indicator GCaMP3 in Schwann cells, we show that both terminal/perisynaptic Schwann cells at the diaphragm neuromuscular junction and axonal Schwann cells along the phrenic nerve exhibit activity-induced calcium responses early in development, mediated by the vesicular release of ATP from the axons of motor neurons acting on P2Y1 receptors. These ex vivo findings corroborate classic in vitro studies demonstrating transmitter release by developing axons, and thus represent a tool to study the mechanisms and significance of this process during embryonic development., (Copyright © 2018 the authors 0270-6474/18/388650-16$15.00/0.)- Published
- 2018
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17. Activity-induced Ca 2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y 1 receptors and regulates muscle fatigue.
- Author
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Heredia DJ, Feng CY, Hennig GW, Renden RB, and Gould TW
- Subjects
- Animals, Mice, Mice, Transgenic, Receptors, Purinergic P2Y1 deficiency, Calcium Signaling, Muscle Fatigue, Receptors, Purinergic P2Y1 metabolism, Schwann Cells physiology
- Abstract
Perisynaptic glial cells respond to neural activity by increasing cytosolic calcium, but the significance of this pathway is unclear. Terminal/perisynaptic Schwann cells (TPSCs) are a perisynaptic glial cell at the neuromuscular junction that respond to nerve-derived substances such as acetylcholine and purines. Here, we provide genetic evidence that activity-induced calcium accumulation in neonatal TPSCs is mediated exclusively by one subtype of metabotropic purinergic receptor. In P2ry1 mutant mice lacking these responses, postsynaptic, rather than presynaptic, function was altered in response to nerve stimulation. This impairment was correlated with a greater susceptibility to activity-induced muscle fatigue. Interestingly, fatigue in P2ry1 mutants was more greatly exacerbated by exposure to high potassium than in control mice. High potassium itself increased cytosolic levels of calcium in TPSCs, a response which was also reduced P2ry1 mutants. These results suggest that activity-induced calcium responses in TPSCs regulate postsynaptic function and muscle fatigue by regulating perisynaptic potassium., Competing Interests: DH, CF, GH, RR, TG No competing interests declared, (© 2018, Heredia et al.)
- Published
- 2018
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18. SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.
- Author
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Sarathy A, Wuebbles RD, Fontelonga TM, Tarchione AR, Mathews Griner LA, Heredia DJ, Nunes AM, Duan S, Brewer PD, Van Ry T, Hennig GW, Gould TW, Dulcey AE, Wang A, Xu X, Chen CZ, Hu X, Zheng W, Southall N, Ferrer M, Marugan J, and Burkin DJ
- Subjects
- Animals, Cell Differentiation drug effects, Cell Line, Disease Models, Animal, Disease Progression, Female, Fibrosis, Humans, Integrins agonists, Mice, Mice, Inbred mdx, Models, Biological, Muscle Development drug effects, Muscle Strength, Muscle, Skeletal drug effects, Muscle, Skeletal metabolism, Muscular Dystrophy, Duchenne drug therapy, Myoblasts, Skeletal cytology, Myoblasts, Skeletal drug effects, Myoblasts, Skeletal metabolism, NF-kappa B metabolism, Protein Serine-Threonine Kinases metabolism, Regeneration drug effects, Signal Transduction drug effects, Imidazoles pharmacology, Indoles pharmacology, Integrins metabolism, Muscular Dystrophy, Duchenne metabolism, Muscular Dystrophy, Duchenne pathology
- Abstract
Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD., (Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.)
- Published
- 2017
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19. A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology.
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Heredia DJ, Schubert D, Maligireddy S, Hennig GW, and Gould TW
- Abstract
The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one (BHC), previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as N-benzyl-p-toluene sulphonamide (BTS), which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined in whole populations of muscle cells expressing the genetically-encoded calcium indicator GCaMP3. Most importantly, BHC allowed for the first time a detailed analysis of synaptic transmission, calcium signaling and fatigue in embryonic mice, such as in Vamp2 mutants reported here, that die before or at birth. Together, these studies illustrate the wide utility of BHC in allowing stable measurements of neuromuscular function.
- Published
- 2016
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20. Structural and Functional Abnormalities of the Neuromuscular Junction in the Trembler-J Homozygote Mouse Model of Congenital Hypomyelinating Neuropathy.
