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TOXICOLOGY

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

Neuroscience Program and Department of Pharmacology/Toxicology, Michigan State University, East Lansing, Michigan

Received May 16, 2007; accepted October 30, 2007.

	Abstract

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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 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 ₆ subunit-containing cerebellar granule cells and ₆ subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express ₆ subunits. They express the same 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 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 ₆ or ₁ subunit-containing GABA_A receptors, time to block of I_GABA by MeHg was comparable. Thus, the presence of the 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.

MeHg has a high affinity for sulfhydryl groups numerous on cysteine-containing proteins. Thus, it has the potential to bind to cell membrane proteins and interfere with many cellular processes (Chang, 1977; Atchison and Hare, 1994). Among these are disruption of excitatory and inhibitory synaptic transmission (Yuan and Atchison, 1993, 1997, 1999, 2003). GABA_A receptor-mediated synaptic transmission is inhibited by MeHg. In dorsal root ganglion neurons in culture, high concentrations of MeHg (100 µM) suppress the peak currents induced by GABA (Arakawa et al., 1991). In hippocampal slice, MeHg gradually decreases GABA_A receptor-mediated inhibitory postsynaptic potential amplitudes to complete block (Yuan and Atchison, 1995). Block of inhibitory synaptic transmission in hippocampal slice by low concentrations of MeHg occurs earlier than does block of glutamatergic synaptic transmission (Yuan and Atchison, 1995, 1997), suggesting that inhibitory synaptic transmission is more sensitive to block by MeHg than is excitatory transmission. Block of inhibitory postsynaptic currents (IPSCs) induced by bath-applied MeHg in cerebellar granule cells in brain slice also occurs much earlier than does block in neighboring Purkinje cells (Yuan and Atchison, 2003). GABA_A receptors, especially those containing the ₆ subunit, play a crucial role in regulating granule cell excitability by controlling a tonic inhibitory conductance (Brickley et al., 1996). Therefore, it is possible that differential sensitivity of GABAergic responses to MeHg between granule and Purkinje cells may contribute to differential pathological effect of MeHg on these two types of cerebellar neurons. However, the mechanisms by which MeHg differentially affects GABAergic synaptic transmission in cerebellar cells remain unknown.

One possibility for this differential sensitivity to MeHg may be differential expression of GABA_A receptor phenotypes in granule and Purkinje cells. Purkinje cells only express the receptor containing the ₁ subunit isoform. Granule cells, on the other hand, are the only neurons in the cerebellum that express the ₆ subunit-containing GABA_A receptor (Lüddens et al., 1990; Varecka et al., 1994), although they too express the ₁ subunit (Nusser et al., 1995; Siegel, 1998), both alone and in combination with ₆. Furthermore, ₆-containing receptors can also contain the subunit; ₁-only receptors do not. Thus, granule cells express a wide range of GABA_A receptors.

Expression of the ₆ subunit is regulated developmentally both in vivo and in vitro, and studies have shown that there is an increased expression of ₆ subunits in rats during maturation (Laurie et al., 1992b; Thompson and Stephenson, 1994). GABA_A receptors containing the ₆ or ₁ subunit have unique pharmacological and biophysical properties, including differential sensitivity to agonists, such as benzodiazepines (Lüddens and Wisden, 1991; Sieghart, 1992; Makela et al., 1997) and barbiturates (Fisher et al., 1997; Cestari et al., 2000) and to antagonists such as Zn²⁺ (Draguhn et al., 1990; Saxena and Macdonald, 1994, 1996; Zempel and Steinbach, 1995), furosemide (Korpi et al., 1995), and La³⁺ (Saxena et al., 1997; Makela et al., 1999). As such, expression of distinct subunits can alter significantly the pharmacological sensitivity of the receptor. However, whether or not the expression of the ₆ subunit-containing GABA_A receptor contributes to the differential sensitivity of cerebellar neurons to MeHg has never been examined.

The present study was designed specifically to examine the comparative sensitivity of GABA_A receptors containing ₁ or ₆ subunits to bath-applied MeHg. Whole-cell voltage-clamp technique was used to compare the effects of MeHg on I_GABA in cells expressing either ₁ or ₆ subunit-containing GABA_A receptors. Effects of MeHg on both native and recombinant receptors were examined. The former studies allowed for direct examination of responsiveness in granule cells. The latter permitted analysis of effects on the two receptor subunit phenotypes in isolation.

	Materials and Methods

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Solutions and Chemicals. MeHg (ICN Biomedical Inc., Costa Mesa, CA) was dissolved in deionized water to a final concentration of 10 mM, which served as a stock solution. On the day of experiments, MeHg working solutions (0.1, 1, or 10 µM) were diluted in extracellular solution consisting of 125 mM NaCl, 2.0 mM CaCl₂, 2.5 mM KCl, 1.0 mM MgCl₂, 1.25 mM KH₂PO₄, 26.0 mM NaHCO₃, and 20.0 mM D-glucose, pH-adjusted to 7.4 at room temperature (23–25°C) using 95% O₂/5% CO₂. In the present studies, three concentrations of MeHg (0.1, 1, or 10 µM) were used. They are clinically relevant because they are all within the range of concentrations found in the blood of individuals exposed to MeHg in the mass poisoning that occurred in Iraq in the 1970s (Bakir et al., 1973). The lower concentrations of MeHg (0.1 and 1 µM) were used to determine the concentrations at which the effects of MeHg become evident. Still lower concentrations were not tested, due to the protracted delay associated with actions of MeHg, which would make maintaining a continuous viable seal of electrode with the membrane impossible (see Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2. MeHg causes a time- and concentration-dependent block of IGABA in granule cells. A, MeHg caused a progressive and complete block of IGABA in rat granule cells in primary culture. Whole-cell IGABA were evoked from a holding potential of –60 mV by a 10-ms pulse of GABA (50 µM, black bar), at intervals of 30 s, in the presence of CNQX (10 µM) and APV (50 µM) in the external solution to block glutamatergic currents. Data were collected before (control) and at various time points during exposure to 1 µM MeHg (4, 6, 8, 12, and 20 min). B, time course of effects of MeHg on IGABA recorded under identical conditions to those described above at 0.1 µM (), 1.0 µM (), or 10 µM MeHg (). Data were collected continually before and after MeHg exposure. Each datum point represents the mean value recorded from three to five cells. C, comparative effects of various concentrations of MeHg on time to complete block of IGABA. Each bar represents the mean values obtained from three to five cells. The times to total block of IGABA by MeHg differed significantly between 0.1 and 1.0 µM(*) and 0.1 and 10 µM MeHg (). No significant difference was observed between 1.0 and 10 µM MeHg (p > 0.05).

