Neonatal Toluene Exposure Alters Agonist and Antagonist Sensitivity and NR2B Subunit Expression of NMDA Receptors in Cultured Cerebellar Gra
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《毒物学科学杂志》
Graduate Institute of Pharmacology and Toxicology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan
Department of Pharmacology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan
Graduate Institute of Medical Sciences, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan
1 Correspondence should be addressed to Ming-Huan Chan at Graduate Institute of Pharmacology and Toxicology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan. Fax: 886-3-856-1465. E-mail: ming@mail.tcu.edu.tw.
ABSTRACT
Toluene has been reported to antagonize the function of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of neonatal toluene exposure on NMDA receptors in primarily cultured cerebellar granule neurons were examined. Spargue-Dawley rats were treated with toluene (0, 200, 500, and 1000 mg/kg, i.p.) from postnatal day (PN) 4 to PN 7. Under toluene-free conditions, Ca2+ signals of cultured neurons in response to glutamate and NMDA were measured for up to 14 days. The expression of NMDA receptor subunits (NR1, NR2A, and NR2B) at 5–14 days in vitro (DIV) were also determined. Neonatal toluene exposure dose-dependently reduced intracellular Ca2+ signals in response to glutamate/glycine and NMDA/glycine in cultured cerebellar granule neurons, and these effects were gradually decreased with time. Such toluene exposure did not influence the inhibition of Mg2+ or MK801 on NMDA-evoked responses, but it decreased the potency of ifenprodil (an NR2B preferring antagonist). The protein levels of NMDA receptor subunit NR2B were consistently reduced by toluene exposure at 5 DIV, but not at 14 DIV. These results demonstrate that neonatal toluene exposure induces long-term but reversible changes in the function and composition of NMDA receptors. Such changes during developmental stages may contribute to the cerebellar dysfunction observed in fetal solvent syndrome.
Key Words: toluene; neonatal; NMDA receptor; Ca2+; NR2B; cerebellar granule neurons.
INTRODUCTION
Toluene is an organic solvent widely used in industry. In addition to occupational and environmental exposure, toluene has become popular as a recreational drug; "solvent sniffing" or other inhalation of toluene in commercial products has been reported. Abuse of toluene by pregnant women can lead to toluene embryopathy or fetal solvent syndrome. Growth retardation and microencephaly are the major effects seen in newborns, accompanied by a number of dysmorphic features such as deep-set eyes, low-set ears, flat nasal bridge, micrognathia, and small fingernails. As the children mature, developmental delay, language impairment, hyperactivity, cerebellar dysfunction, and postnatal growth retardation become evident (Costa et al., 2002; Hersh, 1989; Hersh et al., 1985).
The expressions of N-methyl-D-aspartate (NMDA) receptors, which appear very early during development, and their activity are important for neuronal development. The mediator of its effects is the cytoplasmic concentration of free ionic Ca2+, which activates a number of cytosolic and nuclear transcription pathways. The regulation of intracellular Ca2+ is known to have an essential role in central nervous system (CNS) development in the very early stages of neuronal generation and differentiation (Cambray-Deakin and Burgoyne, 1992; Komuro and Rakic, 1993). Toluene has been reported to inhibit the NMDA receptors (Cruz et al., 1998). Because the modulation of Ca2+ entry through NMDA receptors is an essential mediator during brain development, toluene-induced alterations in NMDA-mediated Ca2+ signals could have an important role in the CNS damage leading to fetal solvent syndrome.
To assess the effects of developmental toluene exposure on NMDA-mediated Ca2+ signals in developing cerebellar granule neurons, we used an experimental model that included in vivo toluene exposure and examination of the NMDA-mediated Ca2+ signals by single-cell Ca2+ imaging in cultured cerebellar granule neurons. Because the neonatal period of development in the cerebellum in rats corresponds to the last trimester in humans, toluene was administered to rat pups during postnatal days (PN) 4–7. Cerebella were removed from the animals and the neurons were dissociated and cultured at PN 8. Primary culture of cerebellar granule neurons obtained from 8-day-old rat pups is a widely used experimental model. Importantly, the developmental profile of the NMDA receptor subunits in the cultured cerebellar granule neurons is similar to that in vivo (Cathala et al., 2000; Popp et al., 1999). This treatment paradigm is more reflective of that occurring with human exposure and the dissociated neurons can be maintained in culture for several weeks. Moreover, these experiments allow us to observe the functional changes in NMDA receptors at various time periods of recovery after toluene exposure. In the present study, the toluene-induced changes of NMDA receptor properties was determined by examining the Ca2+ signals in response to glutamate, NMDA, and glycine [a necessary co-activator of NMDA receptors by binding to a distinct recognition site on the NR1 subunit (Hirai et al., 1996)] in developing cerebellar granule neurons at a serial time points in culture. Furthermore, the inhibitory profiles of Mg2+, MK801, and ifenprodil, as well as the levels of NMDA receptor subunits such as NR1, NR2A, and NR2B in cultured cerebellar granule neurons from toluene-exposed and control pups, were also compared.
MATERIALS AND METHODS
Materials.
Toluene (HPLC grade) was purchased from Mallinckrodt Baker (Paris, KY). Fura 2-AM (fura-2 acetoxymethyl ester) was obtained from Fluka Chemical Co. (Milwaukee, WI). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Animals.
Sprague-Dawley rats were supplied from the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan). The experimental protocol was approved by the Review Committee of the Tzu Chi University for the Use of Animal Subjects. The day of birth was considered to be postnatal day (PN) 0. On PN 4, the litters were culled to 10–12 pups, and the pups within the same litter were randomly assigned to toluene or control group. The toluene animals
Cell culture.
Cerebellar granule cells were isolated from control and toluene-treated pups at PN 8 using a slight modification of the procedure described by Zhu and Baker (1996). Briefly, neurons were dissociated from freshly dissected cerebella, first by aspirating through an 18-gauge needle and then through a 22-gauge needle without enzymatic treatment; they were then seeded onto poly-L-lysine–coated 20-mm-diameter coverslips. Cells were seeded at a density of 2 x 106 cells per coverslip in basal essential medium supplemented with 10% fetal calf serum (FCS), 25 mM KCl, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2. Cytosine arabinoside (10 μM) was added to the culture medium 18–24 h after plating to arrest the growth of non-neuronal cells.