- Author
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Scurry AN, Heredia DJ, Feng CY, Gephart GB, Hennig GW, and Gould TW
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- Animals, Animals, Newborn, Diaphragm pathology, Diaphragm ultrastructure, Electric Stimulation, Evoked Potentials genetics, Homozygote, Humans, Mice, Mice, Inbred BALB C, Mice, Transgenic, Microscopy, Electron, Neural Conduction genetics, Neuromuscular Junction pathology, Neuromuscular Junction ultrastructure, Neuromuscular Junction Diseases genetics, Point Mutation genetics, Charcot-Marie-Tooth Disease genetics, Charcot-Marie-Tooth Disease pathology, Disease Models, Animal, Myelin Proteins genetics, Neuromuscular Junction Diseases etiology, Neuromuscular Junction Diseases pathology
- Abstract
Mutations in peripheral myelin protein 22 (PMP22) result in the most common form of Charcot-Marie-Tooth (CMT) disease, CMT1A. This hereditary peripheral neuropathy is characterized by dysmyelination of peripheral nerves, reduced nerve conduction velocity, and muscle weakness. APMP22 point mutation in L16P (leucine 16 to proline) underlies a form of human CMT1A as well as the Trembler-J mouse model of CMT1A. Homozygote Trembler-J mice (Tr(J)) die early postnatally, fail to make peripheral myelin, and, therefore, are more similar to patients with congenital hypomyelinating neuropathy than those with CMT1A. Because recent studies of inherited neuropathies in humans and mice have demonstrated that dysfunction and degeneration of neuromuscular synapses or junctions (NMJs) often precede impairments in axonal conduction, we examined the structure and function of NMJs in Tr(J)mice. Although synapses appeared to be normally innervated even in end-stage Tr(J)mice, the growth and maturation of the NMJs were altered. In addition, the amplitudes of nerve-evoked muscle endplate potentials were reduced and there was transmission failure during sustained nerve stimulation. These results suggest that the severe congenital hypomyelinating neuropathy that characterizes Tr(J)mice results in structural and functional deficits of the developing NMJ., (© 2016 American Association of Neuropathologists, Inc. All rights reserved.)
- Published
- 2016
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21. Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon.
- Author
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Hennig GW, Gould TW, Koh SD, Corrigan RD, Heredia DJ, Shonnard MC, and Smith TK
- Abstract
Genetically encoded Ca(2+) indicators (GECIs) have been used extensively in many body systems to detect Ca(2+) transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca(2+) indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca(2+) imaging. In this preliminary study, we observed neuronal and glial cell Ca(2+) transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100-1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca(2+) transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca(2+) transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca(2+) transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity but continued long after neural activity had waned. With these new tools an unprecedented level of detail can be recorded from the enteric nervous system (ENS) with minimal manipulation of tissue. These techniques can be extended in order to better understand the roles of particular enteric neurons and glia during normal and disordered motility.
- Published
- 2015
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22. A novel class of interstitial cells in the mouse and monkey female reproductive tracts.
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Peri LE, Koh BH, Ward GK, Bayguinov Y, Hwang SJ, Gould TW, Mullan CJ, Sanders KM, and Ward SM
- Subjects
- Animals, Connexin 43 biosynthesis, Connexin 43 genetics, Estrous Cycle, Female, Green Fluorescent Proteins, Interstitial Cells of Cajal, Macaca fascicularis, Mice, Mice, Inbred C57BL, Promoter Regions, Genetic genetics, Receptor, Platelet-Derived Growth Factor alpha genetics, Receptor, Platelet-Derived Growth Factor alpha metabolism, Species Specificity, Genitalia, Female cytology
- Abstract
Growing evidence suggests important roles for specialized platelet-derived growth factor receptor alpha-positive (PDGFRalpha(+)) cells in regulating the behaviors of visceral smooth muscle organs. Examination of the female reproductive tracts of mice and monkeys showed that PDGFRalpha(+) cells form extensive networks in ovary, oviduct, and uterus. PDGFRalpha(+) cells were located in discrete locations within these organs, and their distribution and density were similar in rodents and primates. PDGFRalpha(+) cells were distinct from smooth muscle cells and interstitial cells of Cajal (ICC). This was demonstrated with immunohistochemical techniques and by performing molecular expression studies on PDGFRalpha(+) cells from mice with enhanced green fluorescent protein driven off of the endogenous promoter for Pdgfralpha. Significant differences in gene expression were found in PDGFRalpha(+) cells from ovary, oviduct, and uterus. Differences in gene expression were also detected in cells from different tissue regions within the same organ (e.g., uterine myometrium vs. endometrium). PDGFRalpha(+) cells are unlikely to provide pacemaker activity because they lack significant expression of key pacemaker genes found in ICC (Kit and Ano1). Gja1 encoding connexin 43 was expressed at relatively high levels in PDGFRalpha(+) cells (except in the ovary), suggesting these cells can form gap junctions to one another and neighboring smooth muscle cells. PDGFRalpha(+) cells also expressed the early response transcription factor and proto-oncogene Fos, particularly in the ovary. These data demonstrate extensive distribution of PDGFRalpha(+) cells throughout the female reproductive tract. These cells are a heterogeneous population of cells that are likely to contribute to different aspects of physiological regulation in the various anatomical niches they occupy., (© 2015 by the Society for the Study of Reproduction, Inc.)
- Published
- 2015
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23. A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing.
