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Di (2-ethylhexyl) phthalate modulates cholinergic mini-presynaptic transmission of projection neurons in Drosophila antennal lobe
Food and Chemical Toxicology 50 (2012) 3291–3297
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Di (2-ethylhexyl) phthalate modulates cholinergic mini-presynaptic transmission of projection neurons in Drosophila antennal lobe Dongzhi Ran a,1, Song Cai a,1, Huiling Wu b, Huaiyu Gu a,⇑ a b
Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen, Guangzhou, China Guangdong Prb Bio-Tech Co., Ltd., China
a r t i c l e
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Article history: Received 30 December 2011 Accepted 24 March 2012 Available online 1 April 2012 Keywords: Di (2-ethylhexyl) phthalate Polyvinyl chloride Projection neurons Neurotoxicity Electrophysiology properties Calcium channel activity
a b s t r a c t Di (2-ethylhexyl) phthalate (DEHP) is one of the Phthalic acid esters which are added in polyvinyl chloride (PVC) products. Previous animal studies have showed that exposure to DEHP has a negative effect on the liver, kidneys, lungs, and reproductive system, particularly the developing testes of prenatal and neonatal males, but few can match the dramatic impact on the nervous system. Drosophila melanogaster as a model organism has been widely used in research of the nervous system. In order to examine the modulation of DEHP in excitatory cholinergic transmission, electrophysiological properties of spontaneous activities, spontaneous action potential (sAP), mini excitatory postsynaptic currents (mEPSCs), and calcium currents were measured in projection neurons (PNs) of Drosophila antennal lobe. In this study, DEHP (100 lM) was showed to influence the spontaneous activities of the PNs and DEHP (300 lM) significantly decrease the frequency of sAP. Meanwhile, DEHP (100 and 300 lM) also reduced the frequency and amplitude of mEPSCs. Furthermore, ion channel studies showed DEHP (100 and 300 lM) inhibited the peak current amplitude of calcium channel. These results indicated that the DEHP modulated the cholinergic mini-synaptic transmission of projection neurons in Drosophila antennal lobe, and this modulation might be mediated by inhibiting the calcium channel activities. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Phthalates (phthalic acid esters, PAEs), a family of chemicals, which are used in plastics and many other products, have attracted researchers attention because of their hepatotoxic, teratogenic, and carcinogenic properties (Liang et al., 2008) besides their harm to pregnant women, infants and children (Jurewicz and Hanke, 2011). Therefore, San Francisco have enforced aforbiddenness of sale, distribution and manufacture of baby products which were made with any level of toxic chemical like PAEs. Di-(2-ethylhexyl) phthalate, known as DEHP, is colorless, odorless liquid with a chemical formula of C24H38O4, and is more fatsoluble than water-soluble (Shaz et al., 2011). DEHP, one of the most commonly used phthalates, has been added into the polyvinyl chloride (PVC) to soften and increase the flexibility of the plastic and vinyl products. As a common plasticizer, DEHP is widely used in cosmetics, personal care products, and consumer products such as food cans and food bags. It has been reported that the level of this toxic chemical will drop to 50% if people can avoid using plastic. DEHP and plastic components are not combined with tight ⇑ Corresponding author. Address: Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel.: +86 20 8733 2068; fax: +86 20 8733 0709. E-mail address: [email protected] (H. Gu). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.03.070
covalent chemical bonds but Vander Waals’ force, therefore the widespread use of this kind of products has made the exposure to DEHP unavoidable. It is harmful to the liver, kidneys, lungs, and reproductive system (Shea, 2003). What is more, it has been reported that at an appropriate concentration, DEHP has potential ability to affect the development of central nervous system (CNS) (Hokanson et al., 2006). Meanwhile, researches focused on rodent studies indicate negative effects on both neural development and behavior. DEHP has been shown to alter gene expression of G-protein-coupled receptors, affect the dopaminergic neurotransduction system in rat brain (Ishido et al., 2005), and decrease the activity of neuronal membrane Na+–K+ATPase (Dhanya et al., 2004). The Drosophila melanogaster has been a model organism for about 100 years since the study of complex biological problems. Nowadays, it has been widely used as a system to study neurobiology, neuropharmacology, and neuropathologic mechanisms. This model organism represents a system where electrophysiology recordings, behavior and gene response can be readily examined. The antennal lobe projection neurons (PNs) of Drosophila are known to be cholinergic, and the nicotinic acetylcholine receptors (nAChRs) are reported to participate in most of the spontaneous excitatory drive in the circuit of normal sensory input. They receive the input signal from olfactory interneurons and transduct to higher brain center. PNs are interneuron, which together with other neurons establish a complex synaptic network in the antennal lobe.
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Nevertheless, few studies have been performed on the mechanism of neurotoxicity of DEHP. To achieve this goal, the present study was designed to record from PNs to evaluate DEHP in excitatory cholinergic transmission in neurons of Drosophila antennal lobe. By investigating the properties of spontaneous firing, spontaneous action potential (sAP), mini Excitement Postsynaptic Currents (mEPSCs) and calcium currents, we found that DEHP regulated cholinergic mini-presynaptic transmission of PNs in Drosophila antennal lobe. 2. Materials and methods 2.1. Fly trains Drosophila stocks were reared on standard cornmeal agar medium supplemented with dry yeast at 24 °C and 60% relative humidity, and a previously developed method (Gu and O’Dowd, 2006, 2007) was used to study spontaneous activity, mEPSCs and calcium channel currents of PNs in the whole brain. In our lab, oratory Drosophila melanogaster produces new adults in 14 days. In order to record the flies at accurate time points, all experiments were performed on wild-type Canton-S female flies 2 days before eclosion which were identified by red eyes and transparent wings in the puparium.
performed. Access resistance was continuously monitored during the experiments. Recordings were made at room temperature, and only a single projection neuron was examined in each brain. All electrophysiological recordings were carried out using a BX51WI upright microscope (Olympus, Lehigh Valley, PA). Signals were acquired with EPC10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), and were filtered at 5 kHz using a built-in filter and digitized at 5 kHz. Data analysis was performed by the pClamp10 Clampfit software (Molecular Devices). 2.5. Analysis of synaptic currents The mEPSCs were detected using Mini analysis software (Synaptosoft, Decatur, GA). Events were accepted for analysis only if they were asymmetrical with a rising phase faster than 3 ms and a more slowly decaying phase. In addition, the threshold criterion for inclusion was 3pA. 2.6. Statistics Statistical comparison the magnitude of spontaneous action potential, mEPSCs and calcium currents before and after application of DEHP was made with one-way ANOVA. Results are presented as mean ± SEM, and differences were considered significant if P < 0.05.
