RESUMEN
Spinal motor neurons (MNs) are susceptible to glutamatergic excitotoxicity, an effect associated with lumbar MN degeneration in amyotrophic lateral sclerosis (ALS). MN susceptibility to environmental toxicant exposure, one prospective contributor to sporadic ALS, has not been systematically studied. The goal of this study was to test the ability of a well-known environmental neurotoxicant to induce hyperexcitability in mouse lumbar MNs. Methylmercury (MeHg) causes neurotoxicity through mechanisms involving elevated intracellular Ca2+ concentration ([Ca2+]i), a hallmark of excitotoxicity. We tested whether acute exposure to MeHg induces hyperexcitability in MNs by altering synaptic transmission, using whole cell patch-clamp recordings of lumbar spinal MNs in vitro. Acute MeHg exposure (20 µM) led to an increase in the frequency of both spontaneous excitatory postsynaptic currents (EPSCs) and miniature EPSCs. The frequency of inhibitory postsynaptic currents (IPSCs) was also increased by MeHg. Action potential firing rates, both spontaneous and evoked, were increased by MeHg, despite increases in both EPSCs and IPSCs, indicating a shift toward hyperexcitability. Also consistent with hyperexcitability, fluo 4-AM microfluorimetry indicated that MeHg exposure induced an increase in [Ca2+]i. Spinal cord hyperexcitability is partially mediated by Ca2+-permeable AMPA receptors, as MeHg-dependent increases in EPSCs were blocked by 1-napthyl spermine. Therefore, spinal MNs appear highly susceptible to MeHg exposure, leading to significant increases in spontaneous network excitability and disruption of normal function. Prolonged hyperexcitability could lead to eventual neurodegeneration and loss of motor function as observed in spinal cord after MeHg exposure in vivo and may contribute to MeHg-induced acceleration of ALS symptoms.NEW & NOTEWORTHY Spinal motor neurons (MN) are susceptible to glutamatergic excitotoxicity, an effect associated with lumbar MN degeneration in amyotrophic lateral sclerosis (ALS). This study investigated MN susceptibility to environmental toxicant exposure, one prospective contributor to sporadic ALS. Spinal MNs appear highly susceptible to methylmercury exposure, leading to significant increases in spontaneous network excitability and disruption of normal function. Prolonged hyperexcitability could lead to neurodegeneration and loss of motor function as observed in ALS spinal cord symptoms.
Asunto(s)
Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/metabolismo , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Neuronas Motoras/efectos de los fármacos , Red Nerviosa/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Esclerosis Amiotrófica Lateral/inducido químicamente , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Exposición a Riesgos Ambientales , RatonesRESUMEN
UNLABELLED: Spinal and bulbar muscular atrophy (SBMA) in men is an androgen-dependent neuromuscular disease caused by expanded CAG repeats in the androgen receptor (AR). Whether muscle or motor neuron dysfunction or both underlies motor impairment in SBMA is unknown. Muscles of SBMA mice show significant contractile dysfunction, implicating them as a likely source of motor dysfunction, but whether disease also impairs neuromuscular transmission is an open question. Thus, we examined synaptic function in three well-studied SBMA mouse models-the AR97Q, knock-in (KI), and myogenic141 models-by recording in vitro miniature and evoked end-plate potentials (MEPPs and EPPs, respectively) intracellularly from adult muscle fibers. We found striking defects in neuromuscular transmission suggesting that toxic AR in SBMA impairs both presynaptic and postsynaptic mechanisms. Notably, SBMA causes neuromuscular synapses to become weak and muscles to become hyperexcitable in all three models. Presynaptic defects included deficits in quantal content, reduced size of the readily releasable pool, and impaired short-term facilitation. Postsynaptic defects included prolonged decay times for both MEPPs and EPPs, marked resistance to µ-conotoxin (a sodium channel blocker), and enhanced membrane excitability. Quantitative PCR revealed robust upregulation of mRNAs encoding neonatal isoforms of the AChR (γ-subunit) and the voltage-gated sodium channel (NaV1.5) in diseased adult muscles of all three models, consistent with the observed slowing of synaptic potentials and resistance to µ-conotoxin. These findings suggest that muscles of SBMA patients regress to an immature state that impairs neuromuscular function. SIGNIFICANCE STATEMENT: We have discovered that SBMA is accompanied by marked defects in neuromuscular synaptic transmission involving both presynaptic and postsynaptic mechanisms. For three different mouse models, we find that diseased synapses are weak, having reduced quantal content due to reductions in the size of the readily releasable pool and/or probability of release. Synaptic potentials in diseased adult fibers are slowed, explained by an aberrant upregulation of the neonatal isoform of the acetylcholine receptor. Diseased fibers also show marked resistance to µ-conotoxin, explained by an aberrant upregulation in the neonatal isoform of the sodium channel, and are hyperexcitable, reminiscent of myotonic dystrophy, showing anode-break action potentials. This work identifies several new molecular targets for recovering function in SBMA.
