ToxSci Advance Access originally published online on November 15, 2006
Toxicological Sciences 2007 95(2):443-451; doi:10.1093/toxsci/kfl168
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Effects of Toluene Exposure during Brain Growth Spurt on GABAA Receptor–Mediated Functions in Juvenile Rats
,1
,1,2
* Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien 970, Taiwan R.O.C
Department of Pharmacy Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan R.O.C
Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan R.O.C
2 To whom correspondence should be addressed at Graduate Institute of Pharmacology and Toxicology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan R.O.C. Fax: +886 3-856-1465. E-mail: hwei{at}mail.tcu.edu.tw.
Received September 18, 2006; accepted November 10, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toluene is a commonly abused inhalant. Its neurobiological effects are, at least in part, mediated by gamma-aminobutyric acid (GABAA) receptors. Since GABAA receptor function is critical during brain development, the long-term effects of toluene exposure during brain growth spurt were investigated. Spargue-Dawley male rats were administered with toluene (500 mg/kg, i.p.) on postnatal day (PN) 4–9. Behavioral and electrophysiological measures and the levels of messenger RNA (mRNA) expression of GABAA receptor subunits were examined on PN 28–32. The seizure sensitivity induced by bicuculline (a GABAA receptor antagonist), methyl ß-carboline-3-carboxylate (inverse agonists of the GABAA/benzodiazepine receptor) but not 3-mercaptopropionic acid (a glutamate decarboxylase inhibitor) was enhanced by toluene exposure. Toluene exposure had no effect on the performance in the elevated plus-maze and rotarod test but reduced the responses to diazepam in these two tests. In vitro intracellular electrophysiological recordings employing brain slices from rats treated with toluene demonstrated a significant decrease in GABAA receptor–mediated inhibitory postsynaptic currents in CA1 neurons but an increased response to GABA perfusion. The relative abundance of the mRNAs encoding various subunits of GABAA receptor (
1, 2, 4, 5, 6, ß2, ß3, 
2S, 2L) was examined in four brain regions (hippocampus, striatum, cortex, and cerebellum) by semiquantitative reverse transcription-PCR. These results demonstrated that subunit- and brain area–selective alterations in GABAA receptors after toluene exposure during brain growth spurt. The alterations in GABAA receptors might be associated with the neurobehavioral disturbance in offspring of toluene-abusing women. Key Words: toluene; brain growth spurt; GABAA receptors; electrophysiology; behavior.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhalant abuse has increased dramatically especially in the young over the last decade globally. Survey results consistently show that nearly 20% of children in middle school and high school have experimented with inhaled substances (Anderson and Loomis, 2003
). Because large numbers of inhalant abusers are young women in their childbearing years, there are rising concerns about the potential negative impact of intentionally inhaled organic solvents on the unborns.
Toluene, one of the most commonly abused organic solvents, is present in paints, glues, gasoline, and cleaner. Abuse of toluene by pregnant women can lead to an embryopathy also referred to fetal solvent syndrome. Characteristics of toluene embryopathy include microcephaly, central nervous system dysfunction, attention deficits and hyperactivity, developmental delay with greater language deficits, minor craniofacial and limb anomalies, and variable growth deficiency (Arai et al., 1997; Hersh, 1989; Pearson et al., 1994). Nevertheless, not all exposed offspring show evident physical features and structural damage. Those who exposed lower doses of toluene might still have important but subtle impairment in synaptic circuitry, reflecting as neurobehavioral disturbance. However, neurodevelopmental evaluations of these children have not been reported.