- Author
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Rogers CM, Morris GE, Gould TW, Bail R, Toumpaniari S, Harrington H, Dixon JE, Shakesheff KM, Segal J, and Rose FR
- Subjects
- Animals, Cell Adhesion, Cell Proliferation, Cell Survival, Fibroblasts cytology, Lactic Acid chemistry, Mice, NIH 3T3 Cells, Polyglycolic Acid chemistry, Polylactic Acid-Polyglycolic Acid Copolymer, Electrochemical Techniques methods, Lactic Acid chemical synthesis, Polyglycolic Acid chemical synthesis, Tissue Engineering instrumentation, Tissue Scaffolds chemistry
- Abstract
Electrospinning is a common technique used to fabricate fibrous scaffolds for tissue engineering applications. There is now growing interest in assessing the ability of collector plate design to influence the patterning of the fibres during the electrospinning process. In this study, we investigate a novel method to generate hybrid electrospun scaffolds consisting of both random fibres and a defined three-dimensional (3D) micro-topography at the surface, using patterned resin formers produced by rapid prototyping (RP). Poly(D,L-lactide-co-glycolide) was electrospun onto the engineered RP surfaces and the ability of these formers to influence microfibre patterning in the resulting scaffolds visualized by scanning electron microscopy. Electrospun scaffolds with patterns mirroring the microstructures of the formers were successfully fabricated. The effect of the resulting fibre patterns and 3D geometries on mammalian cell adhesion and proliferation was investigated by seeding enhanced green fluorescent protein labelled 3T3 fibroblasts onto the scaffolds. Following 24 h and four days of culture, the seeded scaffolds were visually assessed by confocal macro- and microscopy. The patterning of the fibres guided initial cell adhesion to the scaffold with subsequent proliferation over the geometry resulting in the cells being held in a 3D micro-topography. Such patterning could be designed to replicate a specific in vivo structure; we use the dermal papillae as an exemplar here. In conclusion, a novel, versatile and scalable method to produce hybrid electrospun scaffolds has been developed. The 3D directional cues of the patterned fibres have been shown to influence cell behaviour and could be used to culture cells within a similar 3D micro-topography as experienced in vivo.
- Published
- 2014
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24. Controlled release of BMP-2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model.
- Author
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Rahman CV, Ben-David D, Dhillon A, Kuhn G, Gould TW, Müller R, Rose FR, Shakesheff KM, and Livne E
- Subjects
- Animals, Cell Line, Mice, Microscopy, Electron, Scanning, Tissue Scaffolds, Bone Morphogenetic Protein 2 metabolism
- Abstract
Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP-2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature-sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP-2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP-2 loaded into the scaffolds was released by the end of this time period. The released BMP-2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP-2-loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP-2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP-2 delivery for bone-regeneration applications., (Copyright © 2012 John Wiley & Sons, Ltd.)
- Published
- 2014
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25. Development of a porous poly(DL-lactic acid-co-glycolic acid)-based scaffold for mastoid air-cell regeneration.
- Author
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Gould TW, Birchall JP, Mallick AS, Alliston T, Lustig LR, Shakesheff KM, and Rahman CV
- Subjects
- Cadaver, Humans, Mastoid diagnostic imaging, Porosity, Tomography, X-Ray Computed, Mastoid cytology, Polyesters chemistry, Regeneration, Tissue Engineering methods, Tissue Scaffolds chemistry
- Abstract
Objectives/hypothesis: To develop a porous, biodegradable scaffold for mastoid air-cell regeneration., Study Design: In vitro development of a temperature-sensitive poly(DL-lactic acid-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) scaffold tailored for this application., Methods: Human mastoid bone microstructure and porosity were investigated using micro-computed tomography. PLGA/PEG-alginate scaffolds were developed, and scaffold porosity was assessed. Human bone marrow mesenchymal stem cells (hBM-MSCs) were cultured on the scaffolds in vitro. Scaffolds were loaded with ciprofloxacin, and release of ciprofloxacin over time in vitro was assessed., Results: Porosity of human mastoid bone was measured at 83% with an average pore size of 1.3 mm. PLGA/PEG-alginate scaffold porosity ranged from 43% to 78% depending on the alginate bead content. The hBM-MSCs proliferate on the scaffolds in vitro, and release of ciprofloxacin from the scaffolds was demonstrated over 7 to 10 weeks., Conclusions: The PLGA/PEG-alginate scaffolds developed in this study demonstrate similar structural features to human mastoid bone, support cell growth, and display sustained antibiotic release. These scaffolds may be of potential clinical use in mastoid air-cell regeneration. Further in vivo studies to assess the suitability of PLGA/PEG-alginate scaffolds for this application are required., (© 2013 The Authors. The Laryngoscope is published by Wiley Periodicals, Inc. on behalf of The American Laryngological, Rhinological and Otological Society, Inc.)
- Published
- 2013
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26. Motor neuron trophic factors: therapeutic use in ALS?
- Author
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Gould TW and Oppenheim RW
- Subjects
- Amyotrophic Lateral Sclerosis metabolism, Animals, Disease Models, Animal, Humans, Mice, Motor Neurons metabolism, Motor Neurons pathology, Nerve Degeneration metabolism, Nerve Growth Factors physiology, Nerve Growth Factors therapeutic use, Amyotrophic Lateral Sclerosis drug therapy, Amyotrophic Lateral Sclerosis pathology, Motor Neurons drug effects, Nerve Degeneration drug therapy, Nerve Degeneration pathology, Nerve Growth Factors pharmacology
- Abstract
The modest effects of neurotrophic factor (NTF) treatment on lifespan in both animal models and clinical studies of Amyotropic Lateral Sclerosis (ALS) may result from any one or combination of the four following explanations: 1.) NTFs block cell death in some physiological contexts but not in ALS; 2.) NTFs do not rescue motoneurons (MNs) from death in any physiological context; 3.) NTFs block cell death in ALS but to no avail; and 4.) NTFs are physiologically effective but limited by pharmacokinetic constraints. The object of this review is to critically evaluate the role of both NTFs and the intracellular cell death pathway itself in regulating the survival of spinal and cranial (lower) MNs during development, after injury and in response to disease. Because the role of molecules mediating MN survival has been most clearly resolved by the in vivo analysis of genetically engineered mice, this review will focus on studies of such mice expressing reporter, null or other mutant alleles of NTFs, NTF receptors, cell death or ALS-associated genes., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
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27. Nestin negatively regulates postsynaptic differentiation of the neuromuscular synapse.