Intracellular Ca2+ image.
The fluorescent probe fura 2-AM (Fluka Chemical Corp. Milwaukee, WI) was used for monitoring intracellular calcium concentrations. Cerebellar granule neurons grown on poly-L-lysine–coated 20-mm-diameter coverslips were initially washed with Mg-free buffer solution (NaCl 140 mM, KCl 3.5 mM, KH2PO4 0.4 mM, Na2HPO4 0.33 mM, CaCl2 2.2 mM, glucose 10 mM, HEPES 10 mM, pH 7.4) and then incubated with 10 μM fura 2-AM at 37°C for 60 min. After the free fura 2-AM was washed out, the coverslip was mounted in a homemade perfusion chamber and then on the stage of an inverted epifluorescence microscope (Olympus IX70, Japan) equipped with a 40x fluorescence oil objective (Dapo 40, Olympus, Japan). During the subsequent calcium-imaging experiments, those cells were continuously superfused with buffer solution at room temperature (20°–25°C). Cells were visualized with a Quantix EEV57 intensified charged-coupled device (CCD) video camera and image intensifier system (Photometrics Ltd., Munich, Germany). Data were collected and stored with an image acquisition program Imaging Workbench 4 (Axon Instruments, Inc.). The data were also analyzed with Imaging Workbench 4 software, which allowed the regions of interest (ROI) drawn around each cell to be moved between frames. The size and shape of the ROI were not changed at any point for any given cell.
The fura 2 cytosolic calcium measurements were performed using the video-based digital calcium imaging system set-up for dual excitation at 340 and 380 nm and emission at 510 nm. The cytosolic calcium results were presented as the relative fluorescent intensities of the 340 and 380 nm images and as the 340:380 ratios (F340/F380).
Drug application.
Cerebellar granule neurons were stimulated with various concentrations of glutamate (0.3–100 μM) and 5 μM glycine, various concentrations of glycine (0.1–10 μM) and 5 μM glutamate, or various concentrations of NMDA (1–100 μM) and 5 μM glycine. During blocking experiments, NMDA (100 μM )/glycine (5 μM) and various concentrations of MgSO4 (1–1000 μM), MK801 (0.1–1 μM), and ifenprodil (0.1–10 μM) were applied by microperfusion.
Data analysis.
Ca2+ signals were quantified by measurement of the peak amplitude (minus baseline) in the somatic region. Data from every experiment were collected from 8 litters; both control and toluene-exposed animals from each litter were used to prepare the respective cultures. Three to four microscopic fields containing approximately 8–10 neurons were measured in each culture. The average value of the neurons from the same litter for each experimental condition was taken as the unit of analysis.
Western blot.
Cells from 100-mm culture dishes were washed and harvested into ice-cold phosphate-buffered saline (pH 7.5). After centrifugation (5000 x g, 20 min), cells were homogenized in ice-cold homogenization buffer (50 mM Tris-HCl, 5 mM EDTA, 10 mM EGTA, 0.3% [w/v] -mercaptoethanol, 10 μg/ml leupetin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). The protein concentrations of homogenates were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were boiled for 5 min after dilution with sample buffer. Proteins were separated by electrophoresis through a 7.5 % SDS-polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. The NMDA receptor subunits were detected by the amplified alkaline phosphatase Immuno-Blot assay system (Bio-Rad). The membranes were incubated with 5% non-fat dry milk in Tris-buffered saline (TBS: 20 mM Tris, 500 mM NaCl, pH 7.5) containing 0.05% Tween-20 (TTBS). The membranes were then incubated with primary antibody (anti-NR1, anti-NR2A, anti-NR2B, Upstate Group, Inc., Charlottesville, VA) at appropriate dilutions in TTBS overnight at room temperature. After the membrane was washed with TTBS, secondary antibody (goat anti-rabbit IgG conjugated with biotin) was added for 1 h. The membranes were washed with TTBS and then incubated with streptavidin-biotinylated alkaline phosphatase complex for 1 h. After extensive washing with TTBS, the immunoreactive bands were visualized with 5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium (BCIP/NBT) phosphatase substrate. Immunoreactive bands were scanned and analyzed with a digital scanning densitometer (personal densitometer, Molecular Dynamics, Sunnyvale, CA). Concentrations of NMDA receptor subunits in analyzed samples were within a linear range of density that increased in a concentration-dependent manner (data not shown). Values for toluene-exposed rats were expressed as values relative to the average of controls, set at 100%.
Statistics.
The mean values of data were expressed as means ± S.E.M. The litter was considered as the unit of analysis. One-way analysis of variance (ANOVA) was used to analyze the brain weight. Two-way ANOVA was performed on data for body weight (toluene treatment x time), cluster size (toluene treatment x days in vitro), the effects of blockers on NMDA-stimulated Ca2+ signals (toluene treatment x concentrations of blocker), and Western blot (toluene treatment x days in vitro). Ca2+ signals in response to glutamate, NMDA, and glycine were analyzed by three-way ANOVA with toluene treatment, concentrations of stimulant, and days in vitro as three factors. The Student-Newman-Keuls test was used for post hoc comparisons; p < 0.05 was taken to be statistically significant.
RESULTS
Effects of Toluene Exposure on Body Weight and Brain Weight of Rat Pups
As shown in Figure 1, the body weight gain from rats exposed to 1000 mg/kg toluene was significantly lower than that from control rats, and no effect was observed in rats exposed to 200 and 500 mg/kg toluene. The brain weight of rats exposed to 500 and 1000 mg/kg toluene at PN 8 were lower than control (control: 0.74 ± 0.02 g; toluene 200 mg/kg: 0.72 ± 0.02 g; toluene 500 mg/kg: 0.68 ± 0.02 g; toluene 1000 mg/kg: 0.62 ± 0.02 g).