Asunto(s)
Trastornos del Movimiento/fisiopatología , Trastornos Musculares Atróficos/fisiopatología , Unión Neuromuscular , Transmisión Sináptica , Animales , Conotoxinas/farmacología , Potenciales Evocados Motores , Expresión Génica/genética , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Transgénicos , Placa Motora/efectos de los fármacos , Trastornos del Movimiento/etiología , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Trastornos Musculares Atróficos/complicaciones , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
Methylmercury (MeHg) disrupts cerebellar function, especially during development. Cerebellar granule cells (CGC), which are particularly susceptible to MeHg by unknown mechanisms, migrate during this process. Transient changes in intracellular Ca(2+) (Ca(2+) i) are crucial to proper migration, and MeHg is well known to disrupt CGC Ca(2+) i regulation. Acutely prepared slices of neonatal rat cerebellum in conjunction with confocal microscopy and fluo4 epifluorescence were used to track changes induced by MeHg in CGC Ca(2+) i regulation in the external (EGL) and internal granule cell layers (IGL) as well as the molecular layer (ML). MeHg caused no cytotoxicity but did cause a time-dependent increase in fluo4 fluorescence that depended on the stage of CGC development. CGCs in the EGL were most susceptible to MeHg-induced increases in fluo4 fluorescence. MeHg increased fluorescence in CGC processes but only diffusely; Purkinje cells rarely fluoresced in these slices. Neither muscimol nor bicuculline alone altered baseline fluo4 fluorescence in any CGC layer, but each delayed the onset and reduced the magnitude of effect of MeHg on fluo4 fluorescence in the EGL and ML. In the IGL, both muscimol and bicuculline delayed the onset of MeHg-induced increases in fluo4 fluorescence but did not affect fluorescence magnitude. Thus, acute exposure to MeHg causes developmental stage-dependent increases in Ca(2+) i in CGCs. Effects are most prominent in CGCs during development or early stages of migration. GABAA receptors participate in an as yet unclear manner to MeHg-induced Ca(2+) i dysregulation of CGCs.
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Movimiento Celular/efectos de los fármacos , Cerebelo/citología , Cerebelo/metabolismo , Compuestos de Metilmercurio/farmacología , Receptores de GABA-A/efectos de los fármacos , Compuestos de Anilina , Animales , Animales Recién Nacidos , Bicuculina/farmacología , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cerebelo/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Femenino , Colorantes Fluorescentes , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Técnicas In Vitro , Masculino , Muscimol/farmacología , Embarazo , Células de Purkinje/efectos de los fármacos , Ratas , XantenosRESUMEN
ß-Subunits of voltage-gated calcium channels (VGCCs) regulate assembly and membrane localization of the pore-forming α1-subunit and strongly influence channel function. ß4-Subunits normally coassociate with α1A-subunits which comprise P/Q-type (Cav2.1) VGCCs. These control acetylcholine (ACh) release at adult mammalian neuromuscular junctions (NMJs). The naturally occurring lethargic (lh) mutation of the ß4-subunit in mice causes loss of the α1-binding site, possibly affecting P/Q-type channel expression or function, and thereby ACh release. End-plate potentials and miniature end-plate potentials were recorded at hemidiaphragm NMJs of 5-7-week and 3-5-month-old lh and wild-type (wt) mice. Sensitivity to antagonists of P/Q- [ω-agatoxin IVA (ω-Aga-IVA)], L- (nimodipine), N- (ω-conotoxin GVIA), and R-type [C192H274N52O60S7 (SNX-482)] VGCCs was compared in juvenile and adult lh and wt mice. Quantal content (m) of adult, but not juvenile, lh mice was reduced compared to wt. ω-Aga-IVA (~60%) and SNX-482 (~ 45%) significantly reduced m in adult lh mice. Only Aga-IVA affected wt adults. In juvenile lh mice, ω-Aga-IVA and SNX-482 decreased m by >75% and ~20%, respectively. Neither ω-conotoxin GVIA nor nimodipine affected ACh release in any group. Immunolabeling revealed α1E and α1A, ß1, and ß3 staining at adult lh, but not wt NMJs. Therefore, in lh mice, when the ß-subunit that normally coassociates with α1A to form P/Q channels is missing, P/Q-type channels partner with other ß-subunits. However, overall participation of P/Q-type channels is reduced and compensated for by R-type channels. R-type VGCC participation is age-dependent, but is less effective than P/Q-type at sustaining NMJ function.