- Author
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Yang J, Dominguez B, de Winter F, Gould TW, Eriksson JE, and Lee KF
- Subjects
- Acetylcholine metabolism, Agrin genetics, Agrin metabolism, Animals, Cell Line, Cyclin-Dependent Kinase 5 metabolism, Enzyme Activation, Intermediate Filament Proteins genetics, Mice, Mice, Knockout, Nerve Tissue Proteins genetics, Nestin, Receptors, Cholinergic genetics, Receptors, Cholinergic metabolism, Cell Differentiation physiology, Intermediate Filament Proteins metabolism, Muscle Development physiology, Nerve Tissue Proteins metabolism, Neuromuscular Junction physiology, Neuromuscular Junction ultrastructure
- Abstract
Positive and negative regulation of neurotransmitter receptor aggregation on the postsynaptic membrane is a critical event during synapse formation. Acetylcholine (ACh) and agrin are two opposing signals that regulate ACh receptor (AChR) clustering during neuromuscular junction (NMJ) development. ACh induces dispersion of AChR clusters that are not stabilized by agrin via a cyclin-dependent kinase 5 (Cdk5)-mediated mechanism, but regulation of Cdk5 activation is poorly understood. We found that the intermediate filament protein nestin physically interacts with Cdk5 and is required for ACh-induced association of p35, the co-activator of Cdk5, with the muscle membrane. Blockade of nestin-dependent signaling inhibited ACh-induced Cdk5 activation and the dispersion of AChR clusters in cultured myotubes. Similar to the effects of Cdk5 gene inactivation, knockdown of nestin in agrin-deficient mouse embryos substantially restored AChR clusters. These results suggest that nestin is required for ACh-induced, Cdk5-dependent dispersion of AChR clusters during NMJ development.
- Published
- 2011
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28. Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms.
- Author
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An MC, Lin W, Yang J, Dominguez B, Padgett D, Sugiura Y, Aryal P, Gould TW, Oppenheim RW, Hester ME, Kaspar BK, Ko CP, and Lee KF
- Subjects
- Acetylation, Acetylcholine agonists, Animals, Carbachol pharmacology, Cell Differentiation, Cholinergic Agonists pharmacology, Mice, Neuromuscular Junction cytology, Neuromuscular Junction drug effects, Receptors, Nicotinic genetics, Receptors, Nicotinic metabolism, Acetylcholine metabolism, Neuromuscular Junction metabolism
- Abstract
Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.
- Published
- 2010
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29. The vesicular acetylcholine transporter is required for neuromuscular development and function.
- Author
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de Castro BM, De Jaeger X, Martins-Silva C, Lima RD, Amaral E, Menezes C, Lima P, Neves CM, Pires RG, Gould TW, Welch I, Kushmerick C, Guatimosim C, Izquierdo I, Cammarota M, Rylett RJ, Gomez MV, Caron MG, Oppenheim RW, Prado MA, and Prado VF
- Subjects
- Animals, Base Sequence, Cell Line, Embryo, Mammalian embryology, Embryo, Mammalian metabolism, Humans, Mice, Mice, Knockout, Molecular Sequence Data, Muscle, Skeletal embryology, Neuromuscular Junction embryology, Synaptic Vesicles metabolism, Vesicular Acetylcholine Transport Proteins deficiency, Vesicular Acetylcholine Transport Proteins genetics, Acetylcholine metabolism, Motor Neurons metabolism, Muscle Development, Muscle, Skeletal metabolism, Neuromuscular Junction growth & development, Neuromuscular Junction metabolism, Vesicular Acetylcholine Transport Proteins metabolism
- Abstract
The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.
- Published
- 2009
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30. Neurotrophic modulation of motor neuron development.
- Author
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Gould TW and Enomoto H
- Subjects
- Animals, Motor Neurons drug effects, Nerve Growth Factors genetics, Nerve Growth Factors pharmacology, Motor Neurons physiology, Nerve Growth Factors metabolism
- Abstract
Neurotrophic factors (NTFs) are a pleiotropic group of secreted growth factors that regulate multiple aspects of neuronal development, including the regressive event of cell death. Skeletal muscleinnervating lower motoneurons (MNs) of the brain stem and spinal cord comprise one population of central neurons in which programmed cell death (PCD) during embryogenesis has been actively investigated, as much for reasons of technical facility as clinical relevance. The precise identity of NTF-dependent MNs has remained unclear, with most studies simply reporting losses or gains across the entire spinal cord or individual brain-stem nuclei. However, MNs are grouped into highly heterogenous populations based on transcriptional identity, target innervation, and physiological function. Therefore, recent work has focused on the effects of NTF overexpression or deletion on the survival of these MN subpopulations. Together with the recent progress attained in the generation of conditional mutant mice, in which the function of an NTF or its receptor can be eliminated specifically in MNs, these recent studies have begun to define the differential trophic requirements for MN subpopulations during PCD. The intent of this review is to summarize these recent findings and to discuss their significance with respect to neurotrophic theory.
