Neurochemistry International 50 (2007) 791–799 www.elsevier.com/locate/neuint Dextromethorphan attenuates trimethyltin-induced neurotoxicity via s1 receptor activation in rats Eun-Joo Shin a,1, Seung-Yeol Nah b,1, Jong Seok Chae a, Guoying Bing c, Seung Woo Shin a, Tran Phi Hoang Yen a, In-Hyuk Baek a, Won-Ki Kim d, Tangui Maurice e, Toshitaka Nabeshima f, Hyoung-Chun Kim a,* a Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, South Korea b Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, South Korea c Department of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington, KY 40536, USA d Division of NanoSciences, Ewha Womans University, Seoul, South Korea e INSERM U 710 Universite de Montpellier II CC 105, place Eugene Bataillon, 34095 Montpellier cedex 5, France f Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466, Japan Received 16 November 2006; received in revised form 26 December 2006; accepted 17 January 2007 Available online February 2007 Abstract We showed that dextromethorphan (DM) provides neuroprotective/anticonvulsant effects and that DM and its major metabolite, dextrorphan, have a high-affinity for s1 receptors, but a low affinity for s2 receptors In addition, we found that DM has a higher affinity than DX for s1 sites, whereas DX has a higher affinity than DM for PCP sites We extend our earlier findings by showing that DM attenuated trimethyltin (TMT)induced neurotoxicity (convulsions, hippocampal degeneration and spatial memory impairment) in rats This attenuation was reversed by the s1 receptor antagonist BD 1047, but not by the s2 receptor antagonist ifenprodil DM attenuates TMT-induced reduction in the s1 receptor-like immunoreactivity of the rat hippocampus, this attenuation was blocked by the treatment with BD 1047, but not by ifenprodil These results suggest that DM prevents TMT-induced neurotoxicity, at least in part, via s1 receptor stimulation # 2007 Elsevier Ltd All rights reserved Keywords: Dextromethorphan; Anticonvulsant; Trimethyltin; s1 Receptor Introduction Intoxication with trimethyltin (TMT) leads to profound behavioral and cognitive deficits in both humans (Fortemps et al., 1978) and experimental animals (Dyer et al., 1982) In rats, TMT induces degeneration of pyramidal neurons in the hippocampus and cortical areas connected to the hippocampus (Brown et al., 1979; Chang and Dyer, 1983; Shin et al., 2005a) TMT intoxication impairs performance in water maze and radial arm maze tests (Alessandri et al., 1994; Earley et al., 1992; Hagan et al., 1988; Ishida et al., 1997) The s1 receptors appear to play an important neuromodulatory role in cholinergic neurotransmission (Maurice et al., * Corresponding author Tel.: +82 33 250 6917; fax: +82 33 255 7865 E-mail address: kimhc@kangwon.ac.kr (H.-C Kim) Both contributed equally to this work 0197-0186/$ – see front matter # 2007 Elsevier Ltd All rights reserved doi:10.1016/j.neuint.2007.01.008 1999, 2001) TMT-induced learning impairment is attributable in part to s receptor dysfunction (Maurice et al., 1999) Interestingly, systemic administration of the selective s1 ligand JO 1784 attenuates TMT intoxication (O’Connell et al., 1996) In the brain, s sites are found primarily in the hippocampus and in regions associated with motor function, such as the red nucleus and substantia nigra (McLean and Weber, 1988) Dextromethorphan (DM; 3-methoxy-17-methylmorphinan) is a non-narcotic morphinan derivative that has been used widely as an antitussive for almost 40 years DM has also attracted attention because of its neuroprotective properties (Choi, 1987; Kim et al., 1996, 2001a,b, 2003a,b; Shin et al., 2004, 2005b; Tortella et al., 1988, 1989, 1994; Zhang et al., 2004) We have demonstrated that the anticonvulsant and neuroprotective effects of DM may be mediated in part by s1 receptor activation (Kamei et al., 1996; Kim et al., 2003a; Maurice et al., 1999; Shin et al., 2005b) 792 E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 In the present study, to extend our previous understanding of the pharmacological effects mediated by DM, we assessed the role of s receptors in the pharmacological actions of DM on neurotoxicity (convulsive behaviors, neuronal