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Envejecimiento/metabolismo , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Canales de Calcio Tipo R/metabolismo , Canales de Calcio/genética , Unión Neuromuscular/metabolismo , Acetilcolina/metabolismo , Potenciales de Acción/efectos de los fármacos , Envejecimiento/genética , Animales , Bloqueadores de los Canales de Calcio/farmacología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Ratones Mutantes , Placa Motora/efectos de los fármacos , Placa Motora/metabolismo , Mutación , Unión Neuromuscular/efectos de los fármacosRESUMEN
INTRODUCTION: Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune presynaptic neuromuscular disorder. Autoantibodies against subunits of voltage-gated calcium channels (VGCCs) associated with acetylcholine release are thought to cause LEMS. METHODS: HEK293 cells expressing specific individual recombinant subunits of α(1A), α(1B), α(1C), and α(1E); ß(3); and α(2)δ of human neuronal VGCCs were exposed to antibodies from 3 LEMS patients, 1 patient with small-cell lung carcinoma, and 1 with myasthenia gravis. RESULTS: All LEMS patient antibodies bound to cells containing any of the α(1) or ß(3) subunits alone or combined with α(2)δ subunits, but not α(2)δ alone. Autoantibodies from the patient with small-cell lung carcinoma but not the myasthenia gravis patient targeted the same VGCC subunits. CONCLUSIONS: Autoantibodies from LEMS patients bind directly to multiple VGCC α(1) subunits as well as the ß(3) subunit. Thus, multiple components of the presynaptic VGCC complex are prospective targets for antibodies in LEMS.
Asunto(s)
Autoanticuerpos/inmunología , Canales de Calcio/inmunología , Canales de Calcio/metabolismo , Síndrome Miasténico de Lambert-Eaton/inmunología , Subunidades de Proteína/metabolismo , Canales de Calcio/genética , Carcinoma de Células Pequeñas/sangre , Carcinoma de Células Pequeñas/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Síndrome Miasténico de Lambert-Eaton/sangre , Subunidades de Proteína/genética , TransfecciónRESUMEN
The exact causes of Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurological disorder due to loss of upper and/or lower motoneurons, remain elusive. Gene-environment interactions are believed to be an important factor in the development of ALS. We previously showed that in vivo exposure of mice overexpressing the human superoxide dismutase 1 (hSOD1) gene mutation (hSOD1G93A; G93A), a mouse model for ALS, to environmental neurotoxicant methylmercury (MeHg) accelerated the onset of ALS-like phenotype. Here we examined the time-course of effects of MeHg on AMPA receptor (AMPAR)-mediated currents in hypoglossal motoneurons in brainstem slices prepared from G93A, hSOD1wild-type (hWT) and non-carrier WT mice following in vivo exposure to MeHg. Mice were exposed daily to 3 ppm (approximately 0.7 mg/kg/day) MeHg via drinking water beginning at postnatal day 28 (P28) and continued until P47, 64 or 84, then acute brainstem slices were prepared, and spontaneous excitatory postsynaptic currents (sEPSCs) or AMPA-evoked currents were examined using whole cell patch-clamp recording technique. Brainstem slices of untreated littermates were prepared at the same time points to serve as control. MeHg exposure had no significant effect on either sEPSCs or AMPA-evoked currents in slices from hWT or WT mice during any of those exposure time periods under our experimental conditions. MeHg also did not cause any significant effect on sEPSCs or AMPA-currents in G93A hypoglossal motoneurons at P47 and P64. However, at P84, MeHg significantly increased amplitudes of both sEPSCs and AMPA-evoked currents in hypoglossal motineurons from G93A mice (p < 0.05), but not the sEPSC frequency, suggesting a postsynaptic action on AMPARs. MeHg exposure did not cause any significant effect on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs). Therefore, MeHg exposure in vivo caused differential effects on AMPARs in hypoglossal motoneurons from mice with different genetic backgrounds. MeHg appears to preferentially stimulate the AMPAR-mediated currents in G93A hypoglossal motoneurons in an exposure time-dependent manner, which may contribute to the AMPAR-mediated motoneuron excitotoxicity, thereby facilitating development of ALS-like phenotype.
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Esclerosis Amiotrófica Lateral , Compuestos de Metilmercurio , Ratones , Humanos , Animales , Superóxido Dismutasa-1 , Esclerosis Amiotrófica Lateral/inducido químicamente , Esclerosis Amiotrófica Lateral/genética , Compuestos de Metilmercurio/toxicidad , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Superóxido Dismutasa/metabolismo , Ratones Transgénicos , Neuronas Motoras/metabolismo , Tronco Encefálico/metabolismo , Mutación , Modelos Animales de Enfermedad , Médula Espinal/metabolismoRESUMEN
Neuropathic pain afflicts a large percentage of the global population. This form of chronic, intractable pain arises when the peripheral or central nervous systems are damaged, either directly by lesion or indirectly through disease. The comorbidity of neuropathic pain with other diseases, including diabetes, cancer, and AIDS, contributes to a complex pathogenesis and symptom profile. Because most patients present with neuropathic pain refractory to current first-line therapeutics, pharmaceuticals with greater efficacy in pain management are highly desired. In this review we discuss the growing application of ω-conotoxins, small peptides isolated from Conus species, in the management of neuropathic pain. These toxins are synthesized by predatory cone snails as a component of paralytic venoms. The potency and selectivity with which ω-conotoxins inhibit their molecular targets, voltage-gated Ca2+ channels, is advantageous in the treatment of neuropathic pain states, in which Ca2+ channel activity is characteristically aberrant. Although ω-conotoxins demonstrate analgesic efficacy in animal models of neuropathic pain and in human clinical trials, there remains a critical need to improve the convenience of peptide drug delivery methods, and reduce the number and severity of adverse effects associated with ω-conotoxin-based therapies.
Asunto(s)
Caracol Conus/metabolismo , Neuralgia/tratamiento farmacológico , omega-Conotoxinas/farmacología , Analgésicos/efectos adversos , Analgésicos/aislamiento & purificación , Analgésicos/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Humanos , Terapia Molecular Dirigida , Neuralgia/fisiopatología , omega-Conotoxinas/efectos adversos , omega-Conotoxinas/aislamiento & purificaciónRESUMEN
Mice expressing the human Cu(2+)/Zn(2+) superoxide dismutase 1 (hSOD1) gene mutation (hSOD1(G93A); G93A) were exposed to methylmercury (MeHg) at concentrations that did not cause overt motor dysfunction. We hypothesized that low concentrations of MeHg could hasten development of the amyotrophic lateral sclerosis (ALS)-like phenotype in G93A mice. MeHg (1 or 3 ppm/day in drinking water) concentration-dependently accelerated the onset of rotarod failure in G93A, but not wild-type, mice. At the time of rotarod failure, MeHg increased Fluo-4 fluorescence (free intracellular calcium concentration [Ca(2+)](i)) in soma of brainstem-hypoglossal nucleus. These motor neurons control intrinsic and some extrinsic tongue function and exhibit vulnerability in bulbar-onset ALS. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione reduced [Ca(2+)](i) in all G93A mice, irrespective of MeHg treatment. N-acetyl spermine, which antagonizes Ca(2+)-permeable AMPA receptors, further reduced [Ca(2+)](i) more effectively in MeHg-treated than untreated G93A mice, suggesting that MeHg-treated mice have a greater Ca(2+)-permeable AMPA receptor contribution. The non-Ca(2+) divalent cation chelator N,N,N',N'-tetrakis(pyridylmethyl)ethylenediamine reduced Fluo-4 fluorescence in all G93A mice; FluoZin-(Zn(2+) indicator) fluorescence was increased in all MeHg-treated mice. Thus in G93A mice Zn(2+) apparently contributed measurably to the MeHg-induced effect. This is the initial demonstration of accelerated onset of ALS-like phenotype in a genetically susceptible organism by exposure to low concentrations of an environmental neurotoxicant. Increased [Ca(2+)](i) induced by the G93A-MeHg interaction apparently was associated with Ca(2+)-permeable AMPA receptors and may contribute to the hastened development of ALS-like phenotypes by subjecting motor neurons to excessive elevation of [Ca(2+)](i), leading to excitotoxic cell death.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Ácido Glutámico/toxicidad , Compuestos de Metilmercurio/toxicidad , Fenotipo , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/inducido químicamente , Animales , Agonistas de Aminoácidos Excitadores/toxicidad , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Superóxido Dismutasa/biosíntesisRESUMEN
α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca2+ [Ca2+]i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca2+]i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 µM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca2+]i, and cytotoxicity, respectively. Contribution of Ca2+-permeable and impermeable AMPARs was compared using either CNQX, or the Ca2+-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca2+]i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg's effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca2+]i, and cytotoxicity, and that both Ca2+-permeable and impermeable AMPARs contribute the elevations in [Ca2+]i.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Neuronas Motoras/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Receptores AMPA/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Receptores AMPA/antagonistas & inhibidoresRESUMEN
Neuroscience as a discipline is rarely covered in educational institutions in Puerto Rico. In an effort to overcome this deficit we developed the Bridge to Neuroscience Workshop (BNW), a full-day hands-on workshop in neuroscience education. BNW was conceived as an auxiliary component of a parent recruitment program called Bridge to the PhD in Neuroscience Program (BPNP). The objectives of BNW are to identify promising students for BPNP, and to increase awareness of neuroscience as a discipline and a career option. BNW introduces basic concepts in neuroscience using a variety of educational techniques, including mini-lectures, interactive discussions, case studies, experimentation, and a sheep brain dissection. Since its inception in 2011 BNW has undergone a series of transformations that continue to improve upon an already successful and influential educational program for underrepresented minorities. As of Fall 2018, we have presented 21 workshops, impacting 200 high school and 424 undergraduate students. BNW has been offered at University of Puerto Rico (UPR)-Arecibo, UPR-Cayey, UPR-Humacao, Pontificia Universidad Católica de Ponce, and Universidad Interamericana de Puerto Rico-Arecibo. A pre-and post evaluation was given to evaluate material comprehension and thus measure effectiveness of our one-day interactive workshop. Our results suggest that both high school and undergraduate students have little prior knowledge of neuroscience, and that participation in BNW improves not only understanding, but also enthusiasm for the discipline. Currently, our assessment has only been able to evaluate short-term effects (e.g. comprehension and learning). Therefore, our current focus is developing methods capable of determining how participation in BNW impacts future academic and career decisions.