Gamma-aminobutyric acid (GABAA) receptors are ligand-gated Cl– channels and mediated fast inhibitory transmission in the mammalian central nervous system (CNS). They are heteromeric complexes of five subunits that belong to various classes: 1–6, ß1–4, 1–3, 
, 
, 
, 
, and 
1–3 (Barnard et al., 1998; Sieghart and Sperk, 2002). These subunits are expressed in a region- and ontogeny-dependent manner in the brain and generate a large number of GABAA receptor subtypes that differ not only in subunit composition but in their physiological and pharmacological properties (Barnard et al., 1998; Hevers and Luddens, 1998). The GABAA receptors have been implicated in controlling the structure and plasticity of developing brain circuitry. The reports associated with the in vitro effect of toluene on GABAA receptors are contradictory. Toluene (420µM) enhances GABAA receptors (1, ß1, and2L subunits) heterologously expressed in Xenopus oocytes (Beckstead et al., 2000), whereas GABAA receptor–mediated currents in IMR-32 neuroblastoma cells (with1, 3, 4, ß1, ß3, 2, and subunits) are inhibited by toluene half maximal inhibitory concentration (IC 50 = 39 ± 6µM) (Meulenberg and Vijverberg, 2003). Prolonged treatment of primary cultures of rat hippocampal neurons with toluene (1mM; 4 days) reduced whole-cell responses to GABA (Bale et al., 2005). In addition, repeated inhalation of toluene (8000 ppm for 10 days, 30 min/day) results in increases in the GABAA receptor 1 subunit in the medial prefrontal cortex and striatum but decreases in the substantia nigra compacta and ventral tegmental area (Williams et al., 2005). Accordingly, it is suggested that GABAA receptor may play an important role in the pathophysiology of toluene-related neurodevelopmental disorders (Costa et al., 2002).
Recent studies have demonstrated that the pathological and behavioral effects of CNS acting chemicals, such as alcohol, MK-801, and diazepam, on the developing animals depend strongly on the developmental stage during exposure (Ikonomidou et al., 1999, 2000). The brain growth spurt is a dynamic period of CNS development that has been shown to be particularly vulnerable to a variety of neurotoxicants. The brain growth spurt occurs largely during the third trimester of human fetal development but occurs during the early postnatal period in the rats (Dobbing and Sands, 1979). Temporary loss or interference with the function of neurons with GABAA receptors following toluene exposure during brain growth spurt is likely to disturb the normal development of CNS, resulting in permanent neurobehavioral impairment. For example, neonatal exposure to diazepam, a GABAA acting reagent, induces a number of behavioral alterations, including hyperactivity/hyperarousal and reduced the level of anxiety at adulthood (Schroeder et al., 1997). We have found that exposure to toluene during brain growth spurt increased the seizure susceptibility induced by picrotoxin and pentylenetetrazole in juveniles (postnatal day [PN] 34–36) (Chen and Lee, 2002).
In order to elucidate the involvement of GABAA receptors in toluene-related neurobehavioral disorders, this study further addressed the changes in behavioral responses to GABAA receptor modulators, the GABAA receptor–mediated neurotransmission, and GABAA receptor subunit messenger RNA (mRNA) expression in juveniles following toluene exposure during brain growth spurt.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
Toluene (high-performance liquid chromatography grade, 99.8%) was obtained from Mallinckrodt Baker (Phillipsburg, NJ). SuperScript First-Strand synthesis system was purchased from Invitrogen (Carlsbad, CA). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO).
Animal treatment.
Pregnant female Sprague-Dawley rats were supplied from the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan). Rats were housed individually on a 12/12 light-dark cycle (lights on 0700 h) at 22°C. All experiments were performed in accordance with the Republic of China animal protection law (Chapter III: Scientific Application of Animals) and approved by Review Committee of the Tzu Chi University.
Toluene exposure during brain growth spurt.
The day of birth was considered to be PN 0. On PN 4, the litters were culled to 10–12 pups and each litter was randomly assigned to toluene or control group. The toluene animals received 500 mg/kg of toluene (0.1 g/ml in corn oil) by intraperitoneal injection and the control animals received corn oil (0.1 ml/10 g) daily over PN 4–9. A modified 26-G needle (6 mm long) was used for the pups to prevent tissue damage. The mother did not reject the pups treated with toluene. All the pups were weaned on PN 21. Seven litters were used for each treatment group. One male animal was randomly selected from each litter for each behavioral test, respectively. Each animal was only used for one test.
