Introduction

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Organotins are widely used as heat stabilizers of polyvinyl chloride polymers, industrial catalysts in a variety of chemical reactions, and industrial and agricultural biocides (Luijten, 1971; Van der Kerk, 1978; Wilkinson, 1984). Of the known organotins, tri-n-butyltin (TBT) is an antifouling agent used in paints that prevents seaweed and shellfish from attaching to vessel bottoms due to its biocidal action, and it is now an environmental pollutant possessing a variety of toxic actions on mammals (Snoeij et al., 1987). The highest concentrations of TBT were found in the organs of wild animals (Yamamoto, 1994; Shawky and Emons, 1998; Shim et al., 1998). As a result, health concerns regarding TBT in both wildlife and humans are increasing. TBT is believed to exert an immunotoxic action rather than a neurotoxic action in mammals (Snoeij et al., 1987; Whalen et al., 1999). However, TBT has also been shown to be toxic to the developing nervous system in neonatal rats (O'Callaghan and Miller, 1988) and it causes significant changes in rat behavior (Ema et al., 1991a,b), thus indicating a neurotoxic action of TBT. In in vitro studies, TBT induces cell death in rat brain neurons at concentrations lower than those needed for the death of rat thymic lymphocytes (Ueha et al., 1996). The nanomolar concentrations of TBT increase the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) in dissociated rat brain neurons by increasing the membrane Ca²⁺ permeability and releasing Ca²⁺ from the intracellular stores (Oyama et al., 1993; Ueha et al., 1996). Ca²⁺ plays a critical role in the brain synaptic transmission (Zucker et al., 1991; Capogna, 1998; Sheng et al., 1998; Kirischunk et al., 1999). As a result, TBT may affect neurotransmission, leading to neurotoxic actions. However, little information is available on the TBT action on synaptic transmission. We herein describe our findings regarding mechanically dissociated neurons to evaluate the effect of TBT on the GABAergic transmission by using rat ventromedial hypothalamus (VMH) neurons, because these neurons have native synaptic nerve terminals called synaptic boutons, which are very suitable for studying the postsynaptic events elicited by native neurotransmitters released from nerve endings under optical voltage control (Akaike et al., 1992; Koyama et al., 1999).

	Materials and Methods

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Solutions. The standard external solution used in this study had the following composition: 150 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose. The pH was adjusted to 7.4 with Tris base. The composition of the internal patch pipette solution was 20 mM N-methyl-D-glucamine methanesulfonate, 20 mM Cs-methanesulfonate, 5 mM MgCl₂, 100 mM CsCl, 10 mM HEPES, and 0.2.mM nystatin. The pH was also adjusted to 7.4 with Tris base. Therefore, the equilibrium potential of Cl was determined to be 9.8 mV based on the Nernst relation.

Preparation. The methods to dissociate rat VMH neurons with native synaptic nerve terminals attached was previously described (Koyama et al., 1999). In brief, the VMH region was dissected from 500-µm-thick brain slices obtained from 2-week-old Wistar rats (Japan Charles River, Shizuoka, Japan). A fire-polished glass pipette was placed on the surface of VMH region perfused with standard external solution. To dissociate the VMH neurons, the pipette tip was horizontally vibrated at 3 to 5 Hz (100-200 µm) by a custom-built vibration device for 2 min. The brain slice was removed and the dissociated neurons were allowed to settle and adhered to the bottom of the culture dish within 20 min.

