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1.
Biomolecules ; 11(10)2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34680132

RESUMO

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066-1315 (emGFP-1066:TetC), 1093-1315 (emGFP-1093:TetC) and 1109-1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.


Assuntos
Microscopia Confocal , Junção Neuromuscular/diagnóstico por imagem , Toxina Tetânica/toxicidade , Animais , Animais Recém-Nascidos , Axônios/efeitos dos fármacos , Axônios/metabolismo , Sítios de Ligação , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Tecido Nervoso/efeitos dos fármacos , Tecido Nervoso/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos
2.
Neuropharmacology ; 49(2): 254-64, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15993447

RESUMO

AMPA receptor potentiating drugs (e.g. ampakines) enhance glutamatergic neurotransmission, and may have potential therapeutic consequences in CNS disorders. The neuroanatomical basis of action for these compounds is at present unclear. This study aimed to identify the effects of two novel ampakines, Org 26576 and Org 24448, on local cerebral glucose use (LCGU) in the mouse. C57BL/6J mice received Org 26576 (0.1, 1, 10 mg/kg i.p.) or Org 24448 (3, 10, 30 mg/kg i.p.) or vehicle and LCGU was assessed using 14C-2-deoxyglucose autoradiography. Both compounds produced dose-dependent increases in LCGU with specific regional activation at low doses. Org 26576 (1 mg/kg) produced significant increases in 9 of the 43 areas examined, including the anteroventral and laterodorsal thalamus, cingulate cortex, dentate gyrus and CA3 subfield of the hippocampus. Org 24448 (3 mg/kg) produced significant increases in LCGU in 4 of the 43 regions examined, including the dorsal raphe nucleus, medial lateral habenula, CA1 subfield of the hippocampus and median forebrain bundle. Furthermore, the increases in LCGU observed with both Org 26576 (10 mg/kg) and Org 24448 (10 mg/kg) were blocked by pre-treatment with the AMPA receptor antagonist NBQX (10 mg/kg). These data demonstrate that both Org 26576 and Org 24448 produce dose-dependent AMPA receptor mediated increases in LCGU and provide an anatomical basis suggestive that these drugs may be of use in the treatment of conditions such as depression or schizophrenia.


Assuntos
Autorradiografia/métodos , Encéfalo/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Desoxiglucose/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Glucose/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Radioisótopos de Carbono/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinoxalinas/farmacologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/agonistas
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