3. Results 2.2. Drugs and solutions DEHP was bought from Sigma–Aldrich Chemicals Co. (St. Louis, MO). DEHP was formulated into stock solution with dimethyl sulfoxide (DMSO). The stock solutions were further diluted in the bath solution, to the appropriate concentrations of DEHP. After the establishment of a whole-cell configuration, the cells were allowed to stabilize for 3–5 min, and then to start a protocol. Each final concentration of DEHP was used once currents and potential were stable to investigate the influence of DEHP on PNs. DMSO and the chemical products used to prepare external and internal solutions were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA) unless otherwise specified in the protocol. The standard external solution contained (in mM) 101 NaCl, 1 CaCl2, 4 MgCl2, 3 KCl, 5 glucose, 1.25 NaH2PO4, and 20.7 NaHCO3, pH 7.2, 250 Osm. An internal solution containing the followings (in mM): 102 K-gluconate, 0.085 CaCl2, 1.7 MgCl2, 17 NaCl, 0.94 EGTA, and 8.5 HEPES with pH of 7.2 and 235 mOsM. 2.3. Confocal images of Drosophila melanogaster brain All brains were obtained from flies 2 days before eclosion. The entire brain, including optic lobes, was resected and prepared for recordings in standard external solution containing 20 units/ml papain with 1 mM l-cysteineas previously described (Gu and O’Dowd, 2006, 2007). Then the dissected brains were mounted in an RC-26 perfusion chamber (Warner Instruments, Hamden, CT, USA) containing the recording solution bubbled with 95% O2 and 5% CO2 (2 ml/min). The soma and the terminals were injected with biocytin in the recording pipette in the whole cell configurations for at least 30 min. After electrophysiological recording, the brain was fixed in phosphate buffered 4% formaldehyde at 4 °C for 10 h and subjected to biocytin staining. Then, the brain was washed in 1% PBS three times, blocked and incubated in blocking buffer (0.1 MPBS, 0.1% Triton X-100, 1% BSA) containing streptavidin-CY3 (Molecular Devices) for 3 h at room temperature. After incubation, the brain was washed three times at 5-min intervals in PBS. A BX51WI microscope with a 40 objective and confocal camera was used to acquire photos of dendritic arborization of the visual projection neurons. Each representative image was randomly sampled 10 times and the counter was blinded to sample identities (fly genotype, age and other experimental conditions). Statistical significance was calculated using Student’s t-test with two-tailed P values.
3.1. Confocal image: the morphology of PNs of Drosophila melanogaster pupae 2 days before eclosion In order to study the effects of DEHP on PNs, we first demonstrated the morphology properties of PNs. Fig. 1 showed the detailed morphology of PNs in the isolated brain, which are members of the neural circuits. Previous studies have reported the PNs that come from antennal lobe to be cholinergic and cholinoceptive and have closely connection with cholinergic input to Kenyon cells in Drosophila (Bicker, 1999; Yasuyama et al., 2002). In this study, Spontaneous activities, sAP, mEPSCs and calcium currents are recorded from this kind of PNs in the prepared brains of fly pupae 2 days before eclosion. 3.2. Electrophysiological recording: effects of DEHP on spontaneous extracellular activity of PNs in the isolated pupae brains 2 days before eclosion To investigate the effects of DEHP on the electrophysiology properties of brain neurons in detail, spontaneous firing, an initial
2.4. Electrophysiological recording from PNs in isolated whole brain All brains were obtained from female flies 2 days before eclosionas previous. Pipettes were targeted to PNs in the dorsal neuron cluster in the antennal lobe. Whole-cell recordings were performed with pipettes (10–15 MX) filled with internal solution. The pipettes were pulled using a micropipette puller. Cholinergic mEPSCs were recorded using the same internal solution as that used for whole-cell recording (Gu et al., 2009), and were performed in the presence of TTX to block sodium channels and PTX to block GABA receptors. The majority of mEPSCs recorded in these conditions could be blocked by curare. APs were recorded in the whole-cell patch–clamp configuration at the holding potential of 70 mV. The number of action potentials was counted, and only overshooting action potentials more positive than 0 mV were included, meanwhile peak amplitude was measured before and after drug applications. Current-clamp and voltage-clamp recordings were performed using patch–clamp electrodes. Giga ohm seals were achieved before recording in on-cell configuration, followed by whole-cell configuration while in voltage-clamp mode. Slow and fast capacitance compensation was automatically
Fig. 1. Confocal images demonstrated the detailed morphology of the recorded projection neurons (PNs). Image of the fruit fly brain with biotin labeled olfactory PNs, showing the detail morphology of the recorded neurons (A). A single neuron has been labeled (B). There is one major branch of the soma stalk of the visual projection neuron, and this branch curves dorso-medially, giving off several small collaterals. 3D reconstruction of the projection neurons, using 3D imaging software (C).
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3.3. Electrophysiological recording: Effects of DEHP on sAP frequency and amplitude of PNs in the isolated pupae brains 2 days before eclosion
Fig. 2. DEHP modulated spontaneous activities recorded from PNs of the Drosophila brain. This figure showed the recordings of PNs in the group of control saline and after application of DEHP (100 lM) in cell-attached current-clamp configuration. Spontaneous potential reduced significantly in the presence of DEHP (100 lM) compared with control.
recording, was recorded in cultures prepared from brains of Canton-S pupae 2 days before eclosion using current-clamp in cell-attached configuration. In this study, electrophysiology recordings of PNs which was recorded at the holding potential of 70 mV have been measured before and after the application of DEHP (100 lM). The spontaneous activities of Drosophila brain in DEHP group were showed much less than the control group (Fig. 2). This conformed that DEHP could influence the membrane properties of PNs in Drosophila model.
Spontaneous action potentials are well known to be a fundamental property of excitable cells in the CNS, responsible for the neurons activity and a manifestation of different ion channels activities. To further investigate the effects of DEHP on the electrophysiology properties of brain neurons, and determine if the electrophysiological differences in ion channel properties alter neuronal excitability, we focused on sAP properties of PNs in cultures prepared from brains of Canton-S pupae 2 days before eclosion, in terms of sAP frequency and amplitude recordings using whole-cell current-clamp (Fig. 3). Both frequency and amplitude decreased at the concentration of 100 and 300 lM (Fig. 3A). At the concentration of 100 lM, neither sAP frequency (n = 6, p = 0.6 > 0.05, Fig. 3B) nor peak amplitude (n = 6, p = 0.735 > 0.05, Fig. 3C) differed significantly from those of control. Whereas, sAP frequency became different from control saline significantly at 300 lM concentration, while peak amplitude did not (n = 6, p = 0.055 > 0.05, Fig. 3C). After several minutes of exposure to 300 lM DEHP, average sAP frequency was decreased significantly from 5.82 ± 0.84 Hz to 2.10 ± 0.61 Hz (p < 0.05, n = 6). Together, these data showed that DEHP altered the sAP activity in PNs of the Drosophila antennal lobe. Furthermore, it also indicated that
Fig. 3. Effects of DEHP of different concentration on sAP frequency and amplitude recorded from PNs of Drosophila brain. In the current-clamp mode, neurons were held at 70 mV, and single action potentials were elicited using a 10 ms depolarizing current pulse before and after the application of DEHP. (A) Representative current traces recorded in the control saline and the presence of DEHP (100 lM and 300 lM). Both sAP frequency and amplitude were influenced by DEHP. (B) sAP frequency was reduced by the application of DEHP at the concentration of 300 lM (n = 6, p < 0.05) significantly, but not 100 lM (n = 6, p > 0.05). (C) In contrast, AP amplitude was not significantly reduced either at the 100 lM (n = 6, p > 0.05) or 300 lM (n = 6, p > 0.05). Each bar indicates the mean ± SE from the indicated number of neurons (n). (⁄ means P < 0.05, ⁄⁄ means P < 0.01 and ⁄⁄⁄ means P < 0.00).