Morphological Features of the Granule Neuron Development in Culture
Granule neurons obtained from PN 8 rat cerebella and plated on poly-L-lysine–coated 20-mm-diameter coverslips showed a distinct developmental pattern of morphological changes in culture. After dissociation, the neurons were round in shape. During the first day in culture they attached to the substrate, extended processes, and started migration. Gradually, clusters of cerebellar granule neurons formed, and by 6 days in vitro (DIV), prominent fiber tracks were observed. The fiber tracks and neuronal clusters increased in size with culture age before 7 DIV. These developmental changes were similar to granule neuron cultures from control and toluene-exposed pups. Neuronal cluster size was also similar to cultures from control and toluene-treated animals (Table 1). Results suggested that toluene treatment prior to culturing did not change sensitivity to the culture procedure.
Ca2+ Signals in Response to Glutamate and NMDA
The Ca2+ signals in response to serial dilutions of glutamate (0.1–10 μM) and NMDA (1–200 μM) were measured in the presence of 5 μM glycine in granule neuron cultures at 5, 7, 9, and 14 DIV. As shown in Figures 2 and 3, glutamate and NMDA significantly increased the Ca2+ signals in a concentration- and time-dependent manner. These observations were confirmed by three-way ANOVA. Analysis of the peak amplitude of the Ca2+ signals stimulated by glutamate indicated a significant difference in the effects of toluene (F3,560 = 31.18, p < 0.001), glutamate (F4,560 = 446.08, p < 0.001) and days in vitro (F3,560 = 15.29, p < 0.001). There is a statistically significant interaction between toluene and glutamate (p 0.001) as well as between toluene and days in vitro (p = 0.013) and glutamate and days in vitro (p = 0.037). Similarly, analysis of the peak amplitude of the Ca2+ signals stimulated by NMDA indicated a significant difference in the effects of toluene (F3,560 = 43.82, p < 0.001) and NMDA (F4,560 = 769.78, p < 0.001), but not days in vitro (F3,560 = 2.0, p = 0.113). There is a statistically significant interaction between toluene and NMDA (p < 0.001) and between toluene and days in vitro (p < 0.001), indicating that the effect of different levels of toluene depends on the level of NMDA and the number of days in vitro.
Further analysis revealed that the Ca2+ signals in response to glutamate were significantly smaller in cultured granule neurons from toluene-exposed pups than in cultured granule neurons from control pups at 5, 7, and 9 DIV. At 14 DIV, the reduction in glutamate-induced and NMDA-induced Ca2+ signal responses was not significant.
Ca2+ Signals in Response to Glycine
The Ca2+ signals in response to various concentrations of glycine (0.1–10 μM) were measured with the presence of 5 μM glutamate in granule neuron cultures at 5, 7, 9, and 14 DIV. A glycine-concentration response curve is shown in Figure 4. Three-way ANOVA indicated a significant difference in the effects of toluene (F3,560 = 6.83, p < 0.001), glycine (F4,560 = 255.56, p < 0.001), and days in vitro (F3,560 = 15.0, p < 0.001). There is also a statistically significant interaction between toluene and days in vitro (p < 0.001). The peak amplitude (resting levels subtracted) from the Ca2+ signals was significantly lower in cultured granule neurons from toluene-exposed pups than in those from control pups at any number of days in vitro.
Effect of Mg2+on NMDA-Stimulated Ca2+ Signals
The voltage-dependent block of NMDA receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The degree of blockade produced by Mg2+ was performed at 5 DIV. As shown in Figure 5A, MgSO4 (1–100 mM) inhibited the NMDA-stimulated Ca2+ signals in a concentration-dependent manner. Two-way ANOVA indicated that there is a significant difference among the different levels of MgSO4 (F3,112 = 401.4, p < 0.001), but no significant difference among the different levels of toluene (F3,112 = 0.84, p = 0.174) and no significant interaction between toluene and MgSO4 (p = 0.93). Thus, the degree of blockade produced by MgSO4 was not different in cultured granule neurons from toluene-exposed pups compared to cultured granule neurons from control pups at 5 DIV.
Effect of MK801 on NMDA-Stimulated Ca2+ Signals
MK801 is a non-competitive NMDA channel antagonist that was found to bind the channel site. As shown in Figure 5B, MK801 inhibited the NMDA-stimulated Ca2+ signals in a concentration-dependent manner. Two-way ANOVA indicated that there is a significant difference among the different levels of MK801 (F1,42 = 260.3, p < 0.001), but no significant difference among the different levels of toluene (F2,42 = 0.104, p = 0.902) and no significant interaction between toluene and MK801 (p = 0.83). Thus, the degree of blockade produced by MK801 was not different in cultured granule neurons from toluene-exposed pups compared to cultured granule neurons from control pups at 5 DIV.
Effect of Ifenprodil on NMDA-Stimulated Ca2+ Signals
Ifenprodil is a non-competitive antagonist that has been found to selectively inhibit the NMDA receptors that contain the NR2B subunit (Williams, 1993). The concentrations of ifenprodil (0.1–10 μM) used in the current studies were chosen to act preferentially at the high-affinity ifenprodil site, i.e., the site associated with the NR2B subunit. As shown in Figure 5C, ifenprodil inhibited the NMDA-stimulated Ca2+ signals in a concentration-dependent manner in cultured granule neurons from control and toluene-exposed pups. Two-way ANOVA indicated that there is a significant difference among the different levels of ifenprodil (F2,63 = 40.69, p < 0.001) and toluene (F2,63 = 9.457, p < 0.001), and significant interaction between toluene and ifenprodil (p = 0.017). Ifenprodil is less potent in cultured granule neurons from toluene-exposed pups than in cultured granule neurons from control pups at 5 DIV.
Protein Levels of NR1, NR2A, and NR2B Subunits
Western blot analyses were used to compare the NR1, NR2A, and NR2B subunits in cultured granule neurons from toluene-exposed pups and in those from control pups at 5 and 14 DIV (Fig. 6). As previously reported (Hoffmann et al., 2000), the levels of NR1 and NR2A, but not NR2B, significantly increased from 5 DIV to 14 DIV. A densitometric analysis showed that NR1 and NR2A levels were not affected by toluene, whereas the expression of NR2B protein was significantly reduced in cultured granule neurons from toluene-exposed pups at 5 DIV (Fig. 6A and 6B) but not at 14 DIV (Fig. 6C).
DISCUSSION
Toluene exposure of rat pups during postnatal development dose-dependently reduced the Ca2+ signals stimulated by glutamate and NMDA in cultured cerebellar granule neurons derived from these animals, and the effects were gradually alleviated over time. Toluene exposure also reduced the Ca2+ signals in response to glycine, but this inhibition did not occur in a dose- and time-dependent manner. In addition, toluene exposure did not affect the inhibition of Mg2+ or MK801 on NMDA/glycine induced response, but it did reduce the inhibitory potency of ifenprodil, a NR2B subunit selective antagonist. Furthermore, a lower level of NR2B subunit was observed in the cultured neurons from toluene-exposed pups. Taken together, these results indicate that toluene exposure at a critical developmental stage would change the function and subunit composition of NMDA receptors.
In the current study, toluene exposure during PN 4–7 was shown to produce lower brain weight in toluene-exposed pups, reflecting a condition commonly observed in humans with fetal solvent syndrome. However, treatment of the pups with toluene did not appear to influence the developmental pattern or the size of the granule neuron clusters formed in culture. This lack of effect was similar to the results of neonatal alcohol exposure studies conducted with the same exposure paradigm (Gruol et al., 1998). The standard growth medium for granule neurons in cultures contains a relatively high concentration of K+. The K+ depolarizes the neurons resulting in activation of the voltage-dependent Ca2+ channels, resulting in Ca2+ influx and increases in intracellular Ca2+ levels. The higher intracellular Ca2+ level is thought to be a substitute for tropic factors needed for growth and development of the granule neurons (Gallo et al., 1987). Whether an alternative medium containing a lower concentration of K+ may influence the toluene's effects remains to be investigated.
The effects of several antagonists including Mg2+, MK801, and ifenprodil on NMDA-stimulated Ca2+ signals were studied. Toluene exposure did not affect Mg2+ and MK801 sensitivity. Only a significant reduction in ifenprodil sensitivity was observed. The binding sites for Mg2+ and MK801 are in the channel pore regions, and the subtype-specific differences of Mg2+ and MK801 blockade are determined by the NR2 subunits (Kuner and Schoepfer 1996; Laurie and Seeburg, 1994). The present data show that toluene did not alter the actions of Mg2+ and MK801 in the presence of 100 μM NMDA and 5 μM glycine, suggesting that the channel pore regions are not influenced when the channels are fully open. It is known that NMDA receptors are composed of the obligatory NR1 subunit and at least one copy of NR2A, NR2B, NR2C, or NR2D. The presence of specific NR2 subunits determines the properties of receptors, including agonist affinity, magnesium sensitivity, deactivation kinetics, modulation by polyamines, and channel conductance (McBain and Mayer, 1994; Mori and Mishina, 1995). The sensitivity of ifenprodil, a NR2B selective antagonist, is positively dependent on the proportion of NR2B in the cultured neurons (Bhave et al., 1999). Consistent with the reduction of ifenprodil sensitivity, we found that toluene exposure produced a decrease in the level of NR2B subunits. In addition, the function of NMDA receptors containing NR2B subunits is most sensitive to toluene (Cruz et al., 1998). Taken together, the NMDA receptors with NR2B subunits may be the main target of toluene during CNS development. Toluene may directly affect their functions during exposure and selectively result in downregulation of the NR2B subunit expression after toluene withdrawal in cultured condition. However, it is still unclear how toluene can cause downregulation of the NR2B subunit expression.
We examined the NMDA-mediated Ca2+ signals at 5, 7, 9, and 14 DIV. The differences between cultured granule neurons from control and toluene-exposed pups were gradually reduced. Previous studies of the developmental profile of NMDA receptor subunit expression in cerebellar granule neurons indicated that there was no change in NR1, a significant increase in NR2A, and a decrease in NR2B on days 4–14 in vitro (Snell et al., 2001; Vallano et al., 1996). Because the reduction of NR2B subunit expression in the cultured cerebellar granule neurons from toluene-exposed pups was earlier than the normal "developmental switch" of the NR2A and NR2B subunits, the gradually reduced influence of toluene on the Ca2+ signals in response to glutamate and NMDA from 5 DIV to 14 DIV may be associated with the decrease in NR2B expression. Alternatively, phosphorylation and the redox state of NMDA receptors and the associated NMDA receptor modulator protein (e.g., PSD-95) are also well-established biochemical mechanisms for modulation of the receptor response (Iwamoto et al., 2004; Koles et al., 2001; Sinor et al., 1997; Yamada et al., 1999). It is possible that changes in phosphorylation and oxidation state of NMDA receptors or NMDA receptor modulator protein may contribute to the toluene-induced alterations in the sensitive response to glutamate or antagonists observed here.
It is noteworthy that granule neurons from toluene-exposed pups also reduced Ca2+ signals in response to glycine in the presence of 5 μM glutamate, but the effect did not occur in a dose- and time-dependent manner. The NMDA receptor glycine binding site is located on the NR1 subunit (Hirai et al., 1996; Ivanovic et al., 1998). There was no change in the NR1 subunit during development of cerebellar granule neurons. Our data also show the lack of effect of toluene on NR1 subunit protein expression. Hence, it may explain why there was no time-dependent effect of toluene on reduction of Ca2+ signals in response to glycine. Furthermore, the presence of 5 μM glutamate under our experimental conditions may be involved in the similar inhibitory effects of distinct doses of toluene on Ca2+ signals.
In summary, our results suggest that a toluene-induced reduction in Ca2+ signals linked to NMDA receptors, probably through altering the subunit compositions of NMDA receptors, could disrupt many aspects of CNS neuronal functions and thereby have an important part in the CNS damage done to infants by maternal toluene abuse.