- Published
- 2009
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31. The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes.
- Author
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Gould TW, Yonemura S, Oppenheim RW, Ohmori S, and Enomoto H
- Subjects
- Animals, Animals, Newborn, Caspase 3 metabolism, Embryo, Mammalian, Gene Expression Regulation, Developmental genetics, Glial Cell Line-Derived Neurotrophic Factor deficiency, Glial Cell Line-Derived Neurotrophic Factor Receptors deficiency, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Hindlimb embryology, Hindlimb growth & development, Hindlimb innervation, Mice, Mice, Knockout, Muscle Spindles embryology, Muscle Spindles growth & development, Muscle Spindles metabolism, Muscle, Skeletal metabolism, Point Mutation, Proto-Oncogene Proteins c-ret deficiency, Proto-Oncogene Proteins c-ret genetics, Vesicular Acetylcholine Transport Proteins genetics, Vesicular Acetylcholine Transport Proteins metabolism, Glial Cell Line-Derived Neurotrophic Factor physiology, Motor Neurons classification, Motor Neurons physiology, Spinal Cord cytology
- Abstract
Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.
- Published
- 2008
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32. Synaptic dysfunction in disease and following injury in the developing and adult nervous system: caveats in the choice of therapeutic intervention.
- Author
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Gould TW and Oppenheim RW
- Subjects
- Animals, Cell Death, Disease Models, Animal, Humans, Mice, Presynaptic Terminals ultrastructure, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis pathology, Nervous System pathology, Neuromuscular Junction pathology, Presynaptic Terminals physiology
- Abstract
A cardinal feature of most developmental and adult onset neurodegenerative diseases is the death of specific populations of neurons. Largely as a result of the progress made in elucidating the cellular and molecular mechanisms underlying the neuronal death that occurs during development, approaches ameliorating them often focus on the manipulation of neuronal death pathways. Recent evidence derived from the study of animal models of various neuropathological conditions, however, has revealed that damage to axons and synapses long precedes the activation of death pathways. We recently extended these findings to the most commonly studied animal model of familial amyotrophic lateral sclerosis (fALS). Inhibiting the cell death pathway by deletion of the pro-apoptotic gene Bax completely rescued spinal MNs yet failed to prevent disease in fALS transgenic mice. However, we observed distinct abnormalities within presynaptic terminals of spinal MNs at the neuromuscular junction (NMJ), as well as profound denervation. These results suggest that therapies aimed at preserving the synapse rather than the soma may be more effective at treating these neuropathologies.
- Published
- 2007
- Full Text
- View/download PDF
33. Neuromuscular development in the absence of programmed cell death: phenotypic alteration of motoneurons and muscle.
- Author
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Buss RR, Gould TW, Ma J, Vinsant S, Prevette D, Winseck A, Toops KA, Hammarback JA, Smith TL, and Oppenheim RW
- Subjects
- Animals, Apoptosis physiology, Axons physiology, Axons ultrastructure, Cell Size, Chick Embryo, Female, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Knockout, Mice, Transgenic, Motor Neurons ultrastructure, Muscle, Skeletal ultrastructure, Myogenin biosynthesis, Myogenin genetics, bcl-2-Associated X Protein biosynthesis, bcl-2-Associated X Protein genetics, Apoptosis genetics, Motor Neurons cytology, Motor Neurons physiology, Muscle, Skeletal cytology, Muscle, Skeletal growth & development, Phenotype
- Abstract
The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from alpha- to gamma-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.
- Published
- 2006
- Full Text
- View/download PDF
34. Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS.
- Author
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Gould TW, Buss RR, Vinsant S, Prevette D, Sun W, Knudson CM, Milligan CE, and Oppenheim RW
- Subjects
- Amyotrophic Lateral Sclerosis genetics, Animals, Axons, Cell Death, Cell Survival, Demyelinating Diseases, Denervation, Gliosis prevention & control, Mice, Mice, Transgenic, Mitochondria ultrastructure, Neuromuscular Junction physiopathology, Neuromuscular Junction ultrastructure, Presynaptic Terminals metabolism, Schwann Cells metabolism, Spinal Nerve Roots physiopathology, Superoxide Dismutase metabolism, Superoxide Dismutase-1, Time Factors, Vacuoles ultrastructure, Amyotrophic Lateral Sclerosis physiopathology, Gene Deletion, Motor Neurons metabolism, Movement, Mutation, Superoxide Dismutase genetics, bcl-2-Associated X Protein genetics
- Abstract
The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.