degeneration, and learning impairment) induced by TMT We observed that DM-induced activation of s1 receptors is important for preventing neurotoxicity induced by TMT in rats Material and methods 2.1 Animals and drug treatment All animals were treated in strict accordance with the National Institutes of Health (NIH) Guide for the Humane Care and Use of Laboratory Animals (NIH Publication No 85-23, 1985; www.dels.nas.edu/ila) The Fischer 344 rat is known to be highly sensitive to TMT-induced neurotoxicity (MacPhail et al., 2003), and the TMT toxicity is enhanced in older rodents (Scallet et al., 2000) Consequently, we used 12-month-old Fischer 344 rats (Bio Genomics, Inc., Charles River Technology, Gapyung-Gun, Gyeonggi-Do, Korea) in this study The rats were maintained on a 12 h light:12 h dark cycle and were fed ad libitum They were allowed to adapt to these conditions for weeks before TMT administration (8 mg/kg, i.p.) DM (12.5 or 25.0 mg/kg, s.c.) was administered twice at an interval of h; at 30 after the second administration, TMT was administered The rats received DM once per day for 26 days following TMT administration and were sacrificed at 30 after the final dose of DM Control rats received an equal volume of saline For antagonism experiments, s1 receptor antagonist BD 1047 dihydrobromide (1 or mg/kg, i.p.; Tocris Cookson Ltd., Bristol, UK) or s2 receptors antagonist ifenprodil (5 or 10 mg/kg, i.p.; Tocris Cookson) was administered 15 before every dose of DM The doses of the drugs used are comparable with those used by Shin et al (2005b) 2.2 Radioligand binding To assay (+) [3H]SKF-10047 and [3H]DTG binding, frozen brains (without the cerebellum) from male Fischer 344 rats were thawed for h in 25 volumes of ice-cold mM Tris–HCl/10 mM K+-EDTA (pH 8.0 at 25 8C) and then homogenized for 15 s Homogenates were centrifuged (45,000  g, 15 min, 8C) The supernatant was discarded, and the pellets were resuspended in fresh ice-cold buffer and recentrifuged once The final pellets were resuspended in ice-cold mM Tris–HCl (pH 8.0 at 25 8C) (Munson and Rodbard, 1980) In a final volume of 0.5 mL, membrane suspensions were incubated with radioligand in the presence of mM Tris–HCl (pH 8.0 at 25 8C) for 60 The s1-binding assays were performed in the presence of 300 nM unlabeled MK801 to block binding of [3H]-(+)-SKF-10047 (final concentration, nM; New England Nuclear, Boston, MA; specific activity, 46.5 Ci/mmol) to PCP receptors In all s2-binding assays, mM unlabeled (+)-SKF 10047 was included to block binding of [3H]-DTG (final concentration, nM; New England Nuclear; specific activity, 40.8 Ci/mmol) to s1 receptors The reactions were terminated by the addition of mL ice-cold mM Tris–HCl (pH 8.0), followed by rapid filtration through Whatman GF/B filter papers (presoaked in 0.5% polyethyleneimine in H2O to reduce non-specific binding) using a Brandel cell harvester (Gaithersburg, MD) The filters were then rinsed twice with mL aliquots of the same buffer Absolute ethanol and Beckman Ready Value scintillation cocktail were added to the vials, which were counted the next day at an efficiency of about 36% The Ki values were determined using LIGAND software (Calderon et al., 1994; Munson and Rodbard, 1980) To assay [3H]TCP binding, whole brains minus the cerebellum were rapidly removed and disrupted in 45 volumes of ice-cold mM Tris–HCl buffer (pH 7.4) using a Brinkmann polytron homogenizer The homogenates were centrifuged (20,000  g, 29 min, 8C) The pellet was washed in fresh buffer, centrifuged twice more, resuspended in 45 volumes of assay buffer, and kept on ice until used (Calderon et al., 1994) Binding to homogenates was determined in a mL incubation volume consisting of 900 mL tissue (containing $0.8 mg protein), 50 mL [3H]TCP (44.8 Ci/mmol; New England Nuclear) for a final concentration of mM, and 50 mL buffer, test compound, or 10 mM TCP (for determination of non-specific binding) After a 90 incubation at 8C, the reaction was terminated by rapid filtration using a Brandel cell harvester The filters were washed with mL aliquots of ice-cold assay buffer, placed in counting vials with mL of CytoScint ES scintillation cocktail (ICN