ACKNOWLEDGMENTS
This work was supported by a grant (NHRI-EX92-9112NC) from the National Health Research Institute, Taiwan, and a grant (TCMRC91129) from the intramural fund of Tzu Chi University.
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Department of Pharmacology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan
Graduate Institute of Medical Sciences, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan
1 Correspondence should be addressed to Ming-Huan Chan at Graduate Institute of Pharmacology and Toxicology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970, Taiwan. Fax: 886-3-856-1465. E-mail: ming@mail.tcu.edu.tw.
ABSTRACT
Toluene has been reported to antagonize the function of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of neonatal toluene exposure on NMDA receptors in primarily cultured cerebellar granule neurons were examined. Spargue-Dawley rats were treated with toluene (0, 200, 500, and 1000 mg/kg, i.p.) from postnatal day (PN) 4 to PN 7. Under toluene-free conditions, Ca2+ signals of cultured neurons in response to glutamate and NMDA were measured for up to 14 days. The expression of NMDA receptor subunits (NR1, NR2A, and NR2B) at 5–14 days in vitro (DIV) were also determined. Neonatal toluene exposure dose-dependently reduced intracellular Ca2+ signals in response to glutamate/glycine and NMDA/glycine in cultured cerebellar granule neurons, and these effects were gradually decreased with time. Such toluene exposure did not influence the inhibition of Mg2+ or MK801 on NMDA-evoked responses, but it decreased the potency of ifenprodil (an NR2B preferring antagonist). The protein levels of NMDA receptor subunit NR2B were consistently reduced by toluene exposure at 5 DIV, but not at 14 DIV. These results demonstrate that neonatal toluene exposure induces long-term but reversible changes in the function and composition of NMDA receptors. Such changes during developmental stages may contribute to the cerebellar dysfunction observed in fetal solvent syndrome.
Key Words: toluene; neonatal; NMDA receptor; Ca2+; NR2B; cerebellar granule neurons.
INTRODUCTION
Toluene is an organic solvent widely used in industry. In addition to occupational and environmental exposure, toluene has become popular as a recreational drug; "solvent sniffing" or other inhalation of toluene in commercial products has been reported. Abuse of toluene by pregnant women can lead to toluene embryopathy or fetal solvent syndrome. Growth retardation and microencephaly are the major effects seen in newborns, accompanied by a number of dysmorphic features such as deep-set eyes, low-set ears, flat nasal bridge, micrognathia, and small fingernails. As the children mature, developmental delay, language impairment, hyperactivity, cerebellar dysfunction, and postnatal growth retardation become evident (Costa et al., 2002; Hersh, 1989; Hersh et al., 1985).
The expressions of N-methyl-D-aspartate (NMDA) receptors, which appear very early during development, and their activity are important for neuronal development. The mediator of its effects is the cytoplasmic concentration of free ionic Ca2+, which activates a number of cytosolic and nuclear transcription pathways. The regulation of intracellular Ca2+ is known to have an essential role in central nervous system (CNS) development in the very early stages of neuronal generation and differentiation (Cambray-Deakin and Burgoyne, 1992; Komuro and Rakic, 1993). Toluene has been reported to inhibit the NMDA receptors (Cruz et al., 1998). Because the modulation of Ca2+ entry through NMDA receptors is an essential mediator during brain development, toluene-induced alterations in NMDA-mediated Ca2+ signals could have an important role in the CNS damage leading to fetal solvent syndrome.
To assess the effects of developmental toluene exposure on NMDA-mediated Ca2+ signals in developing cerebellar granule neurons, we used an experimental model that included in vivo toluene exposure and examination of the NMDA-mediated Ca2+ signals by single-cell Ca2+ imaging in cultured cerebellar granule neurons. Because the neonatal period of development in the cerebellum in rats corresponds to the last trimester in humans, toluene was administered to rat pups during postnatal days (PN) 4–7. Cerebella were removed from the animals and the neurons were dissociated and cultured at PN 8. Primary culture of cerebellar granule neurons obtained from 8-day-old rat pups is a widely used experimental model. Importantly, the developmental profile of the NMDA receptor subunits in the cultured cerebellar granule neurons is similar to that in vivo (Cathala et al., 2000; Popp et al., 1999). This treatment paradigm is more reflective of that occurring with human exposure and the dissociated neurons can be maintained in culture for several weeks. Moreover, these experiments allow us to observe the functional changes in NMDA receptors at various time periods of recovery after toluene exposure. In the present study, the toluene-induced changes of NMDA receptor properties was determined by examining the Ca2+ signals in response to glutamate, NMDA, and glycine [a necessary co-activator of NMDA receptors by binding to a distinct recognition site on the NR1 subunit (Hirai et al., 1996)] in developing cerebellar granule neurons at a serial time points in culture. Furthermore, the inhibitory profiles of Mg2+, MK801, and ifenprodil, as well as the levels of NMDA receptor subunits such as NR1, NR2A, and NR2B in cultured cerebellar granule neurons from toluene-exposed and control pups, were also compared.
MATERIALS AND METHODS
Materials.
Toluene (HPLC grade) was purchased from Mallinckrodt Baker (Paris, KY). Fura 2-AM (fura-2 acetoxymethyl ester) was obtained from Fluka Chemical Co. (Milwaukee, WI). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Animals.
Sprague-Dawley rats were supplied from the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan). The experimental protocol was approved by the Review Committee of the Tzu Chi University for the Use of Animal Subjects. The day of birth was considered to be postnatal day (PN) 0. On PN 4, the litters were culled to 10–12 pups, and the pups within the same litter were randomly assigned to toluene or control group. The toluene animals
Cell culture.
Cerebellar granule cells were isolated from control and toluene-treated pups at PN 8 using a slight modification of the procedure described by Zhu and Baker (1996). Briefly, neurons were dissociated from freshly dissected cerebella, first by aspirating through an 18-gauge needle and then through a 22-gauge needle without enzymatic treatment; they were then seeded onto poly-L-lysine–coated 20-mm-diameter coverslips. Cells were seeded at a density of 2 x 106 cells per coverslip in basal essential medium supplemented with 10% fetal calf serum (FCS), 25 mM KCl, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2. Cytosine arabinoside (10 μM) was added to the culture medium 18–24 h after plating to arrest the growth of non-neuronal cells.
