Yukun Yuan

Methylmercury-Induced Increase of Intracellular Ca2+ Increases Spontaneous Synaptic Current Frequency in Rat Cerebellar Slices

The relationship between increased intracellular calcium concentration ([Ca2+]i) and changes in spontaneous synaptic current frequency caused by the neurotoxicant methylmercury (MeHg) was examined in Purkinje cells of cerebellar slices using confocal microscopy and whole-cell recording. MeHg (10–100 μM) stimulated and then suppressed completely the frequency of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). Current amplitude was also initially increased. The same MeHg concentrations markedly increased fluorescence of the Ca2+ indicator Fluo-4 throughout the molecular layer as well as the granule cells. No changes in fluorescence occurred in Purkinje cell soma, although fluorescence increased in their subplasmalemmal shell. Simultaneous confocal imaging and whole-cell recording revealed that time to onset of MeHg-induced increase in fluorescence in the molecular layer correlated with that of increased sEPSC and sIPSC frequency in Purkinje cells. Pretreatment with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) significantly suppressed the MeHg-induced increase in sIPSC frequency, further suggesting that MeHg-induced elevation of [Ca2+]i is partially responsible for its early stimulatory effects on spontaneous synaptic responses. However when spontaneous synaptic currents ceased with MeHg, Fluo-4 fluorescence remained elevated. Thus synaptic transmission cessation is apparently not related to changes in [Ca2+]i. It may result from effects of MeHg on transmitter release or sensitivity of postsynaptic receptors. The lack of effect of MeHg on Purkinje cell somal fluorescence reinforces that they are more resistant to MeHg-induced elevations of [Ca2+]i than other cells, including cerebellar granule cells.

Methylmercury Differentially Affects GABAA Receptor‐Mediated Spontaneous IPSCs in Purkinje and Granule Cells of Rat Cerebellar Slices

Using whole‐cell recording techniques we compared effects of the environmental cerebellar neurotoxicant methylmercury (MeHg) on spontaneous IPSCs (sIPSCs) of both Purkinje and granule cells in cerebellar slices of the rat. In Purkinje cells, bath application of 10, 20 or 100 μM MeHg initially increased then suppressed the frequency of sIPSCs to zero. In granule cells, the initial increase in frequency was not observed in ≈50 % of cells examined, but suppression of sIPSCs by MeHg occurred in every cell tested. For both cells, time to onset of effects of MeHg was inversely related to the concentration; moreover, the pattern of changes in mIPSCs induced by MeHg in the presence of tetrodotoxin was similar to that in sIPSCs. For each concentration of MeHg, it took 2–3 times longer to block sIPSCs in Purkinje cells than it did in granule cells. MeHg also initially increased then decreased amplitudes of sIPSCs to block in both cells; again the response was more variable in granule cells. In most Purkinje and some granule cells, MeHg induced a giant, slow inward current during the late stages of exposure. Appearance of this current appeared to be MeHg concentration dependent, and the direction of current flow was reversed by changing the holding potentials. Reduction of the [Cl−] in the internal solution caused inwardly directed, but not outwardly directed giant currents to disappear, suggesting that this current is a Cl−‐mediated response. However, bicuculline and picrotoxin failed to block it. MeHg apparently acts at both presynaptic and postsynaptic sites to alter GABAA receptor‐mediated inhibitory synaptic transmission. GABAA receptors in granule cells appear to be more sensitive to block by MeHg than are those in Purkinje cells, although the general patterns of effects on the two cells are similar.

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.

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 Cu2+/Zn2+ superoxide dismutase 1 (hSOD1) gene mutation (hSOD1G93A; 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 [Ca2+]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 [Ca2+]i in all G93A mice, irrespective of MeHg treatment. N-acetyl spermine, which antagonizes Ca2+-permeable AMPA receptors, further reduced [Ca2+]i more effectively in MeHg-treated than untreated G93A mice, suggesting that MeHg-treated mice have a greater Ca2+-permeable AMPA receptor contribution. The non-Ca2+ divalent cation chelator N,N,N′,N′-tetrakis(pyridylmethyl)ethylenediamine reduced Fluo-4 fluorescence in all G93A mice; FluoZin-(Zn2+ indicator) fluorescence was increased in all MeHg-treated mice. Thus in G93A mice Zn2+ 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 [Ca2+]i induced by the G93A-MeHg interaction apparently was associated with Ca2+-permeable AMPA receptors and may contribute to the hastened development of ALS-like phenotypes by subjecting motor neurons to excessive elevation of [Ca2+]i, leading to excitotoxic cell death.

Methylmercury: A potential environmental risk factor contributing to epileptogenesis

Epilepsy or seizure disorder is one of the most common neurological diseases in humans. Although genetic mutations in ion channels and receptors and some other risk factors such as brain injury are linked to epileptogenesis, the underlying cause for the majority of epilepsy cases remains unknown. Gene-environment interactions are thought to play a critical role in the etiology of epilepsy. Exposure to environmental chemicals is an important risk factor. Methylmercury (MeHg) is a prominent environmental neurotoxicant, which targets primarily the central nervous system (CNS). Patients or animals with acute or chronic MeHg poisoning often display epileptic seizures or show increased susceptibility to seizures, suggesting that MeHg exposure may be associated with epileptogenesis. This mini-review highlights the effects of MeHg exposure, especially developmental exposure, on the susceptibility of humans and animals to seizures, and discusses the potential role of low level MeHg exposure in epileptogenesis. This review also proposes that a preferential effect of MeHg on the inhibitory GABAergic system, leading to disinhibition of excitatory glutamatergic function, may be one of the potential mechanisms underlying MeHg-induced changes in seizure susceptibility.

Differential Effects of Methylmercury on γ-Aminobutyric Acid Type A Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture

Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABAA 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 α6 subunit-containing GABAA receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (IGABA) were recorded in response to MeHg in α6 subunit-containing cerebellar granule cells and α6 subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express α6 subunits. They express the same α1-containing GABAA receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. IGABA were obtained using whole-cell recording and symmetrical [Cl–]. MeHg reduced IGABA 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 IGABA were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of α6 subunit-containing GABAA receptors. Effects of MeHg on IGABA were similar in cells at all days. In human embryonic kidney 293 cells expressing either α6 or α1 subunit-containing GABAA receptors, time to block of IGABA by MeHg was comparable. Thus, the presence of the α6 subunit alone may not underlie the differential effects of MeHg on IGABA observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.

Differential Effects of Methylmercury on GABAA Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture

Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABAA 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 α6 subunit-containing GABAA receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (IGABA) were recorded in response to MeHg in α6 subunit-containing cerebellar granule cells and α6 subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express α6 subunits. They express the same α1-containing GABAA receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. IGABA were obtained using whole-cell recording and symmetrical [Cl–]. MeHg reduced IGABA 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 IGABA were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of α6 subunit-containing GABAA receptors. Effects of MeHg on IGABA were similar in cells at all days. In human embryonic kidney 293 cells expressing either α6 or α1 subunit-containing GABAA receptors, time to block of IGABA by MeHg was comparable. Thus, the presence of the α6 subunit alone may not underlie the differential effects of MeHg on IGABA observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.

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

HIGHLIGHTSThe initial effects of methylmercury (MeHg) on IGABA are mediated by &agr;1&bgr;2&ggr;2S or &agr;6&bgr;2&ggr;2S receptors expressed in Xenopus laevis oocytes.Bath application of MeHg initially potentiated IGABA to its more defined later effects.This effect is reversible and appears to be a direct interaction of MeHg with GABAA receptors. ABSTRACT Early onset effects of methylmercury (MeHg) on recombinant &agr;1&bgr;2&ggr;2S or &agr;6&bgr;2&ggr;2S 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 &agr;1&bgr;2&ggr;2S or &agr;6&bgr;2&ggr;2S receptors were compared during continuous bath application of MeHg. A time‐dependent increase in IGABA mediated by both receptor subtypes occurred following the first 25–30 min of MeHg (5 &mgr;M) exposure. In &agr;6&bgr;2&ggr;2S containing receptors, the MeHg‐induced increase in IGABA was less pronounced compared to that mediated by &agr;1&bgr;2&ggr;2S 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 &agr;1&bgr;2&ggr;2S or &agr;6&bgr;2&ggr;2S 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.

Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices

Effects of neurotoxic chemicals on central synaptic function can be assessed using electrophysiological recording techniques in freshly isolated brain slices. The three most commonly used electrophysiological recording methods in brain slices are extracellular microelectrode recording, intracellular microelectrode recording, and whole‐cell patch clamp recording. This unit presents the basic procedures and applications of extra‐ and intracellular recordings in hippocampal slices and whole‐cell recording in cerebellar slices.