- Published
- 2006
- Full Text
- View/download PDF
35. Distinct susceptibility of developing neurons to death following Bax overexpression in the chicken embryo.
- Author
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Sato N, Sakuma C, Sato Y, Gould TW, Oppenheim RW, and Yaginuma H
- Subjects
- Animals, Cell Line, Chick Embryo, Genetic Vectors, Motor Neurons cytology, Mutation, Neurons, Afferent cytology, Retina cytology, Retina embryology, Retroviridae genetics, Spinal Cord cytology, Spinal Cord embryology, bcl-2-Associated X Protein genetics, Apoptosis, Motor Neurons metabolism, Neurons, Afferent metabolism, bcl-2-Associated X Protein metabolism
- Abstract
Bax is a proapoptotic protein that is required for programmed cell death (PCD) of many neuronal populations. Here we show that, during an early period of retinal PCD and in naturally occurring sensory and motor neuron (MN) death in the spinal cord, Bax delivery results in enhanced death of these neural populations. In contrast, Bax overexpression fails to enhance an early phase of MN death that occurs in the cervical spinal cord, although overexpressed Bax appears to be activated in dying MNs. Bax overexpression does not also affect the survival of immature neurons prior to the PCD period. Taken together, these data provide the first in vivo evidence suggesting that Bax appears to act selectively as an executioner only in neurons undergoing PCD. Furthermore, although Bax appears to mediate the execution pathway for PCD, the effect of Bax overexpression on susceptibility to death differs between different neuronal populations.
- Published
- 2006
- Full Text
- View/download PDF
36. Phosphorylation of c-Jun in avian and mammalian motoneurons in vivo during programmed cell death: an early reversible event in the apoptotic cascade.
- Author
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Sun W, Gould TW, Newbern J, Milligan C, Choi SY, Kim H, and Oppenheim RW
- Subjects
- Animals, Cell Count, Chick Embryo, In Vitro Techniques, Limb Buds embryology, Limb Buds innervation, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Phosphorylation, Proto-Oncogene Proteins c-akt metabolism, Proto-Oncogene Proteins c-jun antagonists & inhibitors, Proto-Oncogene Proteins c-jun genetics, Signal Transduction, Spinal Cord cytology, bcl-2-Associated X Protein genetics, Apoptosis, Motor Neurons metabolism, Proto-Oncogene Proteins c-jun metabolism
- Abstract
c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo. We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.
- Published
- 2005
- Full Text
- View/download PDF
37. The function of neurotrophic factor receptors expressed by the developing adductor motor pool in vivo.
- Author
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Gould TW and Oppenheim RW
- Subjects
- Animals, Apoptosis drug effects, Apoptosis physiology, Cell Survival drug effects, Cell Survival physiology, Chick Embryo, Ciliary Neurotrophic Factor antagonists & inhibitors, Ciliary Neurotrophic Factor pharmacology, Glial Cell Line-Derived Neurotrophic Factor, Hindlimb embryology, Hindlimb innervation, Ligands, Motor Neurons cytology, Motor Neurons drug effects, Muscle, Skeletal embryology, Muscle, Skeletal innervation, Nerve Growth Factors antagonists & inhibitors, Nerve Growth Factors pharmacology, Receptors, Nerve Growth Factor antagonists & inhibitors, Receptors, Nerve Growth Factor biosynthesis, Signal Transduction drug effects, Signal Transduction physiology, Spinal Cord cytology, Motor Neurons metabolism, Receptors, Nerve Growth Factor physiology, Spinal Cord embryology, Spinal Cord metabolism
- Abstract
We examined the spatio-temporal relationship between neurotrophic factor receptor (NTF-R) expression and motoneuron (MN) survival in the developing avian spinal cord and observed heterogeneity in the expression of NTF-Rs between, but not within, pools of MNs projecting to individual muscles. We then focused on the role of NTFs in regulating the survival of one motor pool of MNs, all of which innervate a pair of adductor muscles in the thigh and hence compete for survival during the period of programmed cell death (PCD). The complete NTF-R complement of these MNs was analyzed and found to include many, but not all, NTF-Rs. Treatment with exogenous individual NTFs rescued some, but not all, adductor MNs expressing appropriate NTF-Rs. In contrast, administration of multiple NTFs completely rescued adductor MNs from PCD. Additionally, adductor MNs were partially rescued from PCD by NTFs for which they failed to express receptors. NTF-Rs expressed by the nerve but not in the muscle target were capable of mediating survival signals to MNs in trans. Finally, the expression of some NTF-Rs by adductor MNs was not required for MN survival. These studies demonstrate the complexity in NTF regulation of a defined subset of competing MNs and suggest that properties other than NTF-R expression itself can play a role in mediating trophic responses to NTFs.
- Published
- 2004
- Full Text
- View/download PDF
38. Neuromuscular development after the prevention of naturally occurring neuronal death by Bax deletion.
- Author
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Sun W, Gould TW, Vinsant S, Prevette D, and Oppenheim RW
- Subjects
- Animals, Animals, Newborn, Atrophy, Axons physiology, Axons ultrastructure, Cell Division, Ganglia, Spinal cytology, Glial Cell Line-Derived Neurotrophic Factor, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Neurological, Motor Neurons pathology, Motor Neurons ultrastructure, Nerve Growth Factors pharmacology, Nervous System embryology, Nervous System growth & development, Neuromuscular Junction cytology, Neuromuscular Junction growth & development, bcl-2-Associated X Protein, Apoptosis, Motor Neurons cytology, Muscle, Skeletal innervation, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-bcl-2
- Abstract
The removal of excess neurons by programmed cell death (PCD) is believed to be critical for the proper development and function of the nervous system. A major role of this neuronal loss is to attain quantitative matching of neurons with their targets and afferents. Because motoneurons (MNs) in Bax knock-out (Bax KO) mice fail to undergo PCD in the face of normal target muscle development, we asked whether the excess rescued neurons in Bax KO mice can develop normally. We observed many small atrophied MNs in postnatal Bax KO mice, and these failed to innervate limb muscle targets. When examined embryonically during the PCD period, however, these excess MNs had initiated target innervation. To examine whether a limitation in trophic factor availability is responsible for postnatal MN atrophy and loss of innervation, we applied glial cell line-derived neurotrophic factor (GDNF) to neonatal mice. GDNF injection for 7-14 d induced the regrowth and reinnervation of muscle targets by atrophic MNs in Bax KO mice and prevented the normal postnatal death of MNs in wild-type mice. These results indicate that, although initially all of the MNs, including those rescued by Bax deletion, are able to project to and innervate targets, because of limited target-derived signals required for maintaining innervation and growth, only a subpopulation can grow and retain target contacts postnatally. Although sensory neurons in the dorsal root ganglia are also rescued from PCD by Bax deletion, their subsequent development is less affected than that of MNs.