Intracellular Ca2+ image.
The fluorescent probe fura 2-AM (Fluka Chemical Corp. Milwaukee, WI) was used for monitoring intracellular calcium concentrations. Cerebellar granule neurons grown on poly-L-lysine–coated 20-mm-diameter coverslips were initially washed with Mg-free buffer solution (NaCl 140 mM, KCl 3.5 mM, KH2PO4 0.4 mM, Na2HPO4 0.33 mM, CaCl2 2.2 mM, glucose 10 mM, HEPES 10 mM, pH 7.4) and then incubated with 10 μM fura 2-AM at 37°C for 60 min. After the free fura 2-AM was washed out, the coverslip was mounted in a homemade perfusion chamber and then on the stage of an inverted epifluorescence microscope (Olympus IX70, Japan) equipped with a 40x fluorescence oil objective (Dapo 40, Olympus, Japan). During the subsequent calcium-imaging experiments, those cells were continuously superfused with buffer solution at room temperature (20°–25°C). Cells were visualized with a Quantix EEV57 intensified charged-coupled device (CCD) video camera and image intensifier system (Photometrics Ltd., Munich, Germany). Data were collected and stored with an image acquisition program Imaging Workbench 4 (Axon Instruments, Inc.). The data were also analyzed with Imaging Workbench 4 software, which allowed the regions of interest (ROI) drawn around each cell to be moved between frames. The size and shape of the ROI were not changed at any point for any given cell.
The fura 2 cytosolic calcium measurements were performed using the video-based digital calcium imaging system set-up for dual excitation at 340 and 380 nm and emission at 510 nm. The cytosolic calcium results were presented as the relative fluorescent intensities of the 340 and 380 nm images and as the 340:380 ratios (F340/F380).
Drug application.
Cerebellar granule neurons were stimulated with various concentrations of glutamate (0.3–100 μM) and 5 μM glycine, various concentrations of glycine (0.1–10 μM) and 5 μM glutamate, or various concentrations of NMDA (1–100 μM) and 5 μM glycine. During blocking experiments, NMDA (100 μM )/glycine (5 μM) and various concentrations of MgSO4 (1–1000 μM), MK801 (0.1–1 μM), and ifenprodil (0.1–10 μM) were applied by microperfusion.
Data analysis.
Ca2+ signals were quantified by measurement of the peak amplitude (minus baseline) in the somatic region. Data from every experiment were collected from 8 litters; both control and toluene-exposed animals from each litter were used to prepare the respective cultures. Three to four microscopic fields containing approximately 8–10 neurons were measured in each culture. The average value of the neurons from the same litter for each experimental condition was taken as the unit of analysis.
Western blot.
Cells from 100-mm culture dishes were washed and harvested into ice-cold phosphate-buffered saline (pH 7.5). After centrifugation (5000 x g, 20 min), cells were homogenized in ice-cold homogenization buffer (50 mM Tris-HCl, 5 mM EDTA, 10 mM EGTA, 0.3% [w/v] -mercaptoethanol, 10 μg/ml leupetin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). The protein concentrations of homogenates were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were boiled for 5 min after dilution with sample buffer. Proteins were separated by electrophoresis through a 7.5 % SDS-polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. The NMDA receptor subunits were detected by the amplified alkaline phosphatase Immuno-Blot assay system (Bio-Rad). The membranes were incubated with 5% non-fat dry milk in Tris-buffered saline (TBS: 20 mM Tris, 500 mM NaCl, pH 7.5) containing 0.05% Tween-20 (TTBS). The membranes were then incubated with primary antibody (anti-NR1, anti-NR2A, anti-NR2B, Upstate Group, Inc., Charlottesville, VA) at appropriate dilutions in TTBS overnight at room temperature. After the membrane was washed with TTBS, secondary antibody (goat anti-rabbit IgG conjugated with biotin) was added for 1 h. The membranes were washed with TTBS and then incubated with streptavidin-biotinylated alkaline phosphatase complex for 1 h. After extensive washing with TTBS, the immunoreactive bands were visualized with 5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium (BCIP/NBT) phosphatase substrate. Immunoreactive bands were scanned and analyzed with a digital scanning densitometer (personal densitometer, Molecular Dynamics, Sunnyvale, CA). Concentrations of NMDA receptor subunits in analyzed samples were within a linear range of density that increased in a concentration-dependent manner (data not shown). Values for toluene-exposed rats were expressed as values relative to the average of controls, set at 100%.
Statistics.
The mean values of data were expressed as means ± S.E.M. The litter was considered as the unit of analysis. One-way analysis of variance (ANOVA) was used to analyze the brain weight. Two-way ANOVA was performed on data for body weight (toluene treatment x time), cluster size (toluene treatment x days in vitro), the effects of blockers on NMDA-stimulated Ca2+ signals (toluene treatment x concentrations of blocker), and Western blot (toluene treatment x days in vitro). Ca2+ signals in response to glutamate, NMDA, and glycine were analyzed by three-way ANOVA with toluene treatment, concentrations of stimulant, and days in vitro as three factors. The Student-Newman-Keuls test was used for post hoc comparisons; p < 0.05 was taken to be statistically significant.
RESULTS
Effects of Toluene Exposure on Body Weight and Brain Weight of Rat Pups
As shown in Figure 1, the body weight gain from rats exposed to 1000 mg/kg toluene was significantly lower than that from control rats, and no effect was observed in rats exposed to 200 and 500 mg/kg toluene. The brain weight of rats exposed to 500 and 1000 mg/kg toluene at PN 8 were lower than control (control: 0.74 ± 0.02 g; toluene 200 mg/kg: 0.72 ± 0.02 g; toluene 500 mg/kg: 0.68 ± 0.02 g; toluene 1000 mg/kg: 0.62 ± 0.02 g).