Biomaterials, Inc., Irvine, CA), and allowed to stand overnight before counting The inhibition constants (Ki) for the various compounds were calculated using GraphPAD software (ISI Software, Philadelphia, PA) (Calderon et al., 1994) The protein content was determined by the Coomassie protein assay (Pierce, Rockford, IL), using bovine serum albumin as the standard 2.3 Convulsive behavior Using a convulsion meter (CONVULS-1 Columbus Instruments, Columbus, OH), convulsive impulse counts were measured over a 20 period at h, h, h, day, days, days, days, week, weeks, and weeks after TMT treatment (Shin et al., 2005a) 2.4 Spatial reference memory test: Morris water maze task A hidden platform test was started at 21 days after TMT injection The apparatus was a circular water tank, 140 cm in diameter and 60 cm in height During testing, the tank was filled with water (23 Ỉ 8C) that was clouded with powdered milk A transparent platform was set inside the tank with its top submerged cm below the water surface in the center of one of the four quadrants of the maze, at a constant position throughout the test The tank was located in a large room with four extra-maze cues that were constant throughout the study (Jhoo et al., 2004; Zou et al., 2006) The movements of the animal in the tank were monitored with a video tracking system (EthoVision, Noldus, Netherlands) For each trial, the rat was put into the pool at one of the five positions, with the sequence of positions being selected randomly, and the time taken to reach the hidden platform was recorded Each rat was allowed 120 s to find the platform; if the rat was unable to find it within 120 s, the trial was terminated, and a maximum score of 120 s was assigned Trials were conducted for consecutive days, four times a day The results were analyzed statistically using a two-way ANOVA test for repeated measures, followed by Bonferroni’s test 2.5 Associative memory test: passive avoidance task Passive avoidance was measured using a Gemini Avoidance System (San Diego Instruments, San Diego, CA), which consists of two-compartment shuttle chambers with a constant current shock generator On an acquisition trial (21 days after TMT administration), each rat was placed into the start chamber, which remained darkened After 20 s, the chamber light was illuminated and the door was opened for the rat to move into the dark chamber freely Immediately after the rat entered the dark chamber, the door was closed and an inescapable scrambled electric shock (0.8 mA, s, once) was delivered through the floor grid The rat was then returned to its home cage Twenty-four hours later (22 days after TMT administration), each rat was again placed in the start chamber (retention trial) The interval between the placement in the lighted chamber and the entry into the dark chamber was measured as the step-through latency in the retention trial (maximum 300 s) (Jhoo et al., 2004) 2.6 Fluorescent Nissl staining At 26 days after TMT injection, the rats were perfused transcardially with 50 mL of 50 mM phosphate-buffered saline (PBS), followed by 500 mL of 4% paraformaldehyde in PBS, under pentobarbital anesthesia The brains were removed, post-fixed for 24 h in the same fixative at 8C, and then immersed in 30% sucrose in PBS until they sank The brains were cut into 35-mm transverse free-floating sections, using a horizontal sliding microtome Fluorescent Nissl staining was performed using NeuroTrace 530/615 red fluorescent Nissl stain solution (N-21482, Invitrogen), which is considered to be a specific marker for neurons (Rao et al., 2003) Briefly, sections were incubated in PBS containing 0.1% Triton1 X-100 (Sigma–Aldrich, St Louis, MO) for 10 and incubated in a 100-fold dilution of NeuroTrace solution for 20 The sections were then washed in PBS for h and mounted with an anti-fade agent (Fluoromount-G, Southern Biotech, Birmingham, AL) Digital images of fluorescent Nissl-stained E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 neurons were acquired at 100 and 300 magnifications, using a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Inc., Oberkochen, Germany) 2.7 Immunohistochemistry Immunohistochemistry for the s1 receptor