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Curriculum , Neurociencias/educación , Instituciones Académicas , Universidades , Hispánicos o Latinos , Humanos , Puerto Rico , EstudiantesRESUMEN
Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABA(A) receptor function. This is manifested as a more rapid block of inhibitory postsynaptic currents/inhibitory postsynaptic potentials than for Purkinje cells. The underlying mechanism(s) for differential sensitivity of GABAergic transmission to MeHg in cerebellar neurons is unknown. Differential expression of alpha(6) subunit-containing GABA(A) receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (I(GABA)) were recorded in response to MeHg in alpha(6) subunit-containing cerebellar granule cells and alpha(6) subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express alpha(6) subunits. They express the same alpha(1)-containing GABA(A) receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. I(GABA) were obtained using whole-cell recording and symmetrical [Cl(-)]. MeHg reduced I(GABA) to complete block in both cell types in a time- and concentration-dependent manner. This effect was faster in granule cells than cortical cells. Effects of MeHg on I(GABA) were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of alpha(6) subunit-containing GABA(A) receptors. Effects of MeHg on I(GABA) were similar in cells at all days. In human embryonic kidney 293 cells expressing either alpha(6) or alpha(1) subunit-containing GABA(A) receptors, time to block of I(GABA) by MeHg was comparable. Thus, the presence of the alpha(6) subunit alone may not underlie the differential effects of MeHg on I(GABA) observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.
Asunto(s)
Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Compuestos de Metilmercurio/farmacología , Neuronas/metabolismo , Receptores de GABA-A/biosíntesis , Animales , Línea Celular , Células Cultivadas , Cerebelo/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Femenino , Antagonistas de Receptores de GABA-A , Humanos , Masculino , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genéticaRESUMEN
Migration of cerebellar granule cells (CGCs) from the external germinal cell layer (EGL) to the internal granule cell layer (IGL) within the cerebellar cortex is a crucial developmental process. Antagonists to NMDA receptors impair CGC migration significantly, but studies to determine which subunit subtypes control or affect migration have been controversial. Migrating CGCs transiently express NMDA receptor subunit subtypes NR1a plus NR2B. Grafted NR1-/- subunit knockout cells continue to migrate, indicating that the NR1 subunit is not necessary for migration. In the present study, the functional importance of the NR2B subtype in developing cerebellum was investigated using organotypic slice cultures prepared from postnatal day 8 (P8) rats. Slice cultures were labeled with bromodeoxyuridine (BrdU) during the first 20h and then continuously treated with the NR2B-subtype-specific NMDA antagonist, ifenprodil, or the non-specific NMDA antagonist, APV, for 7 days. Cultures were incubated with fluorescently tagged anti-BrdU IgG and the percent of BrdU-labeled CGCs that migrated from the EGL to the IGL during treatment was analyzed using laser confocal microscopy. Migration into the IGL was significantly impaired by treatment with 0.5 and 1.0 microM ifenprodil. Fewer cells had migrated to the IGL in 1.0 microM ifenprodil than in 0.5 microM ifenprodil; there was no significant difference between the percent impairment caused by 1.0 microM ifenprodil and 50 microM APV. Untreated controls had few, if any, CGCs in the EGL at DIV 8. The percent of CGCs remaining in the EGL following treatment with antagonists significantly increased, indicating impairment of migration. In conclusion, the NR2B subunit appears to be necessary for CGC migration.