Rats subjected to a similar toluene exposure dose and paradigm (PN 4–9) manifested increasing N-methyl-D-aspartic acid (NMDA)-induced seizure susceptibility, reducing behavioral responses to NMDA antagonists, and blood toluene concentrations, from blood sample taken 1 and 3 h following the last injection of toluene, were 27.4 ± 5.1 µg/ml and 7.8 ± 1.5 µg/ml, respectively (Chien et al., 2005; Lee et al., 2005). These levels are in the range obtained from toluene abusers (0.1–74.7 µg/ml) (Garriott et al., 1981; Park et al., 1998; Zabedah et al., 2001). In addition, the placenta penetration efficiency for toluene is greater than 90% (Shumilina, 1991). Furthermore, the dose of toluene (500 mg/kg) used in the present studies produced locomotor hyperactivity, motor incoordination, and occasional ataxia in the adult rats similar to the behavioral signs evoked by toluene inhalation (5000–8000 ppm) (Beyer et al., 2001; Hinman, 1987). Thus, our treatment protocol is able to mimic the toluene exposure during synapotogenesis for the fetus of pregnant toluene abuser.
Seizure induction.
Control and toluene-exposed rats were taken from their home cages for seizure induction. The experimenter was blinded to the neonatal treatment at the time of seizure induction. Convulsants were administered via a lateral tail vein. The infusion pump (L-1800, Kd Scientific Co., MA) was used for infusion and the infusion rate was 0.5 ml/min. The convulsants and concentrations infused were bicuculline (0.2 mg/ml), methyl ß-carboline-3-carboxylate (ß-CCM, 0.5 mg/ml), and 3-mercaptopropionic acid (3-MPA, 24 mg/ml). The animals were weighed and placed in an acrylic chamber with numerous holes for ventilation. The tail of the rat was warmed for 1 min in warm (45°C) water. The scalp vein sets (Nipro Co., Japan) with 25-gauge butterfly infusion needle were used. The needle was inserted in the lateral tail vein and correct placement was verified by the appearance of blood in the infusion tubing. The needle was fixed to the tail with an adhesive tape, and the animal was then released into a 23 x 8x 6 cm3 (l x wx h) plastic cage to allow free movement. The animal was observed throughout infusion and the time between the start of infusion and onset of four convulsion signs was record in seconds and subsequently converted to threshold convulsant dosage (i.e., milligram of drug per kilogram of body weight), based on infusion rate, body weight, and latency. Threshold dosage (mg/kg) = latency (min) x infusion rate (ml/min) x convulsant concentration (mg/ml) /body weight (kg).
Timed tail vein infusion allows for observation and qualitative analysis of several different convulsion end points. Briefly, clonus indicates rapid rhythmic movements due to alternating contraction and relaxation of muscles, whereas tonus indicates rigidity due to contraction of muscles. Four convulsion signs, which occur in progression, characterize bicuculline-induced seizures: myoclonic (MC) twitch (sudden involuntary muscle jerk); face and forelimb (FF) clonus (rapid writhing movements of the head and neck); running and bouncing (RB) clonus (whole-body clonus, including running and jumps); and tonic hindlimb extension (THE) (extreme rigidity, with forelimbs and hindlimbs extended caudally). ß-CCM only resulted in MC twitch and FF clonus. On the other hand, two convulsion signs which reliably characterize MPA-induced seizures are RB clonus and THE.
Elevated plus-maze.
The plus-maze was constructed of Plexiglas and consisted of two open and two closed arms (10 cm wide x 50 cm long, 50 cm walls for closed, 2 cm walls for open), intersected by a center platform (100 cm2), elevated 50 cm off the floor. Rats were brought to the testing room for 1 h before testing. Each rat was tested for 5 min on the maze and videotaped; a rat was placed on the central platform of the maze. The following indices were recorded: the total number of entries into open arm and closed arm and the total time spent in each type of arm. An entry was defined as the entry of all four feet into one arm. From these values, the percentage of open-arm entries and time and the total number of entries were calculated for each animal. Between tests, the maze was wiped clean.
Diazepam (2 mg/kg) anxiolytic effect was tested on the elevated plus-maze. Control and toluene-exposed rats were injected (i.p.) with diazepam 30 min before testing; injection volume was 2 ml/kg. Diazepam was dissolved in saline with Tween 80 (one drop/5 ml). Rats were tested for 5 min each.