Morphological Features of the Granule Neuron Development in Culture
Granule neurons obtained from PN 8 rat cerebella and plated on poly-L-lysine–coated 20-mm-diameter coverslips showed a distinct developmental pattern of morphological changes in culture. After dissociation, the neurons were round in shape. During the first day in culture they attached to the substrate, extended processes, and started migration. Gradually, clusters of cerebellar granule neurons formed, and by 6 days in vitro (DIV), prominent fiber tracks were observed. The fiber tracks and neuronal clusters increased in size with culture age before 7 DIV. These developmental changes were similar to granule neuron cultures from control and toluene-exposed pups. Neuronal cluster size was also similar to cultures from control and toluene-treated animals (Table 1). Results suggested that toluene treatment prior to culturing did not change sensitivity to the culture procedure.
Ca2+ Signals in Response to Glutamate and NMDA
The Ca2+ signals in response to serial dilutions of glutamate (0.1–10 μM) and NMDA (1–200 μM) were measured in the presence of 5 μM glycine in granule neuron cultures at 5, 7, 9, and 14 DIV. As shown in Figures 2 and 3, glutamate and NMDA significantly increased the Ca2+ signals in a concentration- and time-dependent manner. These observations were confirmed by three-way ANOVA. Analysis of the peak amplitude of the Ca2+ signals stimulated by glutamate indicated a significant difference in the effects of toluene (F3,560 = 31.18, p < 0.001), glutamate (F4,560 = 446.08, p < 0.001) and days in vitro (F3,560 = 15.29, p < 0.001). There is a statistically significant interaction between toluene and glutamate (p 0.001) as well as between toluene and days in vitro (p = 0.013) and glutamate and days in vitro (p = 0.037). Similarly, analysis of the peak amplitude of the Ca2+ signals stimulated by NMDA indicated a significant difference in the effects of toluene (F3,560 = 43.82, p < 0.001) and NMDA (F4,560 = 769.78, p < 0.001), but not days in vitro (F3,560 = 2.0, p = 0.113). There is a statistically significant interaction between toluene and NMDA (p < 0.001) and between toluene and days in vitro (p < 0.001), indicating that the effect of different levels of toluene depends on the level of NMDA and the number of days in vitro.
Further analysis revealed that the Ca2+ signals in response to glutamate were significantly smaller in cultured granule neurons from toluene-exposed pups than in cultured granule neurons from control pups at 5, 7, and 9 DIV. At 14 DIV, the reduction in glutamate-induced and NMDA-induced Ca2+ signal responses was not significant.
Ca2+ Signals in Response to Glycine
The Ca2+ signals in response to various concentrations of glycine (0.1–10 μM) were measured with the presence of 5 μM glutamate in granule neuron cultures at 5, 7, 9, and 14 DIV. A glycine-concentration response curve is shown in Figure 4. Three-way ANOVA indicated a significant difference in the effects of toluene (F3,560 = 6.83, p < 0.001), glycine (F4,560 = 255.56, p < 0.001), and days in vitro (F3,560 = 15.0, p < 0.001). There is also a statistically significant interaction between toluene and days in vitro (p < 0.001). The peak amplitude (resting levels subtracted) from the Ca2+ signals was significantly lower in cultured granule neurons from toluene-exposed pups than in those from control pups at any number of days in vitro.
Effect of Mg2+on NMDA-Stimulated Ca2+ Signals
The voltage-dependent block of NMDA receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The degree of blockade produced by Mg2+ was performed at 5 DIV. As shown in Figure 5A, MgSO4 (1–100 mM) inhibited the NMDA-stimulated Ca2+ signals in a concentration-dependent manner. Two-way ANOVA indicated that there is a significant difference among the different levels of MgSO4 (F3,112 = 401.4, p < 0.001), but no significant difference among the different levels of toluene (F3,112 = 0.84, p = 0.174) and no significant interaction between toluene and MgSO4 (p = 0.93). Thus, the degree of blockade produced by MgSO4 was not different in cultured granule neurons from toluene-exposed pups compared to cultured granule neurons from control pups at 5 DIV.
Effect of MK801 on NMDA-Stimulated Ca2+ Signals
MK801 is a non-competitive NMDA channel antagonist that was found to bind the channel site. As shown in Figure 5B, MK801 inhibited the NMDA-stimulated Ca2+ signals in a concentration-dependent manner. Two-way ANOVA indicated that there is a significant difference among the different levels of MK801 (F1,42 = 260.3, p < 0.001), but no significant difference among the different levels of toluene (F2,42 = 0.104, p = 0.902) and no significant interaction between toluene and MK801 (p = 0.83). Thus, the degree of blockade produced by MK801 was not different in cultured granule neurons from toluene-exposed pups compared to cultured granule neurons from control pups at 5 DIV.
Effect of Ifenprodil on NMDA-Stimulated Ca2+ Signals
Ifenprodil is a non-competitive antagonist that has been found to selectively inhibit the NMDA receptors that contain the NR2B subunit (Williams, 1993). The concentrations of ifenprodil (0.1–10 μM) used in the current studies were chosen to act preferentially at the high-affinity ifenprodil site, i.e., the site associated with the NR2B subunit. As shown in Figure 5C, ifenprodil inhibited the NMDA-stimulated Ca2+ signals in a concentration-dependent manner in cultured granule neurons from control and toluene-exposed pups. Two-way ANOVA indicated that there is a significant difference among the different levels of ifenprodil (F2,63 = 40.69, p < 0.001) and toluene (F2,63 = 9.457, p < 0.001), and significant interaction between toluene and ifenprodil (p = 0.017). Ifenprodil is less potent in cultured granule neurons from toluene-exposed pups than in cultured granule neurons from control pups at 5 DIV.
Protein Levels of NR1, NR2A, and NR2B Subunits
Western blot analyses were used to compare the NR1, NR2A, and NR2B subunits in cultured granule neurons from toluene-exposed pups and in those from control pups at 5 and 14 DIV (Fig. 6). As previously reported (Hoffmann et al., 2000), the levels of NR1 and NR2A, but not NR2B, significantly increased from 5 DIV to 14 DIV. A densitometric analysis showed that NR1 and NR2A levels were not affected by toluene, whereas the expression of NR2B protein was significantly reduced in cultured granule neurons from toluene-exposed pups at 5 DIV (Fig. 6A and 6B) but not at 14 DIV (Fig. 6C).
DISCUSSION
Toluene exposure of rat pups during postnatal development dose-dependently reduced the Ca2+ signals stimulated by glutamate and NMDA in cultured cerebellar granule neurons derived from these animals, and the effects were gradually alleviated over time. Toluene exposure also reduced the Ca2+ signals in response to glycine, but this inhibition did not occur in a dose- and time-dependent manner. In addition, toluene exposure did not affect the inhibition of Mg2+ or MK801 on NMDA/glycine induced response, but it did reduce the inhibitory potency of ifenprodil, a NR2B subunit selective antagonist. Furthermore, a lower level of NR2B subunit was observed in the cultured neurons from toluene-exposed pups. Taken together, these results indicate that toluene exposure at a critical developmental stage would change the function and subunit composition of NMDA receptors.
In the current study, toluene exposure during PN 4–7 was shown to produce lower brain weight in toluene-exposed pups, reflecting a condition commonly observed in humans with fetal solvent syndrome. However, treatment of the pups with toluene did not appear to influence the developmental pattern or the size of the granule neuron clusters formed in culture. This lack of effect was similar to the results of neonatal alcohol exposure studies conducted with the same exposure paradigm (Gruol et al., 1998). The standard growth medium for granule neurons in cultures contains a relatively high concentration of K+. The K+ depolarizes the neurons resulting in activation of the voltage-dependent Ca2+ channels, resulting in Ca2+ influx and increases in intracellular Ca2+ levels. The higher intracellular Ca2+ level is thought to be a substitute for tropic factors needed for growth and development of the granule neurons (Gallo et al., 1987). Whether an alternative medium containing a lower concentration of K+ may influence the toluene's effects remains to be investigated.
The effects of several antagonists including Mg2+, MK801, and ifenprodil on NMDA-stimulated Ca2+ signals were studied. Toluene exposure did not affect Mg2+ and MK801 sensitivity. Only a significant reduction in ifenprodil sensitivity was observed. The binding sites for Mg2+ and MK801 are in the channel pore regions, and the subtype-specific differences of Mg2+ and MK801 blockade are determined by the NR2 subunits (Kuner and Schoepfer 1996; Laurie and Seeburg, 1994). The present data show that toluene did not alter the actions of Mg2+ and MK801 in the presence of 100 μM NMDA and 5 μM glycine, suggesting that the channel pore regions are not influenced when the channels are fully open. It is known that NMDA receptors are composed of the obligatory NR1 subunit and at least one copy of NR2A, NR2B, NR2C, or NR2D. The presence of specific NR2 subunits determines the properties of receptors, including agonist affinity, magnesium sensitivity, deactivation kinetics, modulation by polyamines, and channel conductance (McBain and Mayer, 1994; Mori and Mishina, 1995). The sensitivity of ifenprodil, a NR2B selective antagonist, is positively dependent on the proportion of NR2B in the cultured neurons (Bhave et al., 1999). Consistent with the reduction of ifenprodil sensitivity, we found that toluene exposure produced a decrease in the level of NR2B subunits. In addition, the function of NMDA receptors containing NR2B subunits is most sensitive to toluene (Cruz et al., 1998). Taken together, the NMDA receptors with NR2B subunits may be the main target of toluene during CNS development. Toluene may directly affect their functions during exposure and selectively result in downregulation of the NR2B subunit expression after toluene withdrawal in cultured condition. However, it is still unclear how toluene can cause downregulation of the NR2B subunit expression.
We examined the NMDA-mediated Ca2+ signals at 5, 7, 9, and 14 DIV. The differences between cultured granule neurons from control and toluene-exposed pups were gradually reduced. Previous studies of the developmental profile of NMDA receptor subunit expression in cerebellar granule neurons indicated that there was no change in NR1, a significant increase in NR2A, and a decrease in NR2B on days 4–14 in vitro (Snell et al., 2001; Vallano et al., 1996). Because the reduction of NR2B subunit expression in the cultured cerebellar granule neurons from toluene-exposed pups was earlier than the normal "developmental switch" of the NR2A and NR2B subunits, the gradually reduced influence of toluene on the Ca2+ signals in response to glutamate and NMDA from 5 DIV to 14 DIV may be associated with the decrease in NR2B expression. Alternatively, phosphorylation and the redox state of NMDA receptors and the associated NMDA receptor modulator protein (e.g., PSD-95) are also well-established biochemical mechanisms for modulation of the receptor response (Iwamoto et al., 2004; Koles et al., 2001; Sinor et al., 1997; Yamada et al., 1999). It is possible that changes in phosphorylation and oxidation state of NMDA receptors or NMDA receptor modulator protein may contribute to the toluene-induced alterations in the sensitive response to glutamate or antagonists observed here.
It is noteworthy that granule neurons from toluene-exposed pups also reduced Ca2+ signals in response to glycine in the presence of 5 μM glutamate, but the effect did not occur in a dose- and time-dependent manner. The NMDA receptor glycine binding site is located on the NR1 subunit (Hirai et al., 1996; Ivanovic et al., 1998). There was no change in the NR1 subunit during development of cerebellar granule neurons. Our data also show the lack of effect of toluene on NR1 subunit protein expression. Hence, it may explain why there was no time-dependent effect of toluene on reduction of Ca2+ signals in response to glycine. Furthermore, the presence of 5 μM glutamate under our experimental conditions may be involved in the similar inhibitory effects of distinct doses of toluene on Ca2+ signals.
In summary, our results suggest that a toluene-induced reduction in Ca2+ signals linked to NMDA receptors, probably through altering the subunit compositions of NMDA receptors, could disrupt many aspects of CNS neuronal functions and thereby have an important part in the CNS damage done to infants by maternal toluene abuse.
ACKNOWLEDGMENTS
This work was supported by a grant (NHRI-EX92-9112NC) from the National Health Research Institute, Taiwan, and a grant (TCMRC91129) from the intramural fund of Tzu Chi University.
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