was performed as described previously (Alonso et al., 2000), with minor modifications Prior to incubation with the primary antibodies, the sections were preincubated with 0.3% hydrogen peroxide in PBS for 30 min, then in PBS containing 0.4% Triton X-100 for 20 min, and then in 1% normal serum for 20 After a 48 h incubation with the anti-s1 receptor antibody (1:100; Alonso et al., 2000) at 8C, the sections were i ncubated with biotinylated anti-rabbit IgG (1:1000) for h and then immersed in Vectastain ABC reagents (Vector Laboratories, Burlingame, CA) for h The signal was detected using the chromogen 3,30 -diaminobenzidine (Sigma–Aldrich; St Louis, MO) Digital images of s1 receptor-immunoreactive cell populations in the hippocampus were acquired at 40 and 100 magnifications using an Olympus microscope (Olympus, Tokyo, Japan) with an attached Polaroid digital microscope camera (Polaroid, Cambridge, MA) and an IBM PC A region of interest (ROI) was created, and the level of immunoreactivity was calculated using Optimas software, Version 6.51 (Media Cybernetics, Inc Silver Spring, MD) 793 Fischer 344 rats were more pronounced than our previous findings in Sprague–Dawley rats (Shin et al., 2005b) 3.2 Role of s receptors in the pharmacological action of DM on TMT-induced convulsive behaviors Results Thirty-eight of 42 rats (90%) exhibited seizures after TMT injection Rats in the presence of saline, DM, DM + BD, or DM + ifenprodil showed no specific convulsive behaviors Two-way ANOVA for repeated measures revealed significance between group [F (7, 320) = 14.63, P = 2.85  10À9], time [F (8, À63 ] and group  time interaction 320) = 66.39, P = 1.83  10 [F (56, 320) = 2.83, P = 4.91  10À9] Post hoc Bonferroni’s test showed significant seizure activity induced by TMT (P < 0.05) Seizure activity was peaked at days and days after TMT, but almost no seizures were observed at weeks Although it did not alter the ratio of convulsions after TMT administration, 25 mg/kg of DM significantly inhibited the seizure activity induced by TMT (P < 0.05) BD 1047 counteracted the anticonvulsant effects of DM in a dosedependent manner (25 mg/kg of DM + ‘‘1 mg/kg of BD 1047’’ or ‘‘2 mg/kg of BD 1047’’ + TMT versus 25 mg/kg of DM + Saline + TMT, P < 0.05; 25 mg/kg of DM + mg/kg of BD 1047 + TMT versus 25 mg/kg of DM + mg/kg of BD 1047 + TMT, P < 0.05) Ifenprodil did not alter the anticonvulsant effects of DM (Fig 1) 3.1 The affinities of DM and dextrorphan (DX) for s1, s2, and phencyclidine (PCP) receptors 3.3 Role of s receptors in the pharmacological actions of DM on TMT-induced neuronal loss The affinities of DM and its major metabolite, DX, for s1, s2, and phencyclidine (PCP) receptors are shown in Table The Ki values for reference compounds are consistent with previously reported values (Calderon et al., 1994) DM and DX exhibited high-affinity and selectivity for s1 over s2 receptors DM had a higher affinity than DX for s1 sites, whereas DX had a higher affinity than DM for PCP sites These findings in Treatment with DM, BD 1047, ifenprodil, DM + BD 1047, or DM + ifenprodil did not change the cellular architecture of 2.8 Statistics The data were analyzed using a one-way ANOVA with Duncan’s new multiple range (DMR) test or a two-way ANOVA for repeated measures followed by Bonferroni’s test P values of less than 0.05 were deemed statistically significant Table The Ki values of compounds for s1, s2 and PCP receptors in rat brain membranes Compound DM DX Haloperidol (+)-SKF 10047 TCP Receptors s1 (nM) s2 (nM) PCP (nM) 142 Ỉ 38 344 Æ 47 68 Æ 16 84 Æ 22 ND 16873 Æ 2234 12987 Æ 1875 487 Æ 72 18785 Æ 4356 ND 8945 Ỉ 867 486 Ỉ 68 32423 Ỉ 4769 867 Ỉ 217 25 Ỉ s- and PCP-receptor binding experiments were performed on male Fischer 344 rat brain membranes The dissociation constant (Kd, nM) and the density of receptor sites (Bmax, fmole/mg protein) values for the radioligands used in this study are as follows; s1 receptor ([3H](+)-N-allylnormetazocine ((+)-SKF 10047), 48.7 Ci/mmole), Kd = 29 Ỉ 4, Bmax = 494 Ỉ 16), s2 receptor ([3H](+)1,3-di(2-tolyl)guanidine (DTG), 46.3 Ci/mmole), Kd = 28 Ỉ 4, Bmax = 673 Ỉ 45), PCP receptor (([3H]1-[1-(2-thienyl)-cyclohexyl] piperidine (TