Preferential potentiation of AMPA-mediated currents in brainstem hypoglossal motoneurons by subchronic exposure of mice expressing the human superoxide dismutase 1 G93A gene mutation to neurotoxicant methylmercury in vivo.

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.

AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca2+ concentration in human induced pluripotent stem cell motor neurons.

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.

Acute neurotoxicant exposure induces hyperexcitability in mouse lumbar spinal motor neurons.

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.

Bridge to neuroscience workshop: An effective educational tool to introduce principles of neuroscience to Hispanics students.

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.

Evaluating a Gene-Environment Interaction in Amyotrophic Lateral Sclerosis: Methylmercury Exposure and Mutated SOD1.

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.

Effects of methylmercury on spinal cord afferents and efferents-A review.

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.

Methylmercury induces an initial increase in GABA-evoked currents in Xenopus oocytes expressing α1 and α6 subunit-containing GABAA receptors.

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.

Defects in Neuromuscular Transmission May Underlie Motor Dysfunction in Spinal and Bulbar Muscular Atrophy.

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.

Multiple Sources of Ca2+ Contribute to Methylmercury-Induced Increased Frequency of Spontaneous Inhibitory Synaptic Responses in Cerebellar Slices of Rat.

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.

Methylmercury-Dependent Increases in Fluo4 Fluorescence in Neonatal Rat Cerebellar Slices Depend on Granule Cell Migrational Stage and GABAA Receptor Modulation.

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.

Methylmercury impairs canonical dopamine metabolism in rat undifferentiated pheochromocytoma (PC12) cells by indirect inhibition of aldehyde dehydrogenase.

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.

Age-dependent contribution of P/Q- and R-type Ca2+ channels to neuromuscular transmission in lethargic mice.

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.

Lambert-Eaton syndrome antibodies target multiple subunits of voltage-gated Ca2+ channels.

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.

Omega-conotoxins as experimental tools and therapeutics in pain management.

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.

The role of de novo catecholamine synthesis in mediating methylmercury-induced vesicular dopamine release from rat pheochromocytoma (PC12) cells.

The purpose of this study was to characterize methylmercury (MeHg)-induced dopamine (DA) release from undifferentiated pheochromocytoma (PC12) cells and to examine the potential role for DA synthesis in this process. MeHg caused a significant increase in DA release that was both concentration- and time-dependent. DA release was significantly increased by 2µM MeHg at 60min and by 5µM MeHg at 30min; 1µM MeHg was without effect. Because DA release induced by 5µM MeHg was associated with a significant percentage of cell death at 60 and 120min, 2µM MeHg was chosen for further characterization of release mechanisms. MeHg-induced DA release was attenuated but not abolished in the absence of extracellular calcium, whereas the vesicular content depleting drug reserpine (50nM) abolished release. Thus, MeHg-induced DA release requires vesicular exocytosis but not extracellular calcium. MeHg also increased intracellular DA and the rate of DA storage utilization, suggesting a role for DA synthesis in MeHg-induced DA release. The tyrosine hydroxylase inhibitor α-methyltyrosine (300µM, 24h) completely abolished MeHg-induced DA release. MeHg significantly increased DA precursor accumulation in cells treated with 3-hydroxybenzylhydrazine (10µM), revealing that MeHg increases tyrosine hydroxylase activity. Overall, these data demonstrate that MeHg facilitates DA synthesis, increases intracellular DA, and augments vesicular exocytosis.

Ca2+ entry pathways in mouse spinal motor neurons in culture following in vitro exposure to methylmercury.

Methylmercury (MeHg) is a widespread environmental toxicant with major actions on the central nervous system. Among the neurons reportedly affected in cases of Hg poisoning are motor neurons; however, the direct cellular effects of MeHg on motor neurons have not been reported. Ratiometric fluorescence imaging, using the Ca(2+)-sensitive fluorophore fura-2, was used to examine the effect of MeHg on Ca(2+) homeostasis in primary cultures of mouse spinal motor neurons. In vitro MeHg exposure at concentrations (0.1-2 µM) known to affect other neurons in culture differentially, induced a biphasic rise in fura-2 fluorescence ratio indicating an increase in [Ca(2+)](i). The time-to-onset of these fura-2 fluorescence ratio changes was inversely correlated with MeHg concentration. TPEN (20 µM), a non-Ca(2+), divalent cation chelator, reduced the amplitude of the increase in fura-2 fluorescence induced by MeHg in the first phase, indicating that both Ca(2+) and non-Ca(2+) divalent cations contribute to the MeHg-induced effect. When examining various Ca(2+) entry pathways as possible targets contributing to Ca(2+) influx, we found that excitatory amino acid receptor blockers MK-801 (15 µM), and AP-5 (100 µM)-both NMDA receptor-operated ion channel blockers, CNQX (20 µM), a non-NMDA receptor blocker, and the voltage-dependent Ca(2+) channel blockers nifedipine (1 µM) and ω-conotoxin-GVIA (1 µM) all significantly delayed the development of increased Ca(2+) caused by MeHg. The voltage-dependent Na(+) channel blocker tetrodotoxin (TTX, 1 µM) did not alter the MeHg-induced increases in fura-2 fluorescence ratio. Thus, MeHg alters Ca(2+) homeostasis in mouse spinal motor neurons through excitatory amino acid receptor-mediated pathways, and nifedipine and ω-conotoxin-GVIA-sensitive pathways. Spinal motor neurons are highly sensitive to this effect of acute exposure to MeHg.

Channelopathies: summary of the hot topic keynotes session.

The "Hot Topic Keynotes: Channelopathies" session of the 26th International Neurotoxicology Conference brought together toxicologists studying interactions of environmental toxicants with ion channels, to review the state of the science of channelopathies and to discuss the potential for interactions between environmental exposures and channelopathies. This session presented an overview of chemicals altering ion channel function and background about different channelopathy models. It then explored the available evidence that individuals with channelopathies may or may not be more sensitive to effects of chemicals.

Exposure to an environmental neurotoxicant hastens the onset of amyotrophic lateral sclerosis-like phenotype in human Cu2+/Zn2+ superoxide dismutase 1 G93A mice: glutamate-mediated excitotoxicity.

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.

Allethrin differentially modulates voltage-gated calcium channel subtypes in rat PC12 cells.

Pyrethroid insecticides are one of the most widely used classes of insecticides. Previous studies revealed that pyrethroids potently affect the insect voltage-gated sodium (Na(+)) channel (VGSC), resulting in prolonged channel open time. However, recent findings have suggested that pyrethroids may affect targets other than the VGSC. In particular, several studies have shown that pyrethroids can modulate the activity of voltage-gated calcium (Ca(2+)) channels (VGCCs). However, these studies often reported conflicting results; some studies observed stimulatory effects, whereas others observed inhibitory effects of pyrethroids on VGCCs. This study investigated whether allethrin (AL), a well-characterized type I pyrethroid, altered VGCC characteristics measured by whole-cell recording in rat pheochromocytoma cells (PC12) differentiated with nerve growth factor (NGF). AL (5 microM) increased peak, end, and tail composite VGCC current independent of its effects on VGSCs. After blocking VGCC subtype-specific current with omega-conotoxin GVIA (GVIA, an N-type VGCC antagonist) or nimodipine (NIM, an L-type VGCC antagonist), our data further suggest that AL differentially affects VGCC subtypes. Thus, AL apparently stimulated GVIA-insensitive current while inhibiting NIM-insensitive current. AL also significantly altered the voltage dependency of activation and inactivation of L-type VGCCs. The differential modulation of VGCC subtypes by AL may explain some of the conflicting observations of other studies.

The role of environmental mercury, lead and pesticide exposure in development of amyotrophic lateral sclerosis.

Exposure to an environmental toxicant as a risk factor in the development of amyotrophic lateral sclerosis (ALS) was first hinted at (demonstrated) in the Chamorro indigenous people of Guam. During the 1950s and 1960s these indigenous people presented an extremely high incidence of ALS which was presumed to be associated with the consumption of flying fox and cycad seeds. No other strong association between ALS and environmental toxicants has since been reported, although circumstantial epidemiological evidence has implicated exposure to heavy metals such as lead and mercury, industrial solvents and pesticides especially organophosphates and certain occupations such as playing professional soccer. Given that only approximately 10% of all ALS diagnosis have a genetic basis, a gene-environmental interaction provides a plausible explanation for the other 90% of cases. This mini-review provides an overview of our current knowledge of environmental etiologies of ALS with emphasis on the effects of mercury, lead and pesticides as potential risk factors in developing ALS. Epidemiologic and experimental evidence from animal models investigating the possible association between exposure to environmental toxicant and ALS disease has proven inconclusive. Nonetheless, there are indications that there may be causal links, and a need for more research.