Rotarod motor coordination test.
Motor coordination was assessed by means of an automated rotarod (TSE systems, Germany). The rotarod apparatus was accelerated gradually from 4 to 75 rpm (0.5 rpm/s). Each rat was placed on the rod and the trial ended when the rat fell. Four trials per rat were conducted on each day for 3 days. A computer recorded the latency to fall (in seconds).
The effects of neonatal toluene exposure on diazepam-induced motor incoordination were examined using a constant speed of 15 rpm. Before drug testing, the control and toluene-exposed rats with previous 3-day assessing were able to spend on the rod at least for 3 min. All the rats were tested again 30 min after diazepam (3 mg/kg, i.p.) treatment.
In vitro electrophysiology, stimulation, and drug application.
Experiments were performed on hippocampal slices obtained from control or toluene-exposed rats aged 28–32 PN days (PN 28–32). The brain was quickly removed from the skull and placed in an ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl 120, KCl 3.5, MgCl2 1.2, CaCl2 2.5, NaH2PO4 1.2, glucose 11.5, NaHCO3 25, saturated with 95% O2 and 5% CO2, pH 7.4. Transverse hippocampal slices (500 µm) were cut with a vibratome and stored at room temperature in holding the same ACSF solution as above. After a recovery period of at least 1 h, an individual slice was transferred to the recording chamber where it was continuously superfused with oxygenated ACSF at a rate 2–3 ml/min. CA1 pyramidal neurons were voltage clamped at 0 mV to record GABAA receptor–mediated inhibitory postsynaptic currents (IPSCs). Patch pipettes were filled with a solution containing (in mM): K gluconate 122, NaCl 5, CaCl2 0.3; MgCl2 2, ethylene glycol tetra acetic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Na2-adenosine triphosphate 5, Na-guanosine triphosphate 2, amphotericin B 0.4 (pH 7.25, resistance 9–12 M
).
Orthodromic stimuli were delivered with square-wave pulses (4–16 V; 0.1 ms) via a concentric bipolar electrode placed in stratum radiatum to activated Schaffer collaterals. Current signals and applied voltages were generated and recorded with an Axoclamp 200B amplifier (Axon Instruments, CA). Whole-cell recording are acquired with a switch frequency of 5–6 kHz (30% duty cycle), gain of 3–8 nA/mV, time constant 20 ms. Tracings shown in figures represent the average of three consecutive sweep. Output signals were stored on an IBM-compatible computer after digitalization with a DigiData-1200 Series Interface using acquisition and analysis software (pClamp, v. 8.10). DNQX (50µM), D-APV (25µM), and CGP35348 (200µM) were present together as a cocktail in order to isolate GABAA receptor–mediated monosynaptic fast IPSCs. GABA applied by bath superfusion to achieve steady-state concentrations within the 1.0-ml recording chamber.
Isolation of total RNA from various brain areas.
Immediately after seizure test, rats were decapitated and brains were quickly removed. Hippocampus, cortex, striatum, and cerebellum were rapidly dissected on ice. Total RNA of each brain area from different control and toluene-treated rats was isolated using acid guanidinium/phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). It was precipitated twice in ethanol and then dissolved in diethlpyrocarbonate-treated water.
Reverse transcription-PCR quantification.