Preparation of Primary Cerebellar Granule Cell Culture. Primary cultures of rat cerebellar granule neurons were prepared from 7-day-old Sprague-Dawley rats of either gender (Charles River Laboratories, Wilmington, MA) following procedures described by Gallo et al. (1987). Following extraction of cerebella, cells were digested for 13 min at 37°C with 0.025% trypsin (w/v) and plated at a density of 1 x 10⁶ cells/ml on 35-mm Petri dishes (Corning Inc., Corning, NY) coated with poly-L-lysine (0.1 mg/ml). Cells were cultured in basal Eagle's medium supplemented with 10% (w/v) fetal bovine serum, 2 mM glutamine, and 100 µg/ml gentamicin. The final concentration of KCl in the culture medium was adjusted to 25 mM. To achieve functional synapse formation (Chen et al., 1999; Prybylowski et al., 2002), at 4 days in vitro (DIV), the medium was replaced with 5 mM KCl-containing modified Eagle's medium supplemented with 5 mg/ml D-glucose, 0.1 mg/ml transferrin, 0.025 mg/ml insulin, 2 mM glutamine, and 20 µg/ml gentamicin, in addition to Ara-C (10 µM) to inhibit glial cell proliferation. Cells were maintained in a 37°C incubator with 95% O₂ and 5% CO₂. They were used for recordings and immunocytochemistry at DIV 4, 6, and 8 after plating, depending upon the aim of the experiment, to allow for maturation and expression of ₆ and ₁ subunit-containing GABA_A receptors (Laurie et al., 1992a).

Preparation of Primary Cerebral Cortical Cell Culture. For several technical reasons, cortical cells were used as a substitute for cerebellar Purkinje cells in this study. Purkinje cells in culture produce a low yield of cells and are difficult to maintain. Furthermore, they are difficult to record from in the whole-cell configuration due to their extensive arborization which reduces effectiveness of space clamp of the membrane. Their cultures also frequently contain other cell types, particularly glial cells, and MeHg is known to have inhibitory effects on astrocytes (Aschner et al., 2000). Importantly, there are other cellular differential responses to MeHg, including sensitivity to increases in [Ca²⁺]_i between granule and Purkinje cells (Edwards et al., 2005). In contrast, cortical cells are readily maintained in culture, and they contain the same type of GABA_A receptor phenotype as do Purkinje cells. Moreover, they are closer in size to granule cells than are the extremely large Purkinje cells. Thus, to simplify experimental design, cortical cells were substituted for Purkinje cells for this study (Hansen et al., 2001).

Primary cultures of cortical neurons were obtained from newborn Sprague-Dawley rat pups of either gender (Charles River Laboratories) following methods described by Inglefield et al. (2001). Briefly, following removal of the brain and isolation of cortex, cells were digested for 4.5 min at 37°C with trypsin 0.025% (w/v) in buffer that contained 5.0 mM KCl, 0.205 mM KH₂PO₄, 137.0 mM NaCl, 0.17 mM Na₂HPO₄, 5.0 mM D-glucose, 59.0 mM sucrose, and 0.1 mg/ml gentamicin, pH 7.3. Trypsin was inactivated by addition of the above-described buffer containing 0.016% (w/v) DNase I for 4.5 min at 37°C. To this mixture, warmed DMEM supplemented with 10% (w/v) horse serum, 10 mM HEPES, 2 mM glutamine, and gentamicin (0.1 mg/ml) was added, and the cells were centrifuged (500g, 5 min). The resulting pellet was resuspended in DMEM-containing DNase I and recentrifuged. The cell suspension was then resuspended in DMEM and triturated with a Pasteur pipette. Cells were plated at a density of 1 x 10⁶ cells/dish onto 35-mm Petri dishes (Corning Inc.) coated with poly-L-lysine. They were maintained in a 37°C incubator with 95% O₂ and 5% CO₂. DMEM was replaced every 2 days. Ara-C (5 µM) was added to cell cultures 72 h after plating to inhibit glial cell proliferation. Cells were used for recordings 10 to 14 days after plating. All experiments were done in compliance with National Institutes of Health and university standards and were approved by the Michigan State University Institutional Animal Care and Use Committee.

Culture and Transfection of HEK-293 Cells. As described previously (Peng et al., 2002), HEK-293 cells (no. CRL-1573; American Type Culture Collection, Rockville, MD) were trypsinized, centrifuged, and plated at 50 to 100,000 cells in 2 ml of DMEM fortified with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10% (w/v) fetal bovine serum, antibiotics, and antimycotics and grown overnight at 37°C in 5% CO₂. The transient expression of GABA_A receptors was achieved by transfecting the cells one day after plating with 1 µg of total DNA (1:1:1 M ratio of , β, and subunits and 1:5 of a GFP plasmid, all cloned in pCDNA3.1). Plasmids containing rat ₁, β₂, and ₂ or ₆ subunit cDNA were generously provided by Drs. Cynthia Czajkowski (University of Wisconsin, Madison, WI) and William Wisden (University of Heidelberg, Heidelberg, Germany), respectively. All plasmids were purified using QIAGEN kits (endotoxin free). Optimem (96 µl) was added together with Fugene 6 (3 µl) and DNA (1 µg) to maintain an anion/cation ratio of 1:1. The mixture was incubated for 20 min and then added to dishes. Green fluorescent cells were seen starting 24 h after treatment, and robust expression was detected approximately 40 to 72 h post-treatment. On the day of recording, the cells were trypsinized, centrifuged, and replated at one third to one sixth dilution on poly-L-lysine-coated coverglasses in 35-mm culture dishes at a low density with good spatial separation to permit whole-cell recordings. These were made 2 to 8 h following plating. Consistent with the ratio of GFP to GABA_A receptor cDNA (1:5), approximately 20% of HEK cells fluoresced green.

Immunocytochemistry. Cerebellar granule cells on coverslips at DIV 4, 6, or 8, and cortical cells (DIV 10) were rinsed in ice-cold phosphate-buffered saline (PBS; containing 137 mM NaCl, 2.7 mM KCl, 1.4 mM NaH₂PO₄, and 4.3 mM Na₂HPO₄, pH 7.4) three times by immersion and then fixed in cold 1.0% (v/v) p-formaldehyde in PBS for 30 min. Cells were rinsed in PBS and then treated for 30 min with 0.1% Triton-X in PBS containing 20% glycerol. After three rinses in PBS, the cells were labeled overnight at 4°C in either primary rabbit anti-GABA_A receptor ₁ at a concentration of 1:7000 (Novus Biological, Littleton, CO or Abcam, Cambridge, MA) or rabbit anti-GABA_A receptor ₆ at a concentration of 1:1000 (Novus) in PBS containing 20% glycerol. The cells were then rinsed in PBS three times and labeled with FITC-conjugated anti-rabbit (1:200)-labeled secondary antibody in PBS containing 20% glycerol. Cells were rinsed in PBS, mounted on glass slides with an antifade mounting medium (Vectashield Hard Set; Vector, Burlingame, CA), and examined.

Coverslips containing HEK-293 cells were immersed in ice-cold PBS and then, without permeabilizing their membranes, incubated overnight. PBS contained 0.02% (w/v) NaN₃ to kill cells. Following two rinses with PBS, HEK-293 cells were labeled overnight in the cold room with primary anti-₁ (1:7000) or anti-₆ (1:1000) antibodies (Abcam), both of which were generated in rabbits. Cells were then incubated with a TRITC (1:500)-conjugated anti-rabbit or FITC (1: 500)-conjugated anti-rabbit secondary antibody for 2 h at 4°C.

