Differential Effects of Methylmercury on -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 6 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 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 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 timeand 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 6 subunit-containing GABAA receptors. Effects of MeHg on IGABA were similar in cells at all days. In human embryonic kidney 293 cells expressing either 6 or 1 subunit-containing GABAA receptors, time to block of IGABA by MeHg was comparable. Thus, the presence of the 6 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. Methylmercury (MeHg) is a well known environmental neurotoxicant. The cerebellum is especially sensitive to acute and chronic MeHg exposure; ataxia and impaired language development have both been described following MeHg poisoning (Bakir et al., 1973; Grandjean et al., 1998). In the cerebellar cortex, MeHg preferentially affects cerebellar granule cells over other cerebellar neurons including their neighboring Purkinje cells. This effect is not due to a difference in MeHg accumulation. Pathological examination showed that granule cells are grossly degenerated and lost, whereas Purkinje cells are less affected. However, the mechanisms underlying these differential pathological changes and sensitivity remain unclear. 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). GABAA receptor-mediated synaptic transmission is inhibited by MeHg. In dorsal root ganglion neurons in culture, high concentrations of MeHg (100 M) suppress the peak currents induced by GABA (Arakawa et al., 1991). In hippocampal slice, MeHg gradually decreases GABAA 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), sugThis study was supported by National Institutes of Health Grant R01ES11662. 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; IGABA, GABAA receptor-mediated currents; CNQX, 6-cyano-7nitroquinoxaline-2,3-dione disodium salt; APV, DL-2-amino-5-phosphonopentanoic acid; Ara-C, cytosine -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. 0022-3565/08/3242-517–528$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 324, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 123976/3294550 JPET 324:517–528, 2008 Printed in U.S.A. 517 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from gesting that inhibitory synaptic transmission is more sensitive to block by MeHg than is excitatory transmission. Block of inhibitory postsynaptic currents (IPSCs) induced by bathapplied MeHg in cerebellar granule cells in brain slice also occurs much earlier than does block in neighboring Purkinje cells (Yuan and Atchison, 2003). GABAA 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 GABAA 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 GABAA 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 GABAA 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). GABAA 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 Zn (Draguhn et al., 1990; Saxena and Macdonald, 1994, 1996; Zempel and Steinbach, 1995), furosemide (Korpi et al., 1995), and La (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 GABAA 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 GABAA receptors containing 1 or 6 subunits to bath-applied MeHg. Whole-cell voltage-clamp technique was used to compare the effects of MeHg on IGABA in cells expressing either 1 or 6 subunit-containing GABAA 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 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 M) 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 M) 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 M) 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