Quantification of relative mRNA expression of subunit and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control was done by reverse-transcription PCR (RT-PCR). Two micrograms of total RNA from each animal was reverse transcribed to complementary DNA (cDNA) using SuperScript First-Strand synthesis system for RT-PCR using random hexamer primer. The nucleotide sequences and expected RT-PCR product sizes from primer sets for GADPH and GABAR subunits 1, 2, 4, 5, 6, ß2, ß3, 2S, 2L, and 3 were designed using Primer 3 software and given in Table 1. Each PCR reaction was carried out in a volume of 20 µl using bulk master mixes except template cDNA prepared from multiple reactions. The concentration of starting cDNA to be amplified for each subunit was determined by building a standard curve for each gene plotting the density of PCR product against the amount of template cDNA. There was a linear region in which the density of PCR was directly proportional to the amount of template cDNA. To relate the expression of the gene of interest to that of the endogenous reference gene, a ratio was determined between the amount of PCR product within the linear amplification range of the target gene and the endogenous reference gene; this ratio, compared among different cDNAs, provided a relative gene expression level. PCR cycle (single hot start at 94°, 8.5 min at 94°, 30 s at 50°, and 45 s at 72°) was conducted for 30 cycles; the series of PCR calculated were always prepared from the same master mix, cDNA stock. Each PCR reaction contains 1.25 units of AmpliTaq Gold, 2.5mM MgCl2, 1x PCR buffer, 200µM deoxy-nucleotide triphosphate mix (Watson Biotech, Taiwan), 12.5 pmol for each primer. After PCR amplification, reactions were run on 2.5% agarose gel in 1x Tris/acetate/ethylenediaminetetraacetic acid buffer stained with 0.5 µg/ml ethidium bromide and then densitometric analysis of bands for specific gene and internal control were done with UN-SCAN-IT gel automated digitizing system software (Silk Scientific Corporation, UT). The ratios of the band intensity of GABAA receptor subunits to GADPH were calculated.
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Data analysis.
The electrophysiological data were analyzed by repeat measures two-way ANOVA with post hoc Bonferroni test. Data from elevated maze were analyzed by Mann-Whitney test. All other data were compared by Student's t-test. A value of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body Weight Gain and Developmental Landmarks
During the time of toluene exposure, the body weight gain of the toluene-exposed (12.6 ± 1.2 g) and control rats (12.7 ± 1.3 g) was similar. As shown in our previous study (Lee et al., 2005
), toluene-exposed rats did not show any significant difference from control rats in the developmental landmarks including lower incisor eruption, eyelid opening, and air righting examined at PN 10, PN 12, and PN 15, respectively (data not shown). Toluene did not affect the locomotor activity tested on PN 30 (data not shown).
Sensitivity to Seizures Induced by GABAA Receptor Acting Agents
As shown in Table 2, rats that had been exposed to toluene during brain growth spurt demonstrated that all seizure thresholds and lethal doses for bicuculline and FF clonus for ß-CCM were declined. However, the seizure thresholds for MPA were not affected.
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Anxiety on Elevated Plus-Maze: Effect of Diazepam
Using the elevated plus-maze assay, we have compared the basal anxiety of toluene-exposed rats with that of control rats. As shown in Figure 1 there is no significant difference in the time spent in open-arms and total arm entries between two groups. Elevated plus-maze assay was also performed 30 min after animals received diazepam. Since the experience of elevated plus-maze significantly affected the performance, other animals different from basal condition were used. In both groups, diazepam significantly increased the time spent in open arms without effects on total arm entries. However, the diazepam-induced anxiolytic effect (increasing time spent in open arm) in toluene-exposed rats was less effective than that in control rats.
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Rotarod Motor Coordination Test: Effects of Diazepam
There were no significant effects of toluene exposure during brain growth spurt on the performance in the rotarod test over the 3 days of training in the accelerating rotarod from 4 to 75 rpm (0.5 rpm/s) (Fig. 2A). All rats were able to spend on the rod at least for 3 min using a constant speed of 15 rpm before administration of diazepam. Diazepam significantly reduced the latency of rats stay on the rotarod. The diazepam-induced motor incoordination was less severe in toluene-exposed rats compared to control rats (Fig. 2B).
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In Vitro Intracellular Electrophysiological Recordings
The effect of toluene exposure during brain growth spurt on GABAA receptor–mediated inhibitory response was examined at PN 28–32. Two-way repeated measures ANOVA revealed a main effect of group (F1,96 = 4.53, p = 0.049) and stimulus intensity (F6,96 = 15.5, p < 0.001) with no significant interaction (F6,96 = 1.35, p = 0.24). Post hoc analysis between the groups indicated significant decreases in responses to 10–14 mV stimuli in the slice from toluene-exposed rats (Fig. 3). However, as shown in Figure 4, toluene exposure enhanced the response to exogenous application of the GABA (group: F1,52 = 55.1, p < 0.001; concentration: F2,52 = 16.26, p < 0.001; interaction: F2,52 = 0.03, p = 0.97).