To confirm that labeling of anti-₁ and anti-₆ antibodies was localized to the cell membrane, coverslips containing HEK-293 cells were incubated for 1 h in Zamboni fixative containing 15 ml of picric acid, 5.5 ml of 37% formaldehyde solution, and 79.5 ml of PBS. Following fixation, coverslips were rinsed two times with PBS and labeled overnight in the cold room with primary anti-₁ (1:7000) or anti-₆ (1:1000) antibody or with primary anti-pan cadherin (1:500) antibody, generated in mice. Cells were incubated with TRITC (1: 200)-conjugated anti-rabbit, FITC (1:200)-conjugated anti-rabbit, or Pacific Blue (1:200)-conjugated anti-mouse secondary antibody for 2 h at 4°C.

Immunocytochemistry Data Acquisition and Analysis. Cells were examined using either a Nikon Eclipse TE 2000-U Inverted Microscope (Nikon Optics, Tokyo, Japan) equipped with Meta Morph Meta-Imaging software (Molecular Devices, Downingtown, PA) or a Leitz Laborlux S (Wetzlar, Germany) epifluorescence microscope. All images were obtained with the 60x oil immersion (numerical aperture, 1.40) objective using the same acquisition configuration including exposure time, contrast and brightness, and neutral density filters. Fluorescence levels were recorded for each granule cell after subtracting out the background fluorescence near each cell. Background fluorescence was removed, and fluorescence levels were measured using a poly line around the cell. For each subunit treatment and for each day (DIV 4, 6, or 8), 40 granule cells were examined from two or more slides. Background fluorescence was recorded as a check against photobleaching. Negative controls included incubation of cells with primary or secondary antibody only, in addition to labeling cells with antibodies against subunits that they do not express (i.e., labeling cortical cells with anti-₆ antibody or labeling HEK-293 cells expressing only ₆ subunit-containing GABA_A receptors with antibody against the ₁ subunit). The cells used for measurement were chosen by scanning the coverslip from left to right, analyzing the first 20 single, identifiable granule cells observed. Relative fluorescence levels were recorded, and using the Meta Morph software, comparisons were made, and averages of the mean values of relative fluorescence were calculated for each day and each treatment. No fluorescence was seen in any of the control experiments, with the exception that a minute amount of cross-reactivity was detected with the anti-₁ antibody in HEK-293 cells expressing ₆ subunit-containing GABA_A receptors. This cross-reactivity was not detected in granule cells (data not shown). Absorption controls with the peptide and antibodies could not be performed because the peptides were not provided with the antibodies.

Whole-Cell Recordings. Prior to making whole-cell voltage-clamp recordings, the Petri dish containing the cells was rinsed and covered with approximately 1 ml of recording solution. CNQX (10 µM) and APV (50 µM) were added to the extracellular solution to inhibit ionotropic glutamate receptor-mediated responses. Recording electrodes were fabricated from borosilicate capillary glass (o.d., 1.5 mm; i.d., 1.0 mm; Garner Glass Co., Claremont, CA) and had an impedance of 3 to 7 M for cortical cells, 5 to 10 M for granule cells, and 2 to 4 M for HEK-293 cells when filled with a pipette solution consisting of 140 mM CsCl, 4.0 mM NaCl, 0.5 mM CaCl₂, 10.0 mM HEPES, 5.0 mM EGTA-CsOH, and 2.0 mM Mg-ATP, pH adjusted to 7.3 at room temperature. Following acquisition of a cell-attached patch and cancellation of capacitative currents, the whole-cell configuration was obtained by negative pressure applied to the syringe. Cells were voltage clamped at –60 mV so that GABA application produced inward currents under our approximately symmetric [Cl^–] conditions. The zero current potential was 2.5 mV, which is close to the expected Cl^– equilibrium potential under these conditions. Capacitance transient neutralization and series resistance compensation were optimized. All experiments were carried out at room temperature of 23 to 25°C.

GABA (50 µM) was applied onto the target cells by a 10-ms pulse given at intervals of 30 s using a picospritzer (Picospritzer II; General Valve Corporation, Fairfield, NJ) and delivered through a glass pipette (impedance, 900 k) placed close to the cell. The interpulse interval was long enough for GABA_A receptors to recover from desensitization (data not shown). This concentration was chosen to provide an adequate, yet submaximal, current in ₁ subunit-containing cells. As the GABA affinity of ₆-containing receptors is higher than that of ₁-, this concentration provided a maximal amplitude current (see Feng and Macdonald, 2004). The concentration dependence of GABA as a variable in the action of MeHg was also examined in granule cells.

Physiological saline alone or containing MeHg was delivered into the extracellular bathing solution continuously at a rate of approximately 0.45 ml/min via a gravity-powered perfusion system through a series of tubes placed close to the cell. To observe the effects of MeHg on I_GABA, recordings were made in the presence or absence of MeHg. In the absence of MeHg, I_GABA were relatively stable over a period of 40 min. Due to the irreversible nature of action of MeHg, a given coverslip was exposed to only a single [MeHg].

Electrophysiological Data Acquisition and Analysis. Data were acquired every 30 s using a PC computer equipped with a DigiData 1200 interface and pClamp 8.1 software (Axon Instruments Molecular Devices, Union City, CA). I_GABA were recorded using an Axopatch 1-D amplifier and low-pass filtered at 1 kHz with an eight-pole filter before digitization at 10 kHz, storage, and display. Off-line analysis was performed using Clampfit 8.1 software and the MiniAnalysis program 5.6.10 (Synaptosoft Inc., Decatur, GA). The latter was used to examine effects of MeHg on kinetics of I_GABA. Each GABA_A receptor-mediated event was selected manually and marked. The maximum amplitude for each event was measured at the peak of the inward current. The decay phase was best fitted using the Levenberg-Marquardt least-squares method (Levenberg, 1944; Marquardt, 1963) with a double-exponential function of the form _nn, where n is the number of exponential components, is the relative amplitude of the component, and is the time constant.

Data collected before and during application of MeHg were analyzed statistically using a Student's paired t test or a one-way analysis of variance, depending upon whether comparisons were simply made before or after MeHg at a fixed time point (paired Student's t test) or as a function of time or concentration (analysis of variance). The Tukey-Kramer test was used for post hoc comparisons. Values were considered statistically significant at p < 0.05. The data are presented as mean ± S.E.M., and the number of replications is given in the figure legend. The data were obtained from approximately 60 separate cell cultures, and the number of replications refers to individual cells obtained from at least two separate cell cultures.

	Results

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MeHg Blocks I_GABA in Cerebellar Granule Cells. To determine the subunit specificity of effects of MeHg on I_GABA, whole-cell recordings were made from ₆ subunit-containing granule cells and ₁ subunit-containing cortical cells. To verify the GABA_A receptor subunit expression in both cell types of our culture, immunocytochemistry was performed using antibodies against the ₁ or ₆ subunit. As reported previously, both granule and cortical cells labeled positively for the ₁ subunit (Fig. 1, A and B), whereas only granule cells labeled positively for the ₆ subunit (Fig. 1, C and D). In both cell types, the antibody staining appeared to be localized primarily to the cell body and to a lesser extent in neuronal processes. The cell body is stained in a punctate pattern, and in some cells, it was evident that the bulk of the staining is in the membrane periphery, suggesting that staining is primarily localized to the membrane surface.

View larger version (18K): [in this window] [in a new window]   Fig. 1. GABAA receptor 6 and 1 subunit expression in cerebellar granule and cerebral cortical cells in culture. Granule (DIV 6) and cortical cells (DIV 10) were labeled overnight with anti-1 (A and B) or anti-6 (C and D) antibody following acetone fixation. Antibody staining was visualized with TRITC. Note that only cerebellar granule cells express 6 (C), whereas both granule and cortical cells express 1 subunit-containing receptors (A and B).

The effect of changing the GABA concentration on time to onset of MeHg (1 µM) effect was also analyzed (Fig. 3). Over a range of GABA concentrations, block of I_GABA occurred within 25 to 30 min. Thus there was no obvious interaction between MeHg and the agonist concentration.

View larger version (17K): [in this window] [in a new window]   Fig. 3. MeHg-induced block of IGABA in cerebellar granule cells is not [GABA] dependent. Whole-cell IGABA were obtained as described in Fig. 2. Data were collected during exposure to 1 µM MeHg at the concentrations at GABA indicated (10–1000 µM). Each bar represents the mean values obtained from three to five cells. Time-to-complete suppression of IGABA by MeHg did not differ between any concentration of GABA tested (p > 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 4. MeHg causes a time- and concentration-dependent block of IGABA in cortical cells. A, MeHg caused a progressive and complete block of IGABA in rat cortical cells in primary culture. Whole-cell IGABA were recorded under identical conditions to those described in Fig. 2. Data were collected before (control) and at various time points during exposure to 1 µM MeHg (5, 8, 11, 18, and 37 min). B, time course of effects of MeHg on IGABA recorded at 0.1 (), 1.0 µM (), or 10 () µM MeHg. Data were collected continually before and after MeHg exposure. Each datum point represents the mean value recorded from three to five cells. C, comparative effects of different concentrations of MeHg on time to complete block of IGABA. Each bar represents the mean values obtained from three to five cells. The times to total block of IGABA by MeHg differed significantly between 0.1 and 1.0 µM(*) and 0.1 and 10 µM MeHg (). No significant difference was observed between 1.0 and 10 µM MeHg (p > 0.05). D, comparative effects of MeHg on block of IGABA in cortical (dark bars) and granule cells (light bars) in culture. IGABA was blocked significantly faster (p < 0.05) in granule cells as compared with cortical cells for 1.0 and 10 µM MeHg (*).

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effects of MeHg on IGABA are voltage-independent in granule and cortical cells. A, whole-cell IGABA were recorded from cerebellar granule cells before (control) and after 5 min of exposure to 1 µM MeHg at holding potentials ranging from –80 to + 60 mV. Each datum point represents the mean value recorded from three to four cells. MeHg caused a reduction in IGABA amplitude in a linear fashion, such that it did not change at different holding potentials. B, IGABA were recorded from cerebral cortical cells before (control) and after 10 min of exposure to 1 µM MeHg at holding potentials ranging from –80 to + 60 mV. Each datum point represents the mean value recorded from three cells. MeHg caused a reduction in IGABA amplitude in cortical cells in a voltage-independent manner.

Effects of MeHg Do Not Differ in Granule Cells Expressing Only ₁ or a Combination of ₁ and ₆ Subunit-Containing GABA_A Receptors. Because of the developmental regulation of ₆ subunit expression, its levels increase with increasing length of time in culture. We used this feature to try to determine whether increasing granule cell expression of ₆ subunits would lead to increased sensitivity to MeHg. Following cerebellar granule cell culture for differing lengths of time, cells were fixed and labeled with antibodies against GABA_A receptor ₁ or ₆ subunits to determine the time course of subunit expression. As seen for the representative fluorescence micrographs in Fig. 6A, expression of ₁ subunits was observed on the 1st day of antibody labeling (DIV 4) and continued through to the last day of labeling (DIV 8). In contrast, expression of ₆ subunits was not observed until DIV 6, and expression continued through to DIV 8. The staining for either antibody appeared to be primarily localized to the cell body in a punctate pattern. In some cells, the focal plane shows that much of the staining is localized to the periphery of the cell body, suggesting cell surface labeling. Staining was also observed, to a lesser degree, in neuronal processes, and then to a greater extent for ₁ staining.

View larger version (47K): [in this window] [in a new window]   Fig. 6. GABAA receptor 6 and 1 subunit expression in cerebellar granule cells at different days in culture. Granule cells (DIV 4–8) were labeled overnight with anti-1 or anti-6 antibody following acetone fixation. Antibody staining was visualized with TRITC (anti-1) or FITC (anti-6) epifluorescence (60x oil). A, 1 subunit-containing GABAA receptors are expressed in granule cells grown in vitro throughout DIV 4 to 8. B, 6 subunit-containing receptors are not expressed in granule cells until DIV 6 and 8.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Comparative expression of GABAA receptor subunits as a function of postnatal age. A, relative immunofluorescence associated with 1 or 6 subunits expression in cerebellar granule cells was examined in granule cells at DIV 4, 6, and 8. *, significant difference (p < 0.05) from the DIV 4 value for 6. , similar difference from DIV 4 value for 1 staining. B, ratio of immunofluorescence associated with 6 and 1 staining is shown as a function of postnatal age. At DIV 6 and 8, the ratio of 6/1 immunofluorescence was markedly increased from that at DIV 4.

GABA_A receptor-mediated currents were subsequently examined from cells from DIV 4 to represent ₁-containing GABA_A receptor responses and from DIV 6 to 8 to represent primarily ₆-containing responses. At each day in culture tested, MeHg caused a gradual and complete suppression of I_GABA (Fig. 8). This effect was concentration- and time-dependent. The time course of effect by MeHg did not differ between DIV 4 and 6 to 8 as seen in Fig. 8, A to D. For 10 µM MeHg, the mean (±S.E.M.) time to block of I_GABA in granule cells from DIV 4 was 1250 ± 135 s, and for cells, from DIV 6 to 8, it was 1280 ± 200 s. Thus there were no differences in susceptibility of granule cell I_GABA to MeHg as ₆ expression increased.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Effects of MeHg on granule cells at different days in culture. Time course of effects of MeHg on IGABA were obtained under identical conditions to those described in Fig. 1. IGABA were recorded from granule cells grown in culture for 4 or 6 to 8 days in the presence of 0.1 (A), 1.0 (B), or 10 (C) µM MeHg. Data were collected continually before and during MeHg exposure. Each datum point represents the mean value recorded from three to four cells. D, comparative effects of different concentrations of MeHg on time to complete block of IGABA in granule cells grown for 4 or 6 to 8 days in culture. Each bar represents the mean values obtained from three to four cells. The times to total block of IGABA by MeHg did not differ significantly between cells grown for 4 or 6 to 8 days in culture.

View larger version (8K): [in this window] [in a new window]   Fig. 9. GABAA receptor 6 and 1 subunit expression in HEK-293 cells. HEK-293 cells were labeled overnight with anti-1 or anti-6 antibody following NaN3 fixation. Antibody staining was visualized with TRITC epifluorescence (60x oil). HEK-293 cells expressing 1 subunit-containing GABAA receptors labeled positive for the 1 subunit (A) and negative for the 6 subunit (B). HEK-293 cells expressing 6 subunit-containing GABAA receptors labeled negative for the 1 subunit (C) and positive for the 6 subunit (D). Note, a slight amount of fluorescence was detected in C, suggesting that a small amount of cross-reactivity for the anti-1 antibody may exist.

Subunit subtype expression was corroborated pharmacologically using diazepam (1 µM). As seen in Fig. 10A, diazepam prolonged the slow decay time constant of I_GABA in HEK-293 cells expressing the ₁ subunit (Otis and Mody, 1992). However, in HEK-293 cells expressing the ₆ subunit, no effect of diazepam on I_GABA was detected (Fig. 10B).

View larger version (8K): [in this window] [in a new window]   Fig. 10. Effects of diazepam on IGABA in HEK-293 cells. Whole-cell IGABA were evoked from a holding potential of –60 mV by a 10-ms pulse of GABA (500 µM), at intervals of 30 s, under similar recording conditions to those described in Fig. 2. Current traces were collected before (control) and after exposure to diazepam (1 µM) at times indicated by arrows. A, diazepam prolonged IGABA slow decay rate in HEK-293 cells expressing 1 subunit-containing GABAA receptors as can be seen readily by the superimposed control and diazepam traces (right). B, diazepam did not affect IGABA slow decay rate in HEK-293 cells expressing 6 subunit-containing GABAA receptors.

View larger version (14K): [in this window] [in a new window]   Fig. 11. MeHg causes a time- and concentration-dependent block of IGABA in HEK-293 cells. A, time course of effects of MeHg on IGABA recorded under identical conditions to those described in Fig. 2. IGABA were recorded in the presence of [MeHg] of 0.1 (, ), 1.0 (, ), or 10 (, ) µM in HEK-293 cells expressing either 1 (dotted line, open symbols) or 6 (solid line, solid symbols) subunit-containing GABAA receptors. Data were collected continually before and during MeHg exposure. Each datum point represents the mean value recorded from three to four cells. B, comparative effects of 1.0 and 10 µM MeHg on time to complete block of IGABA in HEK-293 cells expressing either 1 (dotted bar) or 6 (solid bar) subunit-containing GABAA receptors. Each bar represents the mean values obtained from three to five cells. The times to total block of IGABA by MeHg did not differ significantly between HEK-293 cells expressing either 6 or 1 subunit-containing GABAA receptors for either [MeHg] tested. C, comparative effects of 0.1 to 10 µM MeHg on time to 40% block of IGABA in HEK-293 cells expressing either 1 (dotted bar) or 6 (solid bar) subunit-containing GABAA receptors. Each bar represents the mean values obtained from three to five cells. The times to 40% block of IGABA by MeHg did not differ significantly between HEK-293 cells expressing either 6 or 1 subunit-containing GABAA receptors for all [MeHg] tested.

View this table: [in this window] [in a new window]   TABLE 1 Comparative effects of MeHg on IGABA block in 1- or 6-containing HEK-293 cells This table summarizes the comparative effects of MeHg on IGABA block for 1 or 6 subunit-containing HEK-293 cells. The time-to-block by each concentration of MeHg did not differ significantly between 1 and 6 receptor subtypes. Note that for 0.1 µM MeHg, complete block of IGABA was not achieved in HEK-293 cells expressing either subtype of receptor [as indicated by the minus (—) symbol] because cells flattened out after 35 min, and recordings could not be maintained.

In addition, stepping the voltage from –80 to +60 mV in the presence of MeHg produced a linear current-voltage relationship in HEK-293 cells expressing both ₁ and ₆ subunit-containing GABA_A receptors (Fig. 12, A and B). This suggests that the effects of MeHg on recombinant I_GABA are also voltage-independent.

View larger version (11K): [in this window] [in a new window]   Fig. 12. Effects of MeHg on IGABA are voltage-independent in HEK-293 cells. A, whole-cell IGABA were recorded from HEK-293 cells expressing 6 subunit-containing GABAA receptors before (control) and after 5 min of exposure to 1 µM MeHg at holding potentials ranging from –80 to + 60 mV. Each datum point represents the mean value recorded from three to four cells. The effects of MeHg on IGABA in these cells were voltage-independent. B, whole-cell IGABA were recorded from HEK-293 cells expressing 1 subunit-containing GABAA receptors before (control) and after 5 min of exposure to 1 µM MeHg at holding potentials ranging from –80 to + 60 mV. Each datum point represents the mean value recorded from four cells. MeHg caused a reduction in IGABA amplitude in a voltage-independent manner.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Previous studies conducted in slice preparations revealed that MeHg impairs inhibitory GABAergic neurons in hippocampus and cerebellum (Yuan and Atchison, 1995, 1997, 2003). Hippocampal GABAergic synaptic transmission was shown to be more sensitive to the effects of MeHg as compared with glutamatergic synaptic transmission (Yuan and Atchison, 2003). Importantly, some cell types appear to be particularly sensitive to the effects of MeHg. Suppression of IPSCs in cerebellar granule cells, for instance, occurred much earlier than did suppression in neighboring Purkinje cells (Yuan and Atchison, 2003). A comparative study of several ion channels with respect to MeHg sensitivity in cultures of granule cells revealed that GABA_A receptors and voltage-gated Ca²⁺ channels were affected at the lowest concentrations of MeHg (Yuan et al., 2005). Thus, GABA_A receptors in the cerebellum appear to be particularly sensitive to the inhibitory effects of MeHg.

The exact mechanism(s) by which MeHg differentially interferes with GABAergic function in different cell types is not known. One possibility for the enhanced sensitivity of GABAergic responses to MeHg in granule cells, as compared with Purkinje cells, is their differential expression of the ₆ subunit-containing GABA_A receptor. GABA_A receptors containing the ₆ subunit are more sensitive than non-₆-containing receptors to inhibition by Zn²⁺ (Draguhn et al., 1990; Saxena and Macdonald, 1994; Zempel and Steinbach, 1995) and La³⁺ (Saxena et al., 1997; Makela et al., 1999), so we hypothesized that a similar enhanced sensitivity might occur to MeHg. To test this possibility, I_GABA was recorded in response to MeHg in granule cells and compared with cortical cells, which contain only the ₁ subunit. At 1 and 10 µM MeHg, I_GABA was inhibited more rapidly in granule cells than in cortical cells. These findings are qualitatively consistent with effects of MeHg on I_GABA reported previously in cerebellar slice but occurred at much lower concentrations of MeHg (Yuan and Atchison, 1999, 2003). Concentration differences between the two systems likely were due to pharmacokinetic factors involving a greater diffusion barrier to MeHg and more nonspecific binding sites in the slice as opposed to low-density monolayer cultures used in the present study. Specifically, the results of these initial experiments supported the possibility that the ₆ subunit may contribute to the differential effects of MeHg on I_GABA observed in granule and Purkinje cells.

To confirm the results obtained in granule and cortical cells, the effects of MeHg on I_GABA were then investigated in granule cells at different lengths of culture. This was done to shift the ratio of ₁ to ₆ subunit expression because granule cells can developmentally express either ₆- and/or ₁-containing GABA_A receptors. Surprisingly, effects of MeHg on I_GABA did not differ in granule neurons expressing ₆-containing GABA_A receptors at either low or high levels. I_GABA was suppressed by MeHg with a similar time- and concentration-dependent manner in granule cells containing either subtype of GABA_A receptor. This suggests that the ₆ subunit alone does not underlie the differential sensitivity of I_GABA to MeHg in granule and cortical cells. Other factors, such as differential GABA_A receptor auxiliary subunit expression in these two cell types, may be involved. In addition to the ₁ and ₆ subunits, cerebellar granule cells express GABA_A receptors containing either β₂ or β₃ subunits in combination with ₂, ₃, or (for ₆-expressing cells only) subunits (Laurie et al., 1992a). Cortical cells (and Purkinje cells), on the other hand, express mainly the ₁β₂₂ subtype of GABA_A receptor, although there is some variability in β subunit expression (Wisden et al., 1996).

Differential GABA_A receptor subunit composition alters the pharmacological responsiveness of the receptor. For instance, the presence of the subunit increases the affinity of ₆-containing receptors for GABA and imparts sensitivity to inhibition by Zn²⁺ (Saxena and Macdonald, 1994). Similarly, sensitivity of the GABA receptor to barbiturates depends on the identity of the β subunit; inclusion of the β₃ subunit makes the receptor insensitive to the effects of pentobarbital (Cestari et al., 2000). Furosemide inhibition is also enhanced when the β₁ subunit of the GABA_A receptor is replaced with either the β₂ or β₃ subunit subtypes (Thompson et al., 1999). Finally, replacement of the β₁ subunit of the receptor with β₂ or β₃, results in enhanced sensitivity to potentiation by ethanol (Mihic et al., 1997) or the general anesthetic etomidate (Belelli et al., 1997). Thus, other subunits may be involved in the differential sensitivity to MeHg. This will need to be examined rigorously using cloned receptors of known subunit composition and expressed heterologously.

To examine the effects of MeHg on ₆-or ₁-containing receptor-mediated currents in isolation, whole-cell recordings were also made from HEK-293 cells containing either the ₆ or ₁ subtype of GABA_A receptor. Effects of MeHg on I_GABA suppression did not differ between cells expressing the individual subunits; time to suppression of I_GABA by MeHg (1 and 10 µM) occurred at similar times. Thus, differential expression of ₆ subunit-containing GABA_A receptors in granule and Purkinje cells does not underlie the differential sensitivities of I_GABA to MeHg observed in granule and Purkinje cells.

There are several possible mechanisms by which MeHg could act differentially to impair GABA_A receptor function. These include increased [Ca²⁺]_i, increased release of Zn²⁺, or interaction with cysteines on the receptor. The two most likely candidates for differential sensitivity include elevation of [Ca²⁺]_i and/or release of Zn²⁺. Both of these effects have been demonstrated to occur with MeHg (Hare et al., 1993; Denny and Atchison, 1994; Marty and Atchison, 1997; Edwards et al., 2005).

Effects of [Ca²⁺]_i on GABA_A receptor function are complex. Several studies report reduction of current amplitude by elevation of [Ca²⁺]_i (Inoue et al., 1986; Martina et al., 1994; Akopian et al., 1998). This effect has been observed with release of Ca²⁺ from both IP₃- and ryanodine receptor-activated stores (Akopian et al., 1998), as well as with use of Ca²⁺-ionophore A23187 [GenBank] (Martina et al., 1994). The effect appears to be voltage-independent, and progressive, much like that seen with MeHg in the present study. Although MeHg increases [Ca²⁺]_i in numerous cell types, granule cells in culture are especially sensitive to this effect, particularly when compared with Purkinje cells (Marty and Atchison, 1997; Edwards et al., 2005). Similarly, in cerebellar slice, granule cells again are more sensitive to MeHg-induced elevation of [Ca²⁺]_i than are Purkinje cells (Yuan and Atchison, 2007). This difference is attributable at least in part to the presence in Purkinje, but not granule cells of calbindin D28k, a Ca²⁺-binding protein. The relative sensitivity of cortical cells to MeHg-induced increase in [Ca²⁺]_i has never been reported. On the other hand, Mody and colleagues (De Koninck and Mody, 1996) reported that in cerebellar slice, kinetics of mIPSCs, but not amplitude, were altered by [Ca²⁺]_i. Certainly if a granule cell-specific increase in [Ca²⁺]_i underlies the enhanced sensitivity of their I_GABA to MeHg, this would likely not show up as a differential response when cells were compared at DIV 4, 6, and 8. Similarly, such an effect would be absent from the HEK cells expressing the recombinant GABA_A receptors. Thus, this remains a viable explanation for the enhanced sensitivity of granule cell I_GABA.

Another interesting possibility relates to the ability of MeHg to increase [Zn²⁺]_i (Hare et al., 1993; Denny et al., 1993; Denny and Atchison, 1994; Edwards et al., 2005). Zn²⁺ is a well described inhibitor of I_GABA in cerebellar granule cells, an effect mediated primarily through the subunit (Draguhn et al., 1990; Kilic et al., 1993; Fisher and Macdonald, 1998). Granule, but not cortical, cells express the subunit, which coexpresses with ₆. Although the recombinant receptors expressed in HEK cells contained the ₆ subunit, they did not contain , instead, expressing with 2. Zn²⁺ has a much lower antagonistic affinity for 2- than for -containing receptors (Fisher and Macdonald, 1998). Granule cells contain glutamatergic vesicles, which contain considerable amounts of Zn²⁺, and MeHg has well described ability to increase release of transmitters, including glutamate (see Yuan and Atchison, 2007). Consequently, if a MeHg-induced release of Zn²⁺ occurred, it would most likely affect granule cells preferentially. This does not explain, however, why there was no difference in sensitivity of granule cells at DIV 4 as compared with DIV 6 and 8, given that the expression of ₆ was clearly increased. Clearly, the actions of MeHg on I_GABA differ from those of conventional receptor blockers such as bicuculline, and intracellular modulatory actions may well contribute to this effect.

In summary, acute bath application of MeHg to rat cerebellar granule and cerebral cortical cells completely and irreversibly suppressed I_GABA. I_GABA in granule cells were more sensitive to the effects of MeHg than were I_GABA in cortical cells. However, expression of the ₆ subunit alone does not underlie the differential effects of MeHg on I_GABA observed in cerebellar granule and Purkinje neurons; additional factors may be involved as well. Further chimeric studies will be needed to determine the relative contribution, if any, of each GABA receptor subunit and its subtypes to the differential sensitivity of GABAergic responses to MeHg in cerebellar cells.

	Acknowledgements

 
The excellent technical assistance of Dawn Autio and Dawn Parsell and word processing and graphics assistance of Jessica Hauptman and Sarah Metzger are especially appreciated. We gratefully acknowledge the generous gifts of plasmids for GABA_A receptor subunits from Cynthia Czajkowski (University of Wisconsin, Madison, WI) and William Wisden (University of Heidelberg, Heidelberg, Germany).

	Footnotes

 
This study was supported by National Institutes of Health Grant R01-ES11662. Portions of this work were submitted by C.J.H. in partial fulfillment of the requirements for the Ph.D. degree in Neuroscience at Michigan State University.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.123976.