- Published
- 2003
39. Cytokines promote motoneuron survival through the Janus kinase-dependent activation of the phosphatidylinositol 3-kinase pathway.
- Author
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Dolcet X, Soler RM, Gould TW, Egea J, Oppenheim RW, and Comella JX
- Subjects
- Animals, Apoptosis drug effects, Apoptosis physiology, Brain-Derived Neurotrophic Factor metabolism, Brain-Derived Neurotrophic Factor pharmacology, Cell Survival drug effects, Cells, Cultured, Chick Embryo, Ciliary Neurotrophic Factor metabolism, Ciliary Neurotrophic Factor pharmacology, Cytokines pharmacology, DNA-Binding Proteins drug effects, DNA-Binding Proteins metabolism, Enzyme Inhibitors pharmacology, Glial Cell Line-Derived Neurotrophic Factor, Hepatocyte Growth Factor metabolism, Hepatocyte Growth Factor pharmacology, Immunohistochemistry, Janus Kinase 1, Janus Kinase 3, MAP Kinase Kinase 1, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinase Kinases metabolism, Mitogen-Activated Protein Kinases drug effects, Mitogen-Activated Protein Kinases metabolism, Motor Neurons cytology, Motor Neurons drug effects, Muscle, Skeletal metabolism, Nerve Tissue Proteins metabolism, Nerve Tissue Proteins pharmacology, Phosphatidylinositol 3-Kinases drug effects, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases drug effects, Proto-Oncogene Proteins drug effects, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, STAT3 Transcription Factor, Signal Transduction drug effects, Spinal Cord cytology, Spinal Cord growth & development, Trans-Activators drug effects, Trans-Activators metabolism, Cell Survival physiology, Cytokines metabolism, Motor Neurons metabolism, Nerve Growth Factors, Phosphatidylinositol 3-Kinases metabolism, Protein-Tyrosine Kinases metabolism, Signal Transduction physiology, Spinal Cord embryology
- Abstract
To determine which intracellular pathways mediate the survival effects of ciliary neurotrophic factor and cardiotrophin-1 cytokines on motoneurons, we studied the activation of the Jak/STAT, the PI 3-kinase/Akt, and the ERK pathways. At shorter time points, cytokines induced the activation of STAT3 and ERK, but not PI 3-kinase. Jak3 inhibitor suppressed cytokine- and muscle extract-induced survival. In contrast, PD 98059, a MEK inhibitor, was not able to prevent cytokine-induced survival, demonstrating that ERK is not involved. Surprisingly, the PI 3-kinase inhibitor LY 294002 prevented the survival-promoting effects of cytokines. When assays of PI 3-kinase activity were performed at later stages following cytokine treatment a significant increase was observed compared to control cultures. This delayed increase of activity could be completely prevented by treatment with protein synthesis or Jak3 inhibitors. Collectively, these results demonstrate that cytokines induce motoneuron survival through a PI 3-kinase activation requiring de novo protein synthesis dependent on Jak pathway.
- Published
- 2001
- Full Text
- View/download PDF
40. Stepping stone to death.
- Author
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Gould TW and Oppenheim RW
- Subjects
- Animals, Axons physiology, Cell Survival, Muscle, Skeletal embryology, Nerve Growth Factors physiology, Zebrafish physiology, Apoptosis physiology, Motor Neurons physiology, Muscle, Skeletal innervation, Zebrafish embryology
- Published
- 2001
- Full Text
- View/download PDF
41. Hepatocyte growth factor/scatter factor is a neurotrophic survival factor for lumbar but not for other somatic motoneurons in the chick embryo.