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RT-PCR Measurements of GABAA Receptor Subunit Composition
To determine the effects of toluene exposure during brain growth spurt on the expression of GABAA receptor subunits, RT-PCR measurements were performed in cortex, hippocampus, striatum, and cerebellum. The expression patterns of GABAA receptor subunits in the rat brain are in a region- and age-specific manner (Fritschy and Mohler, 1995
; Fritschy et al., 1994; Laurie et al., 1992; Wisden et al., 1992). Each mRNA displayed a unique distribution. Some subunits were narrowly confined in certain brain regions, for example, 6 mRNA is present only in cerebellum and cochlea nuclei (Varecka et al., 1994). Therefore, only the abundantly expressed subunits in each brain region were detected and presented. As shown in Figure 5, the abundance of the 1 and 2 subunit mRNAs was significantly increased, whereas ß2 was reduced in the cortex. The abundance of the 5, 2S, 2L, and 3 subunit mRNAs was markedly elevated in the hippocampus. The amounts of the 2, ß2, 2S, and 2L were increased in the striatum. In the cerebellum, the levels of 1, 2, 4, 6, 2S, and 2L were increased.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, the elevation of seizure susceptibility induced by picrotoxin, a blocker binding to a convulsant site within the chloride channel of the GABAA receptor complex, after toluene exposure during brain growth spurt has been observed (Chen and Lee, 2002
). The present study further showed that toluene exposure during brain growth spurt enhanced seizure susceptibility induced by bicuculline, a blocker for GABA binding site in GABAA receptor, and ß-CCM, a benzodiazepine inverse agonist. On the other hand, toluene reduced behavioral sensitivity to anxiolytic and motor impairment effects of diazepam, a benzodiazepine agonist. Based on these remarkable changes in pharmacological responsiveness to GABAA receptor modulators, we speculated that the electrophysiological function of GABAA receptors and the abundance of GABAA receptor subunits might also be changed. Our data have shown that a diminished GABAA receptor–mediated synaptic inhibitory response and enhanced the response to exogenous application of the GABA in hippocampal CA1 neurons and the levels of GABAA receptor subunit mRNAs specifically altered in certain brain regions.
In the seizure susceptibility tests, toluene exposure during brain growth spurt enhanced seizure susceptibility, that is, lowered seizure thresholds, induced by bicuculline and ß-CCM, whereas the seizure sensitivity to 3-MPA, a glutamate decarboxylase inhibitor, was unaffected. Since bicuculline is a blocker for GABA binding site in GABAA receptors, and ß-CCM acts as a benzodiazepine inverse agonist, it appears that toluene exposure during brain growth spurt alters the properties of GABAA receptors. It has been reported that seizure susceptibility to ß-CCM is reduced after treatment of antisense oligodeoxynucleotide directed against the 2 subunit (Zhao et al., 1996). Accordingly, the increased levels of 2 subunit mRNA in the hippocampus, striatum and cerebellum might contribute to the enhanced seizure susceptibility for ß-CCM in toluene-exposed rats. On the other hand, no difference of 3-MPA–induced seizure thresholds between control and toluene-exposed rats suggests that toluene exposure did not alter the availability of synaptic GABA and glutamate decarboxylase activity.
Diazepam is used clinically for its myorelaxant, anxiolytic, sedative, and anticonvulsant properties. Our results showed reduced behavioral responses in the elevated plus-maze and rotarod test in the toluene-exposed rats upon diazepam challenge, indicating reduced sensitivity to the anxiolytic-like and motor impairing effects of diazepam. The possible explanation for this change in diazepam sensitivity might be related to the diminished GABA concentration, GABA/benzodiazepine receptor density, or/and neurosteroid release (Briones-Aranda et al., 2005; Ouardouz and Sastry, 2006). According to the findings that toluene has no effect on 3-MPA–induced seizure thresholds, it appears that toluene exposure might not modify the availability of synaptic GABA and glutamate decarboxylase activity. It is possible but still remains to be clarified whether toluene exposure can influence GABA transports, leading to the changes in endogenous GABA levels or interfere with the neurosteroid release.