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Movimiento Celular/fisiología , Corteza Cerebelosa/crecimiento & desarrollo , Corteza Cerebelosa/metabolismo , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Animales Recién Nacidos , Bromodesoxiuridina , Recuento de Células , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Corteza Cerebelosa/citología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Masculino , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
PURPOSE OF REVIEW: Gene-environment (GxE) interactions likely contribute to numerous diseases, but are often difficult to model in the laboratory. Such interactions have been widely hypothesized for amyotrophic lateral sclerosis (ALS); recent controlled laboratory studies are discussed here and hypotheses related to possible mechanisms of action are offered. Using methylmercury exposure and mutated SOD1 to model the impacts of such an interaction, we interpret evidence about their respective mechanisms of toxicity to interrogate the possibility of additive (or synergistic) effects when combined. RECENT FINDINGS: Recent work has converged on mechanisms of calcium-mediated glutamate excitotoxicity as a likely contributor in one model of a gene-environment interaction affecting the onset and progression of ALS-like phenotype. The current experimental literature on mechanisms of metal-induced neuronal injury and their relevant interactions with genetic contributions in ALS is sparse, but we describe those studies here and offer several integrative hypotheses about the likely mechanisms involved.
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Esclerosis Amiotrófica Lateral/fisiopatología , Interacción Gen-Ambiente , Compuestos de Metilmercurio/toxicidad , Superóxido Dismutasa/toxicidad , Esclerosis Amiotrófica Lateral/inducido químicamente , Calcio , Depuradores de Radicales Libres , Ácido Glutámico , Humanos , Receptores AMPARESUMEN
Methylmercury (MeHg) is an environmental neurotoxicant of public health concern. It readily accumulates in exposed humans, primarily in neuronal tissue. Exposure to MeHg, either acutely or chronically, causes severe neuronal dysfunction in the central nervous system and spinal neurons; dysfunction of susceptible neuronal populations results in neurodegeneration, at least in part through Ca2+-mediated pathways. Biochemical and morphologic changes in peripheral neurons precede those in central brain regions, despite the fact that MeHg readily crosses the blood-brain barrier. Consequently, it is suggested that unique characteristics of spinal cord afferents and efferents could heighten their susceptibility to MeHg toxicity. Transient receptor potential (TRP) ion channels are a class of Ca2+-permeable cation channels that are highly expressed in spinal afferents, among other sensory and visceral organs. These channels can be activated in numerous ways, including directly via chemical irritants or indirectly via Ca2+ release from intracellular storage organelles. Early studies demonstrated that MeHg interacts with heterologous TRP channels, though definitive mechanisms of MeHg toxicity on sensory neurons may involve more complex interaction with, and among, differentially-expressed TRP populations. In spinal efferents, glutamate receptors of the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and possibly kainic acid (KA) classes are thought to play a major role in MeHg-induced neurotoxicity. Specifically, the Ca2+-permeable AMPA receptors, which are abundant in motor neurons, have been identified as being involved in MeHg-induced neurotoxicity. In this review, we will describe the mechanisms that could contribute to MeHg-induced spinal cord afferent and efferent neuronal degeneration, including the possible mediators, such as uniquely expressed Ca2+-permeable ion channels.
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Compuestos de Metilmercurio/toxicidad , Células Receptoras Sensoriales/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Animales , Vías Eferentes/efectos de los fármacos , Vías Eferentes/metabolismo , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células Receptoras Sensoriales/metabolismo , Médula Espinal/metabolismo , Aferentes Viscerales/efectos de los fármacos , Aferentes Viscerales/metabolismoRESUMEN
Early onset effects of methylmercury (MeHg) on recombinant α1ß2γ2S or α6ß2γ2S subunit-containing GABAA receptors were examined. These are two of the most prevalent receptor types found in cerebellum-a consistent target of MeHg-induced neurotoxicity. Heterologously expressed receptors were used in order to: (1) isolate receptor-mediated events from extraneous effects of MeHg due to stimulation of the receptor secondary to increased release of GABA seen with MeHg in neurons in situ and (2) limit the phenotypes of GABAA receptors present at one time. Initial changes in IGABA in Xenopus laevis oocytes expressing either α1ß2γ2S or α6ß2γ2S receptors were compared during continuous bath application of MeHg. A time-dependent increase in IGABA mediated by both receptor subtypes occurred following the first 25-30min of MeHg (5µM) exposure. In α6ß2γ2S containing receptors, the MeHg-induced increase in IGABA was less pronounced compared to that mediated by α1ß2γ2S containing receptors, although the pattern of effects was generally similar. Washing with MeHg-free solution reversed the increase in current amplitude. Application of bicuculline at the time of peak potentiation of IGABA rapidly and completely reversed the MeHg-induced currents. Therefore these MeHg-increased inward currents are mediated specifically by the two subtypes of GABAA receptors and appear to entail direct actions of MeHg on the receptor. However bicuculline did not affect stimulation by MeHg of oocyte endogenous Cl- -mediated current, which presumably results from increased [Ca2+]i. Thus, MeHg initially potentiates IGABA in oocytes expressing either α1ß2γ2S or α6ß2γ2S receptors prior to its more defined later effects, suggesting that MeHg may initially interact directly with GABAA receptors in a reversible manner to cause this potentiation.