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DEHP might influence the ion channels which were related to the process of sAP formation, particularly the calcium channel that makes significant contribution to the regulation of sAP frequency.
and enhanced our hypothesis that DEHP could modulate the cholinergic input circuit, and could make contribution to the formation of synaptic plasticity.
3.4. Electrophysiological recording: effects of DEHP on the mEPSCs frequency and amplitude of PNs in the isolated pupae brains 2 days before eclosion
3.5. Electrophysiological recording: effects of DEHP on calcium currents of PNs in the isolated pupae brains 2 days before eclosion
In this study, spontaneous postsynaptic currents (sPSCs) in control saline, cholinergic mEPSCs in the presence of TTX and PTX, and cholinergic mEPSCs in the presence of TTX, PTX and DEHP (100 and 300 lM) were recorded from PNs in the wild-type Canton-S flies (Fig. 4A). Average mEPSC frequency was 2.22 ± 0.22 Hz and amplitude 8.20 ± 0.51 pA under normal condition. DEHP was observed to inhibit both mEPSC frequency and amplitude at the concentration of 100 and 300 lM. mEPSCs amplitude was not changed significantly at the presence of DEHP neither 100 nor 300 lM (Fig. 4B). However, an addition of 100 lM DEHP to external solution significantly reduced the average mEPSCs frequency from 1.96 ± 0.19 to 0.94 ± 0.54 Hz, and when up to 300 lM concentration, mEPSCs frequency changed significantly from 1.96 ± 0.19 to 0.67 ± 0.33 Hz. mEPSCs recorded in PNs have been proved to be cholinergic (Su and O’Dowd, 2003), mediated by nicotinic acetylcholine receptors (nAChRs), because it can be blocked by curare which is identified to be a competitive antagonist of nicotinic neuromuscular acetylcholine receptors (Wenningmann and Dilger, 2001). Particularly, mEPSCs had shown to reflect the synaptic plasticity. Our results showed the DEHP modulation on mEPSPs frequency of PNs, indicated the ability to regulate the mEPSCs properties of neurons,
Data present has demonstrated that the frequency and amplitude of sAP and mEPSCs of PNs in Drosophila brains have been significantly influenced in the presence of DEHP. To further explore the role of DEHP in modulation, calcium currents were monitored before and after the application of DEHP under good voltage-control. All recordings were made in external saline containing a physiological concentration of calcium (1.8 mM) and peak current amplitude was expressed in terms of density (Dcurrent–current). The peak calcium current amplitude was reduced significantly in the presence of DEHP at both concentration of 100 and 300 lM. Treatment with DEHP (100 lM) caused a significant decrease in the average peak current from 145.62 ± 12.19 pA to 95.58 pA ± 11.46 pA when compared to the control group (Fig. 5E, n = 6, p < 0.05), and DEHP at the concentration of 300 lM caused a significant decrease from 145.62 ± 12.19 pA to 82.97 ± 7.85 pA (Fig. 5E, n = 6, p < 0.05). Consistent with our observations in results 4.3 and 4.4, initial membrane activity level of PNs was inhibited with the application of DEHP. Each data present demonstrated that DEHP (100 and 300 lM) can reduce the calcium currents of PNs in Drosophila antennal lobe and modulate the properties of sAP and mEPSCs due to controlling the calcium activity.
Fig. 4. Effects of DEHP at different concentrations on the frequency and amplitude of mEPSCs recorded from PNs in Drosophila brain. In the voltage-clamp mode, neurons were held at 70 mV (A). PSCs recorded from PNs in control saline. Representative current traces recorded in the control saline and the presence of DEHP (100 and 300 lM) (A). Application of TTX (1 l) was added to block many of the synaptic currents. Application of DEHP at both 100 lM (n = 7, p < 0.05) and 300 lM (n = 7, p < 0.05) reduced mEPSCs frequency significantly (B), but not the mEPSCs amplitude (n = 7, p > 0.05) (C).
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Fig. 5. Effects of DEHP on the peak current amplitude recorded from the PNs in the Drosophila brain. Representative current traces recorded in the control saline and the presence of 100 lM (A) and 300 lM DEHP (B), in whole-cell current-clamp configuration. A series of depolarizing voltage steps from a holding potential of 70 mV elicited rapidly activating inward calcium currents in PNs. Calcium channel I–V curve in the presence of DEHP (100 and 300 lM) compared with control (C and D). The peak calcium currents amplitude was reduced significantly in the presence of DEHP (100 and 300 lM) (E).
4. Discussion The clouding agent, a major food safety incident in China has aroused concern among all walks of life. DEHP, a common plasticizer used in plastic materials, has been listed to be an environmental hormone and a controlled drug by the Environmental Protection Administration. It had been a new marine pollutant from a research of North Atlantic (Giam et al., 1978). Known as an antiandrogenic, DEHP primarily affects the development of the male rats reproductive system (Moore et al., 2001), affect the normal development of testis in rats with the exposure in utero (Tandon et al., 1991) and cause testicular damage in rats (Gray et al., 1977). Nevertheless, it is reported that DEHP is hardly transferred to brain in rats, and seems to produce few directly effects on CNS (Ikeda et al., 1980). Previous studies have showed that DEHP-treated animals did not show any signs of CNS depression and appeared to be normal (Seth et al., 1977). DEHP produced few
adverse effects in reproductive and neurobehavioural parameters in mice at the dose level of 0.01%, 0.03%, and 0.09% in diet (Tanaka, 2002). However, babies will be born with low birth weights and/or skeletal or nervous system developmental problems if the mother is exposed to sufficiently high levels of DEHP during pregnancy (Hauser and Calafat, 2005). Furthermore, there are still some animal experiments which have detected the relationship between DEHP and peroxisome proliferation (Doull et al., 1999) and researches reported that DEHP induce DNA damage in nervous system and oxidative damage in internal organs of mice (Xianghong et al., 2007). Experimental studies investigating the effects of DEHP on calcium channel in experimental animals have yield conflict results. A study in vitro shows that exposure of the rat neurohypophysial nerve terminals and pheochromocytom to DEHP may cause a increase level of intracellular Ca2+ on mammalian nervous tissue (Tully et al., 2000), and increase the intracellular calcium concentration by inducing a Ca2+ influx from the extracellular in human granulocytes (Palleschi et al., 2009). In contrast, DEHP