ABBREVIATIONS: MeHg, methylmercury; IPSC, inhibitory postsynaptic current; I_GABA, GABA_A receptor-mediated currents; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; APV, DL-2-amino-5-phosphonopentanoic acid; Ara-C, cytosine β-D-arabino-furanoside; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine; FITC, fluorescein isothiocyanate; DIV, day(s) in vitro; HEK, human embryonic kidney; GFP, green fluorescent protein; PBS, phosphate-buffered saline.

Address correspondence to: Dr. William D. Atchison, Department of Pharmacology and Toxicology, Michigan State University, B-331 Life Science Building, East Lansing, MI 48824-1317. E-mail: atchiso1{at}msu.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Akopian A, Gabriel R, and Witkovsky P (1998) Calcium released from intracellular stores inhibits GABA_A-mediated currents in ganglion cells of the turtle retina. J Neurophysiol 80: 1105–1115.[Abstract/Free Full Text]

Arakawa O, Nakahiro M, and Narahashi T (1991) Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons. Brain Res 551: 58–63.[CrossRef][Medline]

Aschner M, Yao CP, Allen JW, and Tan KH (2000) Methylmercury alters glutamate transport in astrocytes. Neurochem Int 37: 199–206.[CrossRef][Medline]

Atchison WD and Hare MF (1994) Mechanisms of methylmercury-induced neurotoxicity. FASEB J 8: 622–629.[Abstract]

Bakir F, Damluji S, Amin-Zaki L, Murtadha M, Khalidi A, Al-Rawi NY, Tikriti S, Dhahir HI, Clarkson TW, Smith JC, et al. (1973) Methylmercury poisoning in Iraq. Science 181: 230–240.[Free Full Text]

Belelli D, Lambert JJ, Peters JA, Wafford K, and Whiting PJ (1997) The interaction of the general anesthetic etomidate with the -aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci U S A 94: 11031–11036.[Abstract/Free Full Text]

Bianchi MT and Macdonald RL (2002) Slow phases of GABA_A receptor desensitization: structural determinants and possible relevance for synaptic function. J Physiol (Lond) 544: 3–18.[Abstract/Free Full Text]

Brickley SG, Cull-Candy SG, and Farrant M (1996) Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABA_A receptors. J Physiol (Lond) 497: 753–759.[Abstract/Free Full Text]

Cestari IN, Min KT, Kulli JC, and Yang J (2000) Identification of an amino acid defining the distinct properties of murine β₁ and β₃ subunit-containing GABA_A receptors. J Neurochem 74: 827–838.[CrossRef][Medline]

Chang LW (1977) Neurotoxic effects of mercury. Environ Res 14: 329–373.[Medline]

Chen Q, Moulder K, Tenkova T, Hardy K, Olney JW, and Romano C (1999) Excitotoxic cell death dependent on inhibitory receptor activation. Exp Neurol 160: 215–225.[CrossRef][Medline]

De Koninck Y and Mody I (1996) The effects of raising intracellular calcium on synaptic GABA_A receptor-channels. Neuropharmacology 35: 1365–1374.[CrossRef][Medline]

Denny MF and Atchison WD (1994) Methylmercury-induced elevations in intrasynaptosomal zinc concentrations: an ¹⁹F-NMR study. J Neurochem 63: 383–386.[Medline]

Denny MF, Hare MF, and Atchison WD (1993) Methylmercury alters intrasynaptosomal concentrations of endogenous polyvalent cations. Toxicol App Pharmacol 122: 222–232.[CrossRef][Medline]

Draguhn A, Verdoorn TA, Ewert M, Seeburg PH, and Sakmann B (1990) Functional and molecular distinction between recombinant rat GABA_A receptor subtypes by Zn²⁺. Neuron 5: 781–788.[CrossRef][Medline]

Edwards JR, Marty MS, and Atchison WD (2005) Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury. Toxicol Appl Pharmacol 208: 222–232.[CrossRef][Medline]

Feng HJ and Macdonald RL (2004) Multiple actions of propofol on β and β GABA_A receptors. Mol Pharmacol 66: 1517–1524.[Abstract/Free Full Text]

Fisher JL and Macdonald RL (1998) The role of an subtype M2–M3 His in regulating inhibition of GABA_A receptor current by zinc and other divalent cations. J Neurosci 18: 2944–2953.[Abstract/Free Full Text]

Fisher JL, Zhang J, and Macdonald RL (1997) The role of 1 and 6 subtype amino-terminal domains in allosteric regulation of -aminobutyric acid_a receptors. Mol Pharmacol 52: 714–724.[Abstract/Free Full Text]

Gallo V, Kingsbury A, Balazs R, and Jorgensen OS (1987) The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J Neurosci 7: 2203–2213.[Abstract]

Grandjean P, Weihe P, White RF, and Debes F (1998) Cognitive performance of children prenatally exposed to "safe" levels of methylmercury. Environ Res 77: 165–172.[Medline]

Hansen SL, Ebert B, Fjalland B, and Kristiansen U (2001) Effects of GABA_A receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions. Br J Pharmacol 133: 539–549.[CrossRef][Medline]

Hare MF, McGinnis KM, and Atchison WD (1993) Methylmercury increases intracellular concentrations of Ca⁺⁺ and heavy metals in NG108–15 cells. J Pharmacol Exp Ther 266: 1626–1635.[Abstract/Free Full Text]

Hinkle DJ and Macdonald RL (2003) β subunit phosphorylation selectively increases fast desensitization and prolongs deactivation of 1β12L and 1β32L GABA_A receptor currents. J Neurosci 23: 11698–11710.[Abstract/Free Full Text]

Inglefield JR, Mundy WR, and Shafer TJ (2001) Inositol 1,4,5-triphosphate receptor-sensitive Ca²⁺ release, store-operated Ca²⁺ entry, and cAMP responsive element binding protein phosphorylation in developing cortical cells following exposure to polychlorinated biphenyls. J Pharmacol Exp Ther 297: 762–773.[Abstract/Free Full Text]

Inoue M, Oomura Y, Yakushiji T, and Akaike N (1986) Intracellular calcium ions decrease the affinity of the GABA receptor. Nature 324: 156–158.[CrossRef][Medline]

Jones-Davis DM, Song L, Gallagher MJ, and Macdonald RL (2005) Structural determinants of benzodiazepine allosteric regulation of GABA_A receptor currents. J Neurosci 25: 8056–8065.[Abstract/Free Full Text]

Kilic G, Moran O, and Cherubini E (1993) Currents activated by GABA and their modulation by Zn²⁺ in cerebellar granule cells in culture. Eur J Neurosci 5: 65–72.[CrossRef][Medline]

Korpi ER, Kuner T, Seeburg PH, and Lüddens H (1995) Selective antagonist for the cerebellar granule cell-specific -aminobutyric acid type A receptor. Mol Pharmacol 47: 283–289.[Abstract]

Laurie DJ, Seeburg PH, and Wisden W (1992a) The distribution of thirteen GABA_A receptor subunit mRNAs in the rat brain: II. Olfactory bulb and cerebellum. J Neurosci 12: 1063–1076.[Abstract]

Laurie DJ, Wisden W, and Seeburg PH (1992b) The distribution of thirteen GABA_A receptor subunit mRNAs in the rat brain: III. Embryonic and postnatal development. J Neurosci 12: 4151–4172.[Abstract]

Levenberg KA (1944) Method for the solution of certain problems in least squares. Q Appl Math 2: 164–168.