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Compuestos de Metilmercurio/farmacología , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Bicuculina/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Ácido Niflúmico , Oocitos/efectos de los fármacos , Oocitos/fisiología , Subunidades de Proteína/fisiología , Xenopus laevis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Methylmercury (MeHg) is an important environmental neurotoxicant that is present in seafood and affects the developing and mature nervous system. The neurotoxicity induced by MeHg is a concern, particularly for fish-eating populations and pregnant or nursing women. During MeHg-induced neurotoxicity, degeneration of the granule cell layer in the cerebellum occurs, which leads to deficits in motor function. I suggest that the action of MeHg on specific neurotransmitter receptors contributes to the selective vulnerability of granule cells. MeHg appears to stimulate M(3) muscarinic acetylcholine receptors and to inhibit GABA(A) receptor subtypes preferentially on cerebellar granule cells. This could lead to the loss of tonic inhibition of granule cells as a result of antagonism of GABA(A) receptors, and a M(3)-receptor-mediated increase in the intracellular concentration of Ca(2+) and block of a K(+)-dependent leak current. The net result would be increased spontaneous release of glutamate, which, coupled with a MeHg-induced impairment of glutamate uptake by astrocytes, could cause Ca(2+)-mediated cytotoxicity.
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Cerebelo/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Antagonistas de Receptores de GABA-A , Compuestos de Metilmercurio/toxicidad , Neurotransmisores/toxicidad , Receptor Muscarínico M3/agonistas , Transmisión Sináptica/efectos de los fármacos , Acetilcolina/antagonistas & inhibidores , Acetilcolina/metabolismo , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Cerebelo/metabolismo , Cerebelo/patología , Ácido Glutámico/metabolismo , Humanos , Células de Purkinje/efectos de los fármacos , Receptor Muscarínico M3/metabolismo , Receptores de GABA-A/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We previously showed that elevated intracellular Ca(2+) ([Ca(2+)]i) in the molecular layer and granule cells in cerebellar slices is responsible for the initial increases in frequency of spontaneous or miniature inhibitory postsynaptic currents (sIPSCs or mIPSCs) of Purkinje cells following methylmercury (MeHg) treatment. To identify the contribution of different Ca(2+) sources to MeHg-induced stimulation of spontaneous GABA release, we examined sIPSC or mIPSC frequency of Purkinje cells in acutely prepared cerebellar slices using whole-cell patch-clamp recording techniques under conditions of lowered [Ca(2+)]o, pretreatment with caffeine, cyclopiazonic acid (CPA), thapsigargin or ruthenium red (RR) to deplete ryanodine-sensitive and insensitive intracellular Ca(2+) stores or mitochondria, or a combination of lowering [Ca(2+)]o and increased BAPTA buffering. Lowering [Ca(2+)]o significantly reduced sIPSC or mIPSC frequency and amplitudes, but failed to completely prevent MeHg-induced increase in these events frequency. Caffeine, CPA, or thapisgargin also minimized MeHg-induced increase in sIPSC frequency, yet none of them completely blocked MeHg-induced increase in sIPSC frequency. Similarly, the mitochondrial Ca(2+) transport inhibitor RR, or a combination of lowering [Ca(2+)]o and BAPTA buffering reduced but did not prevent MeHg-induced changes in mIPSC frequency. Consistently, confocal Ca(2+) imaging under low [Ca(2+)]o conditions or in the presence of caffeine or CPA exhibited a marked reduction of MeHg-induced increases in [Ca(2+)]i in both molecular and granule layers. Thus, these results verify that a combination of extracellular Ca(2+) influx and Ca(2+) release from different intracellular Ca(2+) pools all contribute to MeHg-induced increase in [Ca(2+)]i and spontaneous GABA release, although extracellular Ca(2+) appears to be the primary contributor.