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may suppress Ca2+ concentration by inhibiting the nAChR in bovine adrenal chromaffin cells (Liu and Lin, 2002). Additional experiments indicated that DEHP or metabolites may inhibit the membrane Na+–K+ATPase (Dhanya et al., 2004) and has the possibility to cause a long term effects (Tully et al., 2000). Therefore, in consideration of the different mechanisms, such as evoking or inhibiting the ion channel activities, it is necessary for scientists to take steps to explore the DEHP neurotoxicity effects on human CNS and further studies are critically imperative in order to elucidate the neurotoxicity mechanisms of DEHP. In our study, neither sAP nor mEPSCs amplitude could be reduced significantly following the application of DEHP (100 and 300 lM). When at a low concentration of 100 lM, the sAP frequency was not decreased, but the mEPSCs frequency was significantly reduced to 48.0% compared to the control. When at a higher concentration of 300 lM, the sAP frequency was significantly reduced to 36.1% and DEHP demonstrated a larger reduction of mEPSCs to 34.2%. In addition, the calcium current amplitude got a larger reduction to 57% in the high concentration of 300 lM when compared with a lower concentration of 100 lM to 65.6%. Finally, our data demonstrated that DEHP could inhibit sAP and mEPSCs frequency and amplitude in a concentration-dependent manner by modulation calcium channel. Action potentials, a fundamental property of excitable cells in the mammalian CNS, are widely known to reflect the exchange of ions across the neuron membrane, including sodium channel, calcium channel and so on. Meanwhile mEPSCs in Drosophila CNS are explored to be involved in synaptic stability and/or plasticity as they are at some synapses in the mammalian CNS (Yamasaki et al., 2006) and mEPSC may play critical role in functional and/ or structural aspects of the synapses (Zhang et al., 2005). Calcium channels, which progressively substitute voltage-dependent sodium channels during aging, are specifically focused on the mechanisms of Parkinson diseases and Alzheimer’s, and diseases in transgenic Drosophila (Bagatini et al., 2011). Intracellular Ca2+ is investigated to be responsible for the action potential frequency (Jackson et al., 1991), and spontaneous mEPSCs could also be triggered by the release of presynaptic calcium ions (Zhang et al., 2005). In addition, amplitude and frequency changes of mEPSCs are due to a postsynaptic and presynaptic action (Simkus and Stricker, 2002). The amplitude of mEPSCs is characterized to be sensitive to the current which is evoked by single quanta, particularly the frequency to be susceptible to the voltage-gated calcium channels (Su and O’Dowd, 2003). These presented data here demonstrated that DEHP does have modulation in PNs of the Drosophila antennal lobe by regulating electrophysiology properties of cell membrane, supported our hypothesized that DEHP affected CNS by presynaptic calcium channel activities, and showed the susceptibility of the CNS for DEHP. As dysfunctional synaptic plasticity has be implicated in several brain diseases, such as depression, addiction, dementia and anxiety disorders, the anomalous changes of mEPSCs are related to the cognitive impairment characterizing neurodegenerative disease, such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease. Therefore we should pay closer attention to the health effects and the threats associated with the exposure to phthalates, which should evoke dysfunctional synaptic plasticity. Based on this study, it may be postulated that DEHP may play a role in the electrophysiology properties of Drosophila PN membranes, such as Spontaneous firings, sAP and mEPSCs. In addition, this study supports the notion for a role of calcium channel in DEHP neurotoxicity and presents the potential risks of DEHP application for food or environmental usage. However, the detailed mechanisms and effects of DEHP on nervous system are still unknown, and further investigations are imperative.
5. Conclusion We used the cholinergic PNs of Drosophila melanogaster model to identify the neurotoxicity of DEHP. For this purpose, properties of spontaneous firing, spontaneous action potential (sAP), min excitatory postsynaptic currents (mEPSCs), and calcium currents have been detected using cell-attached and whole-cell patch– clamp technique. DEHP was observed to modulate these electrophysiology properties of PNs. DEHP significantly decreased the frequency of sAP at the concentration of 300 lM and decreased the frequency and amplitude of mEPSCs at both 300 and 100 lM significantly. Additionally, in ion channel studies, DEHP (100 and 300 lM) obviously inhibited the peak current amplitude of calcium channel. These data indicated that DEHP would modulate the PNs by inhibiting the calcium channel. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments On behalf of the authors, I would like to thank F. Tandia and Naya Huang for providing language help. I would also like to thank Shengwen Deng and Weicong Li for the help of proof reading article and data analysis. Finally, we thank the support from fundings of NSF 30970980 and Science and Technology Planning Project of Guangdong Province, China(2010B080701102). References Bagatini, P.B., Saur, L., Rodrigues, M.F., Bernardino, G.C., Paim, M.F., Coelho, G.P., Silva, D.V., de Oliveira, R.M., Schirmer, H., Souto, A.A., 2011. The role of calcium channel blockers and resveratrol in the prevention of paraquat-induced parkinsonism in Drosophila melanogaster: a locomotor analysis. Invertebrate Neuroscience 1–9. Bicker, G., 1999. Histochemistry of classical neurotransmitters in antennal lobes and mushroom bodies of the honeybee. Microscopy Research and Technique 45, 174–183. Dhanya, C., Gayathri, N., Mithra, K., Nair, K., Kurup, P., 2004. Vitamin E prevents deleterious effects of di (2-ethyl hexyl) phthalate, a plasticizer used in PVC blood storage bags. Indian Journal of Experimental Biology 42, 871. Doull, J., Cattley, R., Elcombe, C., Lake, B.G., Swenberg, J., Wilkinson, C., Williams, G., van Gemert, M., 1999. A cancer risk assessment of di(2-ethylhexyl)phthalate: application of the new U.S. EPA Risk Assessment Guidelines. Regulatory Toxicology and Pharmacology 29, 327–357. Giam, C., Chan, H., Neff, G., Atlas, E., 1978. Phthalate ester plasticizers: a new class of marine pollutant. Science 199, 419. Gray, T., Butterworth, K., Gaunt, I., Grasso, P., Gangolli, S., 1977. Short-term toxicity study of di-(2-ethylhexyl) phthalate in rats. Food and Cosmetics Toxicology 15, 389–399. Gu, H., O’Dowd, D.K., 2006. Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ. The Journal of Neuroscience 26, 265. Gu, H., O’Dowd, D.K., 2007. Whole cell recordings from brain of adult Drosophila. Journal of Visualized Experiments 6, 248. Gu, H., Jiang, S.A., Campusano, J.M., Iniguez, J., Su, H., Hoang, A.A., Lavian, M., Sun, X., O’Dowd, D.K., 2009. Cav2-type calcium channels encoded by cac regulate APindependent neurotransmitter release at cholinergic synapses in adult Drosophila brain. Journal of Neurophysiology 101, 42. Hauser, R., Calafat, A., 2005. Phthalates and human health. Occupational and Environmental Medicine 62, 806. Hokanson, R., Hanneman, W., Hennessey, M., Donnelly, K., McDonald, T., Chowdhary, R., Busbee, D., 2006. DEHP, bis (2)-ethylhexyl phthalate, alters gene expression in human cells: possible correlation with initiation of fetal developmental abnormalities. Human and Experimental Toxicology 25, 687. Ikeda, G., Sapienza, P., Couvillion, J., Farber, T., 1980. Comparative distribution, excretion and metabolism of di-(2-ethylhexyl) phthalate in rats, dogs and miniature pigs. Food and Cosmetics Toxicology 18, 637–642. Ishido, M., Morita, M., Oka, S., Masuo, Y., 2005. Alteration of gene expression of G protein-coupled receptors in endocrine disruptors-caused hyperactive rats. Regulatory Peptides 126, 145–153. Jackson, M.B., Konnerth, A., Augustine, G.J., 1991. Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals. Proceedings of the National Academy of Sciences 88, 380.
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Di (2-ethylhexyl) phthalate modulates cholinergic mini-presynaptic transmission of projection neurons in Drosophila antennal lobe Dongzhi Ran a,1, Song Cai a,1, Huiling Wu b, Huaiyu Gu a,⇑ a b
Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen, Guangzhou, China Guangdong Prb Bio-Tech Co., Ltd., China
a r t i c l e
i n f o
Article history: Received 30 December 2011 Accepted 24 March 2012 Available online 1 April 2012 Keywords: Di (2-ethylhexyl) phthalate Polyvinyl chloride Projection neurons Neurotoxicity Electrophysiology properties Calcium channel activity
a b s t r a c t Di (2-ethylhexyl) phthalate (DEHP) is one of the Phthalic acid esters which are added in polyvinyl chloride (PVC) products. Previous animal studies have showed that exposure to DEHP has a negative effect on the liver, kidneys, lungs, and reproductive system, particularly the developing testes of prenatal and neonatal males, but few can match the dramatic impact on the nervous system. Drosophila melanogaster as a model organism has been widely used in research of the nervous system. In order to examine the modulation of DEHP in excitatory cholinergic transmission, electrophysiological properties of spontaneous activities, spontaneous action potential (sAP), mini excitatory postsynaptic currents (mEPSCs), and calcium currents were measured in projection neurons (PNs) of Drosophila antennal lobe. In this study, DEHP (100 lM) was showed to influence the spontaneous activities of the PNs and DEHP (300 lM) significantly decrease the frequency of sAP. Meanwhile, DEHP (100 and 300 lM) also reduced the frequency and amplitude of mEPSCs. Furthermore, ion channel studies showed DEHP (100 and 300 lM) inhibited the peak current amplitude of calcium channel. These results indicated that the DEHP modulated the cholinergic mini-synaptic transmission of projection neurons in Drosophila antennal lobe, and this modulation might be mediated by inhibiting the calcium channel activities. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Phthalates (phthalic acid esters, PAEs), a family of chemicals, which are used in plastics and many other products, have attracted researchers attention because of their hepatotoxic, teratogenic, and carcinogenic properties (Liang et al., 2008) besides their harm to pregnant women, infants and children (Jurewicz and Hanke, 2011). Therefore, San Francisco have enforced aforbiddenness of sale, distribution and manufacture of baby products which were made with any level of toxic chemical like PAEs. Di-(2-ethylhexyl) phthalate, known as DEHP, is colorless, odorless liquid with a chemical formula of C24H38O4, and is more fatsoluble than water-soluble (Shaz et al., 2011). DEHP, one of the most commonly used phthalates, has been added into the polyvinyl chloride (PVC) to soften and increase the flexibility of the plastic and vinyl products. As a common plasticizer, DEHP is widely used in cosmetics, personal care products, and consumer products such as food cans and food bags. It has been reported that the level of this toxic chemical will drop to 50% if people can avoid using plastic. DEHP and plastic components are not combined with tight ⇑ Corresponding author. Address: Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China. Tel.: +86 20 8733 2068; fax: +86 20 8733 0709. E-mail address: [email protected] (H. Gu). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.03.070
covalent chemical bonds but Vander Waals’ force, therefore the widespread use of this kind of products has made the exposure to DEHP unavoidable. It is harmful to the liver, kidneys, lungs, and reproductive system (Shea, 2003). What is more, it has been reported that at an appropriate concentration, DEHP has potential ability to affect the development of central nervous system (CNS) (Hokanson et al., 2006). Meanwhile, researches focused on rodent studies indicate negative effects on both neural development and behavior. DEHP has been shown to alter gene expression of G-protein-coupled receptors, affect the dopaminergic neurotransduction system in rat brain (Ishido et al., 2005), and decrease the activity of neuronal membrane Na+–K+ATPase (Dhanya et al., 2004). The Drosophila melanogaster has been a model organism for about 100 years since the study of complex biological problems. Nowadays, it has been widely used as a system to study neurobiology, neuropharmacology, and neuropathologic mechanisms. This model organism represents a system where electrophysiology recordings, behavior and gene response can be readily examined. The antennal lobe projection neurons (PNs) of Drosophila are known to be cholinergic, and the nicotinic acetylcholine receptors (nAChRs) are reported to participate in most of the spontaneous excitatory drive in the circuit of normal sensory input. They receive the input signal from olfactory interneurons and transduct to higher brain center. PNs are interneuron, which together with other neurons establish a complex synaptic network in the antennal lobe.
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Nevertheless, few studies have been performed on the mechanism of neurotoxicity of DEHP. To achieve this goal, the present study was designed to record from PNs to evaluate DEHP in excitatory cholinergic transmission in neurons of Drosophila antennal lobe. By investigating the properties of spontaneous firing, spontaneous action potential (sAP), mini Excitement Postsynaptic Currents (mEPSCs) and calcium currents, we found that DEHP regulated cholinergic mini-presynaptic transmission of PNs in Drosophila antennal lobe. 2. Materials and methods 2.1. Fly trains Drosophila stocks were reared on standard cornmeal agar medium supplemented with dry yeast at 24 °C and 60% relative humidity, and a previously developed method (Gu and O’Dowd, 2006, 2007) was used to study spontaneous activity, mEPSCs and calcium channel currents of PNs in the whole brain. In our lab, oratory Drosophila melanogaster produces new adults in 14 days. In order to record the flies at accurate time points, all experiments were performed on wild-type Canton-S female flies 2 days before eclosion which were identified by red eyes and transparent wings in the puparium.
performed. Access resistance was continuously monitored during the experiments. Recordings were made at room temperature, and only a single projection neuron was examined in each brain. All electrophysiological recordings were carried out using a BX51WI upright microscope (Olympus, Lehigh Valley, PA). Signals were acquired with EPC10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), and were filtered at 5 kHz using a built-in filter and digitized at 5 kHz. Data analysis was performed by the pClamp10 Clampfit software (Molecular Devices). 2.5. Analysis of synaptic currents The mEPSCs were detected using Mini analysis software (Synaptosoft, Decatur, GA). Events were accepted for analysis only if they were asymmetrical with a rising phase faster than 3 ms and a more slowly decaying phase. In addition, the threshold criterion for inclusion was 3pA. 2.6. Statistics Statistical comparison the magnitude of spontaneous action potential, mEPSCs and calcium currents before and after application of DEHP was made with one-way ANOVA. Results are presented as mean ± SEM, and differences were considered significant if P < 0.05.