Lüddens H and Wisden W (1991) Function and pharmacology of multiple GABA_A receptor subunits. Trends Pharmacol Sci 12: 49–51.[CrossRef][Medline]

Lüddens H, Pritchett DB, Kohler M, Killisch I, Keinanen K, Monyer H, Sprengel R, and Seeburg PH (1990) Cerebellar GABA_A receptor selective for a behavioural alcohol antagonist. Nature 346: 648–651.[CrossRef][Medline]

Makela R, Uusi-Okari M, Homanics GE, Quinlan JJ, Firestone LL, Wisden W, and Korpi ER (1997) Cerebellar -aminobutyric acid type A receptors pharmacological subtypes revealed by mutant mouse lines. Mol Pharmacol 53: 380–388.

Makela R, Wisden W, and Korpi ER (1999) Loreclezole and La³⁺ differentiate cerebellar granule cell GABA_A receptor subtypes. Eur J Pharmacol 367: 101–105.[CrossRef][Medline]

Marquardt D (1963) An algorithm for least-squares estimation of nonlinear parameters. SIAM J Appl Math 11: 431–441.[CrossRef]

Martina M, Kilic G, and Cherubini E (1994) The effect of intracellular Ca²⁺ on GABA-activated currents in cerebellar granule cells in culture. J Membr Biol 142: 209–216.[Medline]

Marty MS, and Atchison WD (1997) Elevations of intracellular Ca²⁺ as a probably contributor to decreased viability in cerebellar granule cells following acute exposure to methylmercury. Toxicol App Pharmacol 150: 98–105.[CrossRef]

Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, et al. (1997) Sites of alcohol and volatile anaesthetic action on GABA_A and glycine receptors. Nature 389: 385–389.[CrossRef][Medline]

Nusser Z, Roberts JD, Baude A, Richards JG, and Somogyi P (1995) Relative densities of synaptic and extrasynaptic GABA_A receptors on cerebellar granule cells as determined by a quantitative immunogold method. J Neurosci 15: 2948–2960.[Abstract]

Otis TS and Mody I (1992) Modulation of decay kinetics and frequency of GABA_A receptor-mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons. Neuroscience 49: 13–32.[CrossRef][Medline]

Peng S, Hajela RK, and Atchison WD (2002) Effects of methylmercury on human neuronal L-type Ca²⁺ channels transiently expressed in human embryonic kidney cells (HEK-293). J Pharmacol Exp Ther 302: 424–432.[Abstract/Free Full Text]

Prybylowski K, Fu Z, Losi G, Hawkins LM, Lou J, Chang K, Wenthold RJ, and Vicini S (2002) Relationship between availability of NMDA receptor subunits and their expression at the synapse. J Neurosci 22: 8902–8910.[Abstract/Free Full Text]

Saxena NC (2000) Inhibition of GABA_A receptor (GABAR) currents by arachidonic acid in HEK-293 cells stably transfected with 1β22 GABAR subunits. Pflugers Arch 440: 380–392.[CrossRef][Medline]

Saxena NC and Macdonald RL (1994) Assembly of GABA_A receptor subunits: role of the subunit. J Neurosci 14: 7077–7086.[Abstract]

Saxena NC and Macdonald RL (1996) Properties of putative cerebellar -aminobutyric acid_A receptor isoforms. Mol Pharmacol 49: 567–579.[Abstract]

Saxena NC, Neelands TR, and Macdonald RL (1997) Contrasting actions of lanthanum on different recombinant -aminobutyric acid receptor isoforms expressed in L929 fibroblasts. Mol Pharmacol 51: 328–335.[Abstract/Free Full Text]

Siegel RE (1998) Developmental expression of cerebellar GABA_A-receptor subunit mRNAs. Perspect Dev Neurobiol 5: 207–217.[Medline]

Sieghart W (1992) GABA_A receptors: ligand-gated Cl^– ion channels modulated by multiple drug-binding sites. Trends Pharmacol Sci 13: 446–450.[CrossRef][Medline]

Thompson CL and Stephenson FA (1994) GABA_A receptor subtypes expressed in cerebellar granule cells: a developmental study. J Neurochem 62: 2037–2044.[Medline]

Thompson SA, Arden SA, Marshall G, Wingrove PB, Whiting PJ, and Wafford KA (1999) Residues in transmembrane domains I and II determine -aminobutyric acid type A receptor subtype-selective antagonism by furosemide. Mol Pharmacol 55: 993–999.[Abstract/Free Full Text]

Varecka L, Wu CH, Rotter A, and Frostholm A (1994) GABA_A/benzodiazepine receptor 6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice. J Comp Neurol 339: 341–352.[CrossRef][Medline]

Wisden W, Korpi ER, and Bahn S (1996) The cerebellum a model system for studying GABA_A receptor diversity. Neuropharmacology 35: 1139–1160.[CrossRef][Medline]

Yuan Y and Atchison WD (1993) Disruption by methylmercury of membrane excitability and synaptic transmission of CA1 neurons in hippocampal slices of the rat. Toxicol Appl Pharmacol 120: 203–215.[CrossRef][Medline]

Yuan Y and Atchison WD (1995) Methylmercury acts at multiple sites to block hippocampal synaptic transmission. J Pharmacol Exp Ther 275: 1308–1316.[Abstract/Free Full Text]

Yuan Y and Atchison WD (1997) Action of methylmercury on GABA_A receptor-mediated inhibitory synaptic transmission is primarily responsible for its early stimulatory effects on hippocampal CA1 excitatory synaptic transmission. J Pharmacol Exp Ther 282: 64–73.[Abstract/Free Full Text]

Yuan Y and Atchison WD (1999) Comparative effects of methylmercury on parallel-fiber and climbing-fiber responses of rat cerebellar slices. J Pharmacol Exp Ther 288: 1015–1025.[Abstract/Free Full Text]

Yuan Y and Atchison WD (2003) Methylmercury differentially affects GABA_A receptor-mediated spontaneous IPSCs in Purkinje and granule cells of rat cerebellar slices. J Physiol (Lond) 550: 191–204.[Abstract/Free Full Text]

Yuan Y and Atchison WD (2007) Methylmercury-induced increase of intracellular Ca²⁺ increases spontaneous synaptic current frequency in rat cerebellar slices. Mol Pharmacol 71: 1109–1121.[Abstract/Free Full Text]

Yuan Y, Otero JK, Yao AZ, Herden CJ, Sirois JE, and Atchison WD (2005) Inwardly rectifying and voltage-gated outward potassium channels exhibit low sensitivity to methylmercury. Neurotoxicology 26: 439–454.[CrossRef][Medline]

Zempel JM and Steinbach JH (1995) Neonatal rat cerebellar granule and Purkinje neurons in culture express different GABA_A receptors. Eur J Neurosci 7: 1895–1905.[CrossRef][Medline]

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