- Author
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Novak KD, Prevette D, Wang S, Gould TW, and Oppenheim RW
- Subjects
- Animals, Antibodies pharmacology, Cell Death drug effects, Cell Survival drug effects, Cells, Cultured, Chick Embryo, Cranial Nerves cytology, Cranial Nerves embryology, Gene Expression Regulation, Developmental, Hepatocyte Growth Factor antagonists & inhibitors, In Situ Hybridization, Limb Buds embryology, Limb Buds innervation, Limb Buds physiology, Motor Neurons chemistry, Motor Neurons drug effects, Proto-Oncogene Proteins c-met analysis, Proto-Oncogene Proteins c-met biosynthesis, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Spinal Cord embryology, Hepatocyte Growth Factor genetics, Hepatocyte Growth Factor pharmacology, Motor Neurons cytology, Spinal Cord cytology
- Abstract
Hepatocyte growth factor/scatter factor (HGF/SF) is expressed in the developing limb muscles of the chick embryo during the period of spinal motoneuron (MN) programmed cell death, and its receptor c-met is expressed in lumbar MNs during this same period. Although cultured motoneurons from brachial, thoracic, and lumbar segments are all rescued from cell death by chick embryo muscle extract (CMX) as well as by other specific trophic agents, HGF/SF only promotes the survival of lumbar MNs. Similarly, treatment of embryos in ovo with exogenous HGF/SF rescues lumbar but not other somatic MNs from cell death. Blocking antibodies to HGF/SF (anti-HGF) reduce the effects of CMX on MN survival in vitro and decrease the number of lumbar MNs in vivo. The expression of c-met on MNs in vivo is regulated by a limb-derived trophic signal distinct from HGF/SF. HGF/SF is a potent, select, and physiologically relevant survival factor for a subpopulation of developing spinal MNs in the lumbar segments of the chick embryo.
- Published
- 2000
42. The spatial-temporal gradient of naturally occurring motoneuron death reflects the time of prior exit from the cell cycle and position within the lateral motor column.
- Author
-
Gould TW, Burek MJ, Sosnowski JM, Prevette D, and Oppenheim RW
- Subjects
- Animals, Bromodeoxyuridine, Cell Division, Chick Embryo, Embryo, Nonmammalian innervation, Immunohistochemistry, Time Factors, Apoptosis genetics, Cell Cycle genetics, Motor Neurons metabolism, Spinal Nerves embryology
- Abstract
Embryonic lumbar spinal motoneurons (MNs) are characterized by a period of programmed cell death (PCD) that spans several days and occurs in a rostrocaudal gradient. The generation of these MNs also takes place in a temporal-spatial gradient, such that MNs within rostral lumbar segments exit the cell cycle earlier and MNs within progressively caudal regions are born later. In vitro studies have shown that the latest born spinal MNs, presumably through the possession of endogenous "survival properties," are also the last to acquire their trophic dependence. If the birth date and therefore spinal cord location of lumbar spinal MNs influence the spatial-temporal pattern of PCD, then earlier born MNs should die sooner and be located more rostrally than those generated later. Alternatively, if the time at which MNs die during development is unrelated to their prior exit from the cell cycle, those born at various phases should die throughout the period of PCD. We report here that lumbar MNs generated during the earliest part (embryonic day 2-3) of the proliferative period in the developing chick spinal cord tend to die during the earliest stages of the PCD period and that MNs born in successive 12-h intervals die at correspondingly later periods during PCD. Furthermore, the spatial progression of PCD of these subpopulations of MNs occurs in a rostrocaudal gradient. Finally, while MNs do appear to die in a mediolateral gradient during the period of MN PCD, this pattern is only partly accounted for by MNs born in consecutive intervals. These data support the notion that the timing and rostrocaudal location of MNs undergoing PCD reflect their time of exit from the cell cycle.
- Published
- 1999
- Full Text
- View/download PDF
43. Androgens rescue avian embryonic lumbar spinal motoneurons from injury-induced but not naturally occurring cell death.
- Author
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Gould TW, Burek MJ, Ishihara R, Lo AC, Prevette D, and Oppenheim RW
- Subjects
- Androgen Antagonists pharmacology, Animals, Apoptosis drug effects, Axotomy, Cell Death, Cell Division drug effects, Cell Survival drug effects, Chick Embryo, Dihydrotestosterone pharmacology, Flutamide pharmacology, Lumbosacral Region, Motor Neurons drug effects, Receptors, Androgen analysis, Spinal Cord cytology, Testosterone pharmacology, Androgens pharmacology, Motor Neurons cytology, Motor Neurons physiology, Spinal Cord embryology
- Abstract
The regulation of survival of spinal motoneurons (MNs) has been shown to depend during development and after injury on a variety of neurotrophic molecules produced by skeletal muscle target tissue. Increasing evidence also suggests that other sources of trophic support prevent MNs from undergoing naturally occurring or injury-induced death. We have examined the role of endogenous and exogenous androgens on the survival of developing avian lumbar spinal MNs during their period of programmed cell death (PCD) between embryonic day (E)6 and E11 or after axotomy on E12. We found that although treatment with testosterone, dihydrotestosterone (DHT), or the androgen receptor antagonist flutamide (FL) failed to affect the number of these MNs during PCD, administration of DHT from E12 to E15 following axotomy on E12 significantly attenuated injury-induced MN death. This effect was inhibited by cotreatment with FL, whereas treatment with FL alone did not affect MN survival. Finally, we examined the spinal cord at various times during development and following axotomy on E12 for the expression of androgen receptor using the polyclonal PG-21 antibody. Our results suggest that exogenously applied androgens are capable of rescuing MNs from injury-induced cell death and that they act directly on these cells via an androgen receptor-mediated mechanism. By contrast, endogenous androgens do not appear to be involved in the regulation of normal PCD of developing avian MNs., (Copyright 1999 John Wiley & Sons, Inc.)
- Published
- 1999
- Full Text
- View/download PDF
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