Electrical Measurements and Data Analysis. Electrical measurements were performed using a nystatin perforated patch recording mode under voltage-clamp conditions (Akaike and Harata, 1994). The membrane potential was controlled with a patch-clamp amplifier (CEZ-2300; Nihon Kohden, Tokyo, Japan) and membrane currents filtered at 1 kHz were monitored on both a storage oscilloscope (Textronix 5111A; Sony, Tokyo, Japan) and a pen recorder (Recti-Horiz 8K; Nippondenki San-Ei, Tokyo, Japan). Both the potential and current records were stored for further analyses on a digital audio tape recorder (RD-120; TEAC, Tokyo, Japan). Miniature inhibitory postsynaptic currents (mIPSCs) were analyzed using the pCLAMP software package (Axon Instruments, Burlingame, CA) and Mini Analysis software (Synaptosoft, Leonia, NJ). Amplitude histograms were constructed at 1.5-pA intervals. Cumulative amplitude histograms were compared using the Kolmogorov-Smirnov test (P < 0.05). The time-to-peak and time course of decay of individual mPSCs were analyzed using Mini Analysis. Numerical values are presented as mean ± standard error of the mean. Differences in amplitude and frequency distributions were compared using Student's paired two-sample t test. P < 0.05 was considered to be significant.

Drugs. TBT chloride was purchased from the Tokyo Kasei Co. (Tokyo, Japan). TBT (1 µM-1 mM) was initially dissolved in dimethyl sulfoxide (Wako Pure Chemicals, Osaka, Japan). The solution containing TBT was added into the standard external solution to achieve final concentrations. Dimethyl sulfoxide as a solvent at a final concentration (0.1%) did not affect any electrical measurements. Although the purity of the reagent was 98%, the effects were attributed to TBT because it is much more cytotoxic than possible contaminants such as di-n-butyltin and mono-n-butyltin. Other chemical reagents were purchased from the Sigma Chemical (St. Louis, MO) unless mentioned otherwise. All drugs were applied to VMH neurons by using a rapid "Y-tube" application technique, which allowed for complete solution changes surrounding the cells within 20 ms (Murase et al., 1990).

	Results

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Spontaneous mIPSCs of VMH Neurons. Experiments were conducted under the presence of tetrodotoxin, 2-amino-5-phosphonopentanoic acid, and 6-cyano-7-nitroquinoxaline-2,3-dione to pharmacologically isolate spontaneous GABAergic mIPSCs. Spontaneous mIPSCs were observed in mechanically dissociated rat VMH neurons with synaptic bouton(s) attached. Bicuculine (10 µM), an antagonist for GABA_A receptor, completely and reversibly suppressed the mIPSCs (Fig. 1A). When mIPSCs were recorded at various holding potentials, the current-voltage relationship for these mIPSCs reversed at 9.8 mV, the equilibrium potential of Cl (Fig. 1, B and C). These results suggest that the mIPSCs were elicited via the activation of GABA_A receptor-Cl channel complexes on VMH neurons. As a result, GABA released from the presynaptic bouton(s) participated in the activation of GABAergic mIPSCs.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Miniature postsynaptic currents elicited by the spontaneous release from nerve terminals projecting to the rat VMH neurons. A, recording of miniature postsynaptic currents before, during (as indicated with bar), and after the application of 30 µM bicuculline, a blocker of GABAA receptor. Bicuculline completely blocked the miniature postsynaptic currents in VMH neurons. B, miniature postsynaptic currents at various holding potentials. C, current-voltage relationship for the mean amplitude of miniature postsynaptic currents. The currents were reversed at a membrane potential near the equilibrium potential for Cl (ECl).

Effects of TBT on GABAergic mIPSCs. TBT at a concentration of 100 nM increased the frequency of GABAergic mIPSCs without affecting the current amplitude (Fig. 2, A and B). TBT at 1 µM further increased the frequency of mIPSCs but tended to decrease their amplitude. Furthermore, TBT induced a sustained inward current. This inward current persisted even after washing out the agent. The current was thus most likely independent from the transmitters released from synaptic boutons because the persistent inward current elicited by TBT was observed in neurons that had no synaptic nerve ending (data not shown). The micromolar concentrations of TBT seemed to deteriorate the postsynaptic membranes, thus resulting in a nonspecific increase in the membrane permeability. Figure 2B summarizes the concentration-dependent effects of TBT on the frequency and mean amplitude of mIPSCs. The threshold concentration of TBT to induce the changes in spontaneous mIPSCs was between 10 and 30 nM. TBT at 100 nM substantially increased the mIPSC frequency without altering the mean amplitude of the mIPSCs. TBT at 1 µM profoundly increased the mIPSC frequency, whereas it only slightly decreased the mean amplitude. These results indicate that TBT at nanomolar concentrations facilitates the GABA release by the presynaptic action at nerve terminals on VMH neurons.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Effect of tri-n-butyltin on mIPSCs. A, recordings of mIPSCs before, during (as indicated with bar), and after the application of tri-n-butyltin at 100 nM (top) and 1 µM (bottom). The dotted line (baseline, bottom) shows the control level of holding current. Tri-n-butyltin (1 µM) induced an inward current in nearly half of neurons examined. B, dose-dependent effects of tri-n-butyltin (1 nM-1 µM) on the frequency (left) and mean amplitude (right) of mIPSCs. The results were normalized to the frequency and mean amplitude of control mIPSCs. The symbol and bars show the mean and S.E.M. in four experiments. Dotted line indicates the control level. *P < 0.05 compared with control.

Further Analyses of TBT Actions on mIPSCs. Given that 100 nM TBT is found in the organs of some wild animals (Yamamoto, 1994; Shawky and Emons, 1998; Shim et al., 1998), we focused our attention on the actions on mIPSCs at this concentration. The responses such as Fig. 2A suggested a time-dependent change of the GABAergic mIPSC frequency during the course of the 100 nM TBT response. In more detail, the frequency of the mIPSCs increased throughout the exposure to TBT. The effect of TBT on the mIPSCs was reversible (Fig. 3A). A full recovery was observed at 20 min after the removal of TBT, but the time of recovery was longer after the application in some cells. Cumulative distribution of the mIPSC frequency before and during the application of 100 nM TBT (Fig. 3B) indicates that TBT greatly increased the frequency of mIPSCs.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Time course analysis of the effect of 100 nM tri-n-butyltin on the frequency of mIPSCs. A, tri-n-butyltin-induced change increase in the frequency of mIPSCs in every 10-s duration. The points are 10-s averages. The symbol and bar show the mean frequency of mIPSCs and S.E.M. before (), during (), and after () the application of 100 nM tri-n-butyltin. B, effect of 100 nM tri-n-butyltin on the cumulative frequency distribution.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Distribution analyses of the effects of 100 nM tri-n-butyltin on the mean amplitude of mIPSCs. A, amplitude histograms from the same neuron for the control () and application of 100 nM tri-n-butyltin (). B, effect of 100 nM tri-n-butyltin on the cumulative amplitude distribution.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of 100 nM tri-n-butyltin on the current kinetics of individual mIPSCs. A, superimposed current traces of individual mIPSC in absence (control) and presence of 100 nM tri-n-butyltin (tri-n-butyltin). Dotted line (baseline) indicates the control level of holding current. B, effects of 100 nM tri-n-butyltin on rise time (time to peak), fast and slow decay time constants of individual mIPSCs. Column and bar indicate the mean and S.E.M. of four to six experiments, respectively. Open and filled columns, respectively, indicate the times before and during the application of tri-n-butyltin.