3. Results 2.2. Drugs and solutions DEHP was bought from Sigma–Aldrich Chemicals Co. (St. Louis, MO). DEHP was formulated into stock solution with dimethyl sulfoxide (DMSO). The stock solutions were further diluted in the bath solution, to the appropriate concentrations of DEHP. After the establishment of a whole-cell configuration, the cells were allowed to stabilize for 3–5 min, and then to start a protocol. Each final concentration of DEHP was used once currents and potential were stable to investigate the influence of DEHP on PNs. DMSO and the chemical products used to prepare external and internal solutions were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA) unless otherwise specified in the protocol. The standard external solution contained (in mM) 101 NaCl, 1 CaCl2, 4 MgCl2, 3 KCl, 5 glucose, 1.25 NaH2PO4, and 20.7 NaHCO3, pH 7.2, 250 Osm. An internal solution containing the followings (in mM): 102 K-gluconate, 0.085 CaCl2, 1.7 MgCl2, 17 NaCl, 0.94 EGTA, and 8.5 HEPES with pH of 7.2 and 235 mOsM. 2.3. Confocal images of Drosophila melanogaster brain All brains were obtained from flies 2 days before eclosion. The entire brain, including optic lobes, was resected and prepared for recordings in standard external solution containing 20 units/ml papain with 1 mM l-cysteineas previously described (Gu and O’Dowd, 2006, 2007). Then the dissected brains were mounted in an RC-26 perfusion chamber (Warner Instruments, Hamden, CT, USA) containing the recording solution bubbled with 95% O2 and 5% CO2 (2 ml/min). The soma and the terminals were injected with biocytin in the recording pipette in the whole cell configurations for at least 30 min. After electrophysiological recording, the brain was fixed in phosphate buffered 4% formaldehyde at 4 °C for 10 h and subjected to biocytin staining. Then, the brain was washed in 1% PBS three times, blocked and incubated in blocking buffer (0.1 MPBS, 0.1% Triton X-100, 1% BSA) containing streptavidin-CY3 (Molecular Devices) for 3 h at room temperature. After incubation, the brain was washed three times at 5-min intervals in PBS. A BX51WI microscope with a 40 objective and confocal camera was used to acquire photos of dendritic arborization of the visual projection neurons. Each representative image was randomly sampled 10 times and the counter was blinded to sample identities (fly genotype, age and other experimental conditions). Statistical significance was calculated using Student’s t-test with two-tailed P values.
3.1. Confocal image: the morphology of PNs of Drosophila melanogaster pupae 2 days before eclosion In order to study the effects of DEHP on PNs, we first demonstrated the morphology properties of PNs. Fig. 1 showed the detailed morphology of PNs in the isolated brain, which are members of the neural circuits. Previous studies have reported the PNs that come from antennal lobe to be cholinergic and cholinoceptive and have closely connection with cholinergic input to Kenyon cells in Drosophila (Bicker, 1999; Yasuyama et al., 2002). In this study, Spontaneous activities, sAP, mEPSCs and calcium currents are recorded from this kind of PNs in the prepared brains of fly pupae 2 days before eclosion. 3.2. Electrophysiological recording: effects of DEHP on spontaneous extracellular activity of PNs in the isolated pupae brains 2 days before eclosion To investigate the effects of DEHP on the electrophysiology properties of brain neurons in detail, spontaneous firing, an initial
2.4. Electrophysiological recording from PNs in isolated whole brain All brains were obtained from female flies 2 days before eclosionas previous. Pipettes were targeted to PNs in the dorsal neuron cluster in the antennal lobe. Whole-cell recordings were performed with pipettes (10–15 MX) filled with internal solution. The pipettes were pulled using a micropipette puller. Cholinergic mEPSCs were recorded using the same internal solution as that used for whole-cell recording (Gu et al., 2009), and were performed in the presence of TTX to block sodium channels and PTX to block GABA receptors. The majority of mEPSCs recorded in these conditions could be blocked by curare. APs were recorded in the whole-cell patch–clamp configuration at the holding potential of 70 mV. The number of action potentials was counted, and only overshooting action potentials more positive than 0 mV were included, meanwhile peak amplitude was measured before and after drug applications. Current-clamp and voltage-clamp recordings were performed using patch–clamp electrodes. Giga ohm seals were achieved before recording in on-cell configuration, followed by whole-cell configuration while in voltage-clamp mode. Slow and fast capacitance compensation was automatically
Fig. 1. Confocal images demonstrated the detailed morphology of the recorded projection neurons (PNs). Image of the fruit fly brain with biotin labeled olfactory PNs, showing the detail morphology of the recorded neurons (A). A single neuron has been labeled (B). There is one major branch of the soma stalk of the visual projection neuron, and this branch curves dorso-medially, giving off several small collaterals. 3D reconstruction of the projection neurons, using 3D imaging software (C).
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3.3. Electrophysiological recording: Effects of DEHP on sAP frequency and amplitude of PNs in the isolated pupae brains 2 days before eclosion
Fig. 2. DEHP modulated spontaneous activities recorded from PNs of the Drosophila brain. This figure showed the recordings of PNs in the group of control saline and after application of DEHP (100 lM) in cell-attached current-clamp configuration. Spontaneous potential reduced significantly in the presence of DEHP (100 lM) compared with control.
recording, was recorded in cultures prepared from brains of Canton-S pupae 2 days before eclosion using current-clamp in cell-attached configuration. In this study, electrophysiology recordings of PNs which was recorded at the holding potential of 70 mV have been measured before and after the application of DEHP (100 lM). The spontaneous activities of Drosophila brain in DEHP group were showed much less than the control group (Fig. 2). This conformed that DEHP could influence the membrane properties of PNs in Drosophila model.
Spontaneous action potentials are well known to be a fundamental property of excitable cells in the CNS, responsible for the neurons activity and a manifestation of different ion channels activities. To further investigate the effects of DEHP on the electrophysiology properties of brain neurons, and determine if the electrophysiological differences in ion channel properties alter neuronal excitability, we focused on sAP properties of PNs in cultures prepared from brains of Canton-S pupae 2 days before eclosion, in terms of sAP frequency and amplitude recordings using whole-cell current-clamp (Fig. 3). Both frequency and amplitude decreased at the concentration of 100 and 300 lM (Fig. 3A). At the concentration of 100 lM, neither sAP frequency (n = 6, p = 0.6 > 0.05, Fig. 3B) nor peak amplitude (n = 6, p = 0.735 > 0.05, Fig. 3C) differed significantly from those of control. Whereas, sAP frequency became different from control saline significantly at 300 lM concentration, while peak amplitude did not (n = 6, p = 0.055 > 0.05, Fig. 3C). After several minutes of exposure to 300 lM DEHP, average sAP frequency was decreased significantly from 5.82 ± 0.84 Hz to 2.10 ± 0.61 Hz (p < 0.05, n = 6). Together, these data showed that DEHP altered the sAP activity in PNs of the Drosophila antennal lobe. Furthermore, it also indicated that
Fig. 3. Effects of DEHP of different concentration on sAP frequency and amplitude recorded from PNs of Drosophila brain. In the current-clamp mode, neurons were held at 70 mV, and single action potentials were elicited using a 10 ms depolarizing current pulse before and after the application of DEHP. (A) Representative current traces recorded in the control saline and the presence of DEHP (100 lM and 300 lM). Both sAP frequency and amplitude were influenced by DEHP. (B) sAP frequency was reduced by the application of DEHP at the concentration of 300 lM (n = 6, p < 0.05) significantly, but not 100 lM (n = 6, p > 0.05). (C) In contrast, AP amplitude was not significantly reduced either at the 100 lM (n = 6, p > 0.05) or 300 lM (n = 6, p > 0.05). Each bar indicates the mean ± SE from the indicated number of neurons (n). (⁄ means P < 0.05, ⁄⁄ means P < 0.01 and ⁄⁄⁄ means P < 0.00).