It is now understood that the sedative and anxiolytic effects of benzodiazepine drugs are mainly mediated by 1- and 2-containing GABAA receptors, respectively (Low et al., 2000; McKernan et al., 2000). In fact, the reduced diazepam sensitivity might be associated with reduced levels of 1 and 2 subunits (Bailey and Toth, 2004). Contradictorily, significantly increased 1 and 2 GABAA receptor mRNA levels in the cortex, striatum, and cerebellum were observed in toluene-exposed rats. Further comprehensive studies involved in GABAA receptor subunit protein expression and the levels of GABA and neurosteroids are needed to clarify the mechanisms that are responsible for the diazepam insensitivity induced by toluene exposure during brain growth spurt.
It is noted that toluene exposure during brain growth spurt resulted in a reduced GABA A receptor–mediated postsynaptic response, while yielding an increased response to application of GABA in the hippocampal CA1 pyramidal neurons. This differential functionality of GABAA receptors following toluene exposure could be obtained only if these GABAA receptors were different. Several lines of evidence from electron microscopic and electrophysiological studies show that distinct GABAA receptor isoforms are present at synapses and in the extrasynaptic membrane, and these receptor subpopulations mediate two different forms of inhibition: tonic inhibition and synaptic inhibition, respectively (Semyanov et al., 2004; Soltesz and Nusser, 2001; Yeung et al., 2003). Thus, such a difference might result from the activation of synaptic as well as extrasynaptic receptors by exogenous application of GABA, while orthodromic stimulation would be expected to activate predominately synaptic GABAA receptors. Thus, toluene exposure might cause an adaptation to downregulate the synaptic GABAA receptors, while at the same time upregulating the extrasynaptic receptors. In fact, in hippocampal pyramidal neurons the 5 subunit-containing GABAA receptors are largely extrasynaptic (Caraiscos et al., 2004; Liang et al., 2004; Scimemi et al., 2005). Consistently, the levels of 5 subunit were elevated in the hippocampus after toluene exposure, which may contribute to the increased extrasynaptic activation in response to application of GABA.
Additionally, it has been suggested that altered balance between extrasynaptic and synaptic receptors affects seizure sensitivity to GABAergic convulsants (Sinkkonen et al., 2004). The enhancement in seizure sensitivity to GABAergic convulsants observed in toluene-exposed rats is likely attributed, at least in part, to the changes in extrasynaptic and synaptic GABAA receptor functions in the hippocampus. Further studies on areas implicated more directly in anxiety, such as mesolimbic structures, and motor coordination, such as cerebellum, are needed to elucidate the association between the changes in behaviors and GABAA receptor dysfunction.
It has been demonstrated that toluene exposure during synapotogenesis enhances seizure susceptibility to NMDA and reduces the behavioral responses to NMDA receptor antagonists, such as MK-801 or ketamine (Chien et al., 2005; Lee et al., 2005). The present study also showed enhanced the seizure susceptibilities to GABAA receptor antagonists and reduced the behavioral responses to diazepam, an allosteric modulator at GABAA receptors following toluene exposure during brain growth spurt. Given the multiple action sites of toluene, it is not surprised to find that both glutamatergic and GABAergic systems are implicated in the developmental neurotoxicity of toluene. Our findings provide the first evidence that toluene exposure during brain growth spurt can lead to a long-term effect on GABAA receptors in rats. The observed changes occurred in GABA receptors as well as NMDA receptors might be implicated in toluene-related neurodevelopmental disorders.
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NOTES |
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Conflict of interest: This work was supported by a grant (NHRI-EX91-9112NC) from National Health Research Institute, Taiwan to H.H.C. and a grant (TCMRC92211-01) from the intramural fund of Tzu Chi University to C.L.L.
1 Contributed equally to this work. 
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ACKNOWLEDGMENTS |
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This work was supported by a grant (NHRI-EX91-9112NC) from National Health Research Institute, Taiwan to H.H.C. and a grant (TCMRC92211-01) from the intramural fund of Tzu Chi University to C.L.L.
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