Involvement of External Ca²⁺ in TBT-Induced Facilitation of GABAergic Transmission. To assess the role of external Ca²⁺ in TBT-induced increase in the frequency of GABAergic mIPSCs, the effect of TBT was examined in the Ca²⁺-free external solution. The mIPSC frequency decreased with no change in the current amplitude during exposure to Ca²⁺-free solutions (Fig. 6). The application of 100 nM TBT under Ca²⁺-free conditions failed to change the frequency of mIPSC (Fig. 6). Such results suggest that external Ca²⁺ is required for the TBT-induced increase in mIPSC frequency. Because the Ca²⁺ influx into the presynaptic nerve terminals through voltage-dependent Ca²⁺ channels is thought to play an important role in the TBT action, the effect of Cd²⁺, a nonspecific blocker of voltage-dependent Ca²⁺ channels, was examined. Cd²⁺ at 100 µM reduced both the frequency and the current amplitude of GABAergic mIPSCs (Fig. 7). The application of 100 nM TBT failed to increase the frequency of mIPSCs in the presence of Cd²⁺. These results suggest that TBT facilitates the GABA release by increasing the Ca²⁺ influx through the voltage-dependent Ca²⁺ channels of the nerve terminals.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Effects of 100 nM tri-n-butyltin on the frequency and mean amplitude of mIPSCs under the Ca2+-free condition. A, recordings of mIPSCs before, during (as indicated with bar), and after the application of tri-n-butyltin under Ca2+-free conditions. B, effect of tri-n-butyltin on cumulative amplitude distribution. C, effect of tri-n-butyltin on the mean amplitude (left) and frequency (right) of mIPSCs. The results were normalized to the frequency and mean amplitude of mIPSCs under normal conditions (2 mM Ca2+). The symbol and bar show the mean and S.E.M. in four experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effects of 100 nM tri-n-butyltin on the frequency and mean amplitude of mIPSCs under the suppression of voltage-dependent Ca2+ channels by 100 µM CdCl2. A, recordings of mIPSCs before, during (as indicated with bar), and after the application of tri-n-butyltin under the suppression of voltage-dependent Ca2+ channels. B, effect of tri-n-butyltin on the cumulative amplitude distribution. C, effect of tri-n-butyltin on the mean amplitude (left) and frequency (right) of mIPSCs. The results were normalized to the frequency and mean amplitude of mIPSCs under normal conditions.

	Discussion

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In Vitro Cytotoxicity of TBT. TBT at 30 to 100 nM increases the [Ca²⁺]_i of dissociated rat cerebellar neurons by increasing membrane Ca²⁺ permeability and releasing Ca²⁺ from the intracellular stores (Oyama et al., 1994a; Ueha et al., 1996). Increasing [Ca²⁺]_i seems to be one of common features in TBT-induced cytotoxicity because TBT increases the [Ca²⁺]_i in the lymphocytes (Chikahisa and Oyama, 1992; Chow et al., 1992), hepatocytes (Hechtenberg and Beyersmann, 1993; Reader et al., 1993), muscles (Kang et al., 1998), and cell line cells such as PC12 cells and HEL30 cells (Viviani et al., 1995; Corsini et al., 1997). The nanomolar concentrations of TBT to increase the frequency of mIPSCs (or to facilitate synaptic transmission) (Fig. 2) correspond closely to those to increase [Ca²⁺]_i (Chikahisa and Oyama, 1992; Chow et al., 1992; Hechtenberg and Beyersmann, 1993; Reader et al., 1993; Oyama et al., 1994b; Viviani et al., 1995; Corsini et al., 1997).

Environmentally Relevant Concentrations of TBT and Possible Neurotoxicity of TBT. Surveys of the organs of wild animals found wide-ranging concentrations of TBT: 27 to 202 ng/g in fish muscle, 54 to 223 ng/g in fish liver, 10 to 25 ng/g in common mussels, and 49 to 97 ng/g in bladderwracks (Shawky and Emons, 1998). Furthermore, even higher total concentrations of butyltin compounds, including mono-, di-, and tributyltin are accumulated in various wild animal populations: 115 to 1007 ng/g for common cormorants (Guruge et al., 1996), 8.5 to 2610 ng/g for river otters (Kannan et al., 1999), and 1200 to 2200 ng/g for dolphins (Kannan et al., 1996), respectively. Because the molecular weight of TBT is 290, the concentrations of TBT in organs of some animals presumably exceed 100 nM. Therefore, the nanomolar concentrations of TBT used in this study were relevant to those of TBT commonly accumulated in animal species. The concentration of TBT in human brains is difficult to assess, and no human death has been reported in individuals exposed to TBT. However, the daily human intake of TBT is estimated through market basket surveys (fishes and mollusks) to be 2.29 to 2.4 µg in Japan (Yamamoto, 1994; Sekizawa, 1998). Thus, TBT may be accumulated in some human organs at the nanomolar concentrations to induce some neurotoxic actions.