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Calcio/metabolismo , Cerebelo/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Animales , Cafeína/farmacología , Cerebelo/metabolismo , Femenino , Técnicas In Vitro , Indoles/farmacología , Masculino , Microscopía Confocal , Microscopía Fluorescente , Técnicas de Placa-Clamp , Células de Purkinje/efectos de los fármacos , Células de Purkinje/metabolismo , Ratas Sprague-Dawley , Tapsigargina/farmacologíaRESUMEN
Mechanical forces can alter the gating of several kinds of ion channels in many types of cells, but the mechanisms underlying the mechanosensitivity are not clearly understood. To date, there are very few reports on mechanosensitivity of Ca2+ channels, particularly neuronal Ca2+ channels. We examined the mechanical sensitivity of human recombinant L-type Ca2+ channels in response to fluid flow. Neuronal L-type Ca2+ channels (Ca(v) 1.2) were expressed transiently in HEK293 cells using expression cDNA clones of human alpha1C, alpha2delta, and beta subunits along with green fluorescent protein (GFP) as a reporter protein. Current (I(Ba)) through these heterologously-expressed channels was measured using whole cell recording technique with 20 mM Ba2+ as charge carrier. Transfected cells were exposed to a constant, increased fluid flow from a separate pipette during current recording. The L-type I(Ba) was found to be very sensitive to the flow-induced shear forces. Peak current amplitude increased by as much as approximately 50% during fluid flow as compared to that in the absence of fluid pressure. However, no change was observed in the amplitude of the average current during the final 5 ms of the 150-ms voltage step. Current amplitude promptly returned to normal control levels upon stopping fluid flow. The current-voltage relationship was not altered by fluid flow. The flow-induced increase in current amplitude exhibited an apparent shift in steady-state inactivation toward more negative potentials; inactivation was faster but was not voltage dependent. Activation was slightly faster under flow. Thus, increased mechanical tension associated with fluid flow can alter the fundamental properties of voltage-gated Ca2+ channels, even for channels which might not normally be exposed to fluid flow shear forces in their native environment.
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Bario/metabolismo , Canales de Calcio Tipo L/genética , Membrana Celular/fisiología , Activación del Canal Iónico/genética , Mecanotransducción Celular/fisiología , Bario/farmacocinética , Canales de Calcio Tipo L/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Células Cultivadas , Líquido Extracelular/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The concentration- and time-dependence of effects of methylmercury (MeHg) on voltage-gated outward K(+) (Kv) channels, inwardly rectifying K(+) (Kir) channels, voltage-gated Ca(2+) channels and GABA(A) receptor activated channels were compared in cerebellar granule cells in culture using whole cell patch clamp recording techniques. The objective was to determine if MeHg equally affects different types of ion channels. Under similar experimental conditions, these four ion channel types displayed markedly different sensitivity to MeHg. At 0.1-1 microM, MeHg caused apparent inhibition of Ca(2+)-channel and GABA(A) receptor-mediated currents, but did not cause any significant effect on Kv or Kir channels. Among the four channel types examined, GABA(A) receptors appeared to be the most sensitive to MeHg. The Kv channels, particularly the delayed rectifiers (DRs), appeared to be relatively resistant to MeHg compared with GABA(A) receptors and Ca(2+) channels. Kir channels were virtually unaffected by MeHg in the concentration range of 10-100 microM. The differential sensitivity of GABA(A) receptors and Kv channels to MeHg was also observed in granule and Purkinje cells in freshly isolated cerebellar slices of rat. The insensitivity of Kir channel to MeHg was also seen in Xenopus laevis oocytes expressing cloned Kir7.1 channels. Thus, these appear to be general properties of these channels as opposed to distinct effects associated with granule cells in culture. These results suggest that MeHg does preferentially affect certain types of ion channels. Hence, the effects of MeHg on membrane ion channels are not due simply to nonspecific actions on the membrane. Furthermore, at least certain types of Kir channels appear to be the most resistant type of ion channel reported to date to effects of MeHg.
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Compuestos de Metilmercurio/farmacología , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Animales , Animales Recién Nacidos , Canales de Calcio/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Oocitos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/efectos de los fármacos , Xenopus laevisRESUMEN
The environmental neurotoxicant methylmercury (MeHg) disrupts dopamine (DA) neurochemical homeostasis by stimulating DA synthesis and release. Evidence also suggests that DA metabolism is independently impaired. The present investigation was designed to characterize the DA metabolomic profile induced by MeHg, and examine potential mechanisms by which MeHg inhibits the DA metabolic enzyme aldehyde dehydrogenase (ALDH) in rat undifferentiated PC12 cells. MeHg decreases the intracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC). This is associated with a concomitant increase in intracellular concentrations of the intermediate metabolite 3,4-dihydroxyphenylaldehyde (DOPAL) and the reduced metabolic product 3,4-dihydroxyethanol. This metabolomic profile is consistent with inhibition of ALDH, which catalyzes oxidation of DOPAL to DOPAC. MeHg does not directly impair ALDH enzymatic activity, however MeHg depletes cytosolic levels of the ALDH cofactor NAD(+), which could contribute to impaired ALDH activity following exposure to MeHg. The observation that MeHg shunts DA metabolism along an alternative metabolic pathway and leads to the accumulation of DOPAL, a reactive species associated with protein and DNA damage, as well as cell death, is of significant consequence. As a specific metabolite of DA, the observed accumulation of DOPAL provides evidence for a specific mechanism by which DA neurons may be selectively vulnerable to MeHg.