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DEHP might influence the ion channels which were related to the process of sAP formation, particularly the calcium channel that makes significant contribution to the regulation of sAP frequency.
and enhanced our hypothesis that DEHP could modulate the cholinergic input circuit, and could make contribution to the formation of synaptic plasticity.
3.4. Electrophysiological recording: effects of DEHP on the mEPSCs frequency and amplitude of PNs in the isolated pupae brains 2 days before eclosion
3.5. Electrophysiological recording: effects of DEHP on calcium currents of PNs in the isolated pupae brains 2 days before eclosion
In this study, spontaneous postsynaptic currents (sPSCs) in control saline, cholinergic mEPSCs in the presence of TTX and PTX, and cholinergic mEPSCs in the presence of TTX, PTX and DEHP (100 and 300 lM) were recorded from PNs in the wild-type Canton-S flies (Fig. 4A). Average mEPSC frequency was 2.22 ± 0.22 Hz and amplitude 8.20 ± 0.51 pA under normal condition. DEHP was observed to inhibit both mEPSC frequency and amplitude at the concentration of 100 and 300 lM. mEPSCs amplitude was not changed significantly at the presence of DEHP neither 100 nor 300 lM (Fig. 4B). However, an addition of 100 lM DEHP to external solution significantly reduced the average mEPSCs frequency from 1.96 ± 0.19 to 0.94 ± 0.54 Hz, and when up to 300 lM concentration, mEPSCs frequency changed significantly from 1.96 ± 0.19 to 0.67 ± 0.33 Hz. mEPSCs recorded in PNs have been proved to be cholinergic (Su and O’Dowd, 2003), mediated by nicotinic acetylcholine receptors (nAChRs), because it can be blocked by curare which is identified to be a competitive antagonist of nicotinic neuromuscular acetylcholine receptors (Wenningmann and Dilger, 2001). Particularly, mEPSCs had shown to reflect the synaptic plasticity. Our results showed the DEHP modulation on mEPSPs frequency of PNs, indicated the ability to regulate the mEPSCs properties of neurons,
Data present has demonstrated that the frequency and amplitude of sAP and mEPSCs of PNs in Drosophila brains have been significantly influenced in the presence of DEHP. To further explore the role of DEHP in modulation, calcium currents were monitored before and after the application of DEHP under good voltage-control. All recordings were made in external saline containing a physiological concentration of calcium (1.8 mM) and peak current amplitude was expressed in terms of density (Dcurrent–current). The peak calcium current amplitude was reduced significantly in the presence of DEHP at both concentration of 100 and 300 lM. Treatment with DEHP (100 lM) caused a significant decrease in the average peak current from 145.62 ± 12.19 pA to 95.58 pA ± 11.46 pA when compared to the control group (Fig. 5E, n = 6, p < 0.05), and DEHP at the concentration of 300 lM caused a significant decrease from 145.62 ± 12.19 pA to 82.97 ± 7.85 pA (Fig. 5E, n = 6, p < 0.05). Consistent with our observations in results 4.3 and 4.4, initial membrane activity level of PNs was inhibited with the application of DEHP. Each data present demonstrated that DEHP (100 and 300 lM) can reduce the calcium currents of PNs in Drosophila antennal lobe and modulate the properties of sAP and mEPSCs due to controlling the calcium activity.
Fig. 4. Effects of DEHP at different concentrations on the frequency and amplitude of mEPSCs recorded from PNs in Drosophila brain. In the voltage-clamp mode, neurons were held at 70 mV (A). PSCs recorded from PNs in control saline. Representative current traces recorded in the control saline and the presence of DEHP (100 and 300 lM) (A). Application of TTX (1 l) was added to block many of the synaptic currents. Application of DEHP at both 100 lM (n = 7, p < 0.05) and 300 lM (n = 7, p < 0.05) reduced mEPSCs frequency significantly (B), but not the mEPSCs amplitude (n = 7, p > 0.05) (C).
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Fig. 5. Effects of DEHP on the peak current amplitude recorded from the PNs in the Drosophila brain. Representative current traces recorded in the control saline and the presence of 100 lM (A) and 300 lM DEHP (B), in whole-cell current-clamp configuration. A series of depolarizing voltage steps from a holding potential of 70 mV elicited rapidly activating inward calcium currents in PNs. Calcium channel I–V curve in the presence of DEHP (100 and 300 lM) compared with control (C and D). The peak calcium currents amplitude was reduced significantly in the presence of DEHP (100 and 300 lM) (E).
4. Discussion The clouding agent, a major food safety incident in China has aroused concern among all walks of life. DEHP, a common plasticizer used in plastic materials, has been listed to be an environmental hormone and a controlled drug by the Environmental Protection Administration. It had been a new marine pollutant from a research of North Atlantic (Giam et al., 1978). Known as an antiandrogenic, DEHP primarily affects the development of the male rats reproductive system (Moore et al., 2001), affect the normal development of testis in rats with the exposure in utero (Tandon et al., 1991) and cause testicular damage in rats (Gray et al., 1977). Nevertheless, it is reported that DEHP is hardly transferred to brain in rats, and seems to produce few directly effects on CNS (Ikeda et al., 1980). Previous studies have showed that DEHP-treated animals did not show any signs of CNS depression and appeared to be normal (Seth et al., 1977). DEHP produced few
adverse effects in reproductive and neurobehavioural parameters in mice at the dose level of 0.01%, 0.03%, and 0.09% in diet (Tanaka, 2002). However, babies will be born with low birth weights and/or skeletal or nervous system developmental problems if the mother is exposed to sufficiently high levels of DEHP during pregnancy (Hauser and Calafat, 2005). Furthermore, there are still some animal experiments which have detected the relationship between DEHP and peroxisome proliferation (Doull et al., 1999) and researches reported that DEHP induce DNA damage in nervous system and oxidative damage in internal organs of mice (Xianghong et al., 2007). Experimental studies investigating the effects of DEHP on calcium channel in experimental animals have yield conflict results. A study in vitro shows that exposure of the rat neurohypophysial nerve terminals and pheochromocytom to DEHP may cause a increase level of intracellular Ca2+ on mammalian nervous tissue (Tully et al., 2000), and increase the intracellular calcium concentration by inducing a Ca2+ influx from the extracellular in human granulocytes (Palleschi et al., 2009). In contrast, DEHP
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may suppress Ca2+ concentration by inhibiting the nAChR in bovine adrenal chromaffin cells (Liu and Lin, 2002). Additional experiments indicated that DEHP or metabolites may inhibit the membrane Na+–K+ATPase (Dhanya et al., 2004) and has the possibility to cause a long term effects (Tully et al., 2000). Therefore, in consideration of the different mechanisms, such as evoking or inhibiting the ion channel activities, it is necessary for scientists to take steps to explore the DEHP neurotoxicity effects on human CNS and further studies are critically imperative in order to elucidate the neurotoxicity mechanisms of DEHP. In our study, neither sAP nor mEPSCs amplitude could be reduced significantly following the application of DEHP (100 and 300 lM). When at a low concentration of 100 lM, the sAP frequency was not decreased, but the mEPSCs frequency was significantly reduced to 48.0% compared to the control. When at a higher concentration of 300 lM, the sAP frequency was significantly reduced to 36.1% and DEHP demonstrated a larger reduction of mEPSCs to 34.2%. In addition, the calcium current amplitude got a larger reduction to 57% in the high concentration of 300 lM when compared with a lower concentration of 100 lM to 65.6%. Finally, our data demonstrated that DEHP could inhibit sAP and mEPSCs frequency and amplitude in a concentration-dependent manner by modulation calcium channel. Action potentials, a fundamental property of excitable cells in the mammalian CNS, are widely known to reflect the exchange of ions across the neuron membrane, including sodium channel, calcium channel and so on. Meanwhile mEPSCs in Drosophila CNS are explored to be involved in synaptic stability and/or plasticity as they are at some synapses in the mammalian CNS (Yamasaki et al., 2006) and mEPSC may play critical role in functional and/ or structural aspects of the synapses (Zhang et al., 2005). Calcium channels, which progressively substitute voltage-dependent sodium channels during aging, are specifically focused on the mechanisms of Parkinson diseases and Alzheimer’s, and diseases in transgenic Drosophila (Bagatini et al., 2011). Intracellular Ca2+ is investigated to be responsible for the action potential frequency (Jackson et al., 1991), and spontaneous mEPSCs could also be triggered by the release of presynaptic calcium ions (Zhang et al., 2005). In addition, amplitude and frequency changes of mEPSCs are due to a postsynaptic and presynaptic action (Simkus and Stricker, 2002). The amplitude of mEPSCs is characterized to be sensitive to the current which is evoked by single quanta, particularly the frequency to be susceptible to the voltage-gated calcium channels (Su and O’Dowd, 2003). These presented data here demonstrated that DEHP does have modulation in PNs of the Drosophila antennal lobe by regulating electrophysiology properties of cell membrane, supported our hypothesized that DEHP affected CNS by presynaptic calcium channel activities, and showed the susceptibility of the CNS for DEHP. As dysfunctional synaptic plasticity has be implicated in several brain diseases, such as depression, addiction, dementia and anxiety disorders, the anomalous changes of mEPSCs are related to the cognitive impairment characterizing neurodegenerative disease, such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease. Therefore we should pay closer attention to the health effects and the threats associated with the exposure to phthalates, which should evoke dysfunctional synaptic plasticity. Based on this study, it may be postulated that DEHP may play a role in the electrophysiology properties of Drosophila PN membranes, such as Spontaneous firings, sAP and mEPSCs. In addition, this study supports the notion for a role of calcium channel in DEHP neurotoxicity and presents the potential risks of DEHP application for food or environmental usage. However, the detailed mechanisms and effects of DEHP on nervous system are still unknown, and further investigations are imperative.
5. Conclusion We used the cholinergic PNs of Drosophila melanogaster model to identify the neurotoxicity of DEHP. For this purpose, properties of spontaneous firing, spontaneous action potential (sAP), min excitatory postsynaptic currents (mEPSCs), and calcium currents have been detected using cell-attached and whole-cell patch– clamp technique. DEHP was observed to modulate these electrophysiology properties of PNs. DEHP significantly decreased the frequency of sAP at the concentration of 300 lM and decreased the frequency and amplitude of mEPSCs at both 300 and 100 lM significantly. Additionally, in ion channel studies, DEHP (100 and 300 lM) obviously inhibited the peak current amplitude of calcium channel. These data indicated that DEHP would modulate the PNs by inhibiting the calcium channel. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments On behalf of the authors, I would like to thank F. Tandia and Naya Huang for providing language help. I would also like to thank Shengwen Deng and Weicong Li for the help of proof reading article and data analysis. Finally, we thank the support from fundings of NSF 30970980 and Science and Technology Planning Project of Guangdong Province, China(2010B080701102). References Bagatini, P.B., Saur, L., Rodrigues, M.F., Bernardino, G.C., Paim, M.F., Coelho, G.P., Silva, D.V., de Oliveira, R.M., Schirmer, H., Souto, A.A., 2011. The role of calcium channel blockers and resveratrol in the prevention of paraquat-induced parkinsonism in Drosophila melanogaster: a locomotor analysis. Invertebrate Neuroscience 1–9. Bicker, G., 1999. Histochemistry of classical neurotransmitters in antennal lobes and mushroom bodies of the honeybee. Microscopy Research and Technique 45, 174–183. Dhanya, C., Gayathri, N., Mithra, K., Nair, K., Kurup, P., 2004. Vitamin E prevents deleterious effects of di (2-ethyl hexyl) phthalate, a plasticizer used in PVC blood storage bags. Indian Journal of Experimental Biology 42, 871. Doull, J., Cattley, R., Elcombe, C., Lake, B.G., Swenberg, J., Wilkinson, C., Williams, G., van Gemert, M., 1999. A cancer risk assessment of di(2-ethylhexyl)phthalate: application of the new U.S. EPA Risk Assessment Guidelines. Regulatory Toxicology and Pharmacology 29, 327–357. Giam, C., Chan, H., Neff, G., Atlas, E., 1978. Phthalate ester plasticizers: a new class of marine pollutant. Science 199, 419. Gray, T., Butterworth, K., Gaunt, I., Grasso, P., Gangolli, S., 1977. Short-term toxicity study of di-(2-ethylhexyl) phthalate in rats. Food and Cosmetics Toxicology 15, 389–399. Gu, H., O’Dowd, D.K., 2006. Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ. The Journal of Neuroscience 26, 265. Gu, H., O’Dowd, D.K., 2007. Whole cell recordings from brain of adult Drosophila. Journal of Visualized Experiments 6, 248. Gu, H., Jiang, S.A., Campusano, J.M., Iniguez, J., Su, H., Hoang, A.A., Lavian, M., Sun, X., O’Dowd, D.K., 2009. Cav2-type calcium channels encoded by cac regulate APindependent neurotransmitter release at cholinergic synapses in adult Drosophila brain. Journal of Neurophysiology 101, 42. Hauser, R., Calafat, A., 2005. Phthalates and human health. Occupational and Environmental Medicine 62, 806. Hokanson, R., Hanneman, W., Hennessey, M., Donnelly, K., McDonald, T., Chowdhary, R., Busbee, D., 2006. DEHP, bis (2)-ethylhexyl phthalate, alters gene expression in human cells: possible correlation with initiation of fetal developmental abnormalities. Human and Experimental Toxicology 25, 687. Ikeda, G., Sapienza, P., Couvillion, J., Farber, T., 1980. Comparative distribution, excretion and metabolism of di-(2-ethylhexyl) phthalate in rats, dogs and miniature pigs. Food and Cosmetics Toxicology 18, 637–642. Ishido, M., Morita, M., Oka, S., Masuo, Y., 2005. Alteration of gene expression of G protein-coupled receptors in endocrine disruptors-caused hyperactive rats. Regulatory Peptides 126, 145–153. Jackson, M.B., Konnerth, A., Augustine, G.J., 1991. Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals. Proceedings of the National Academy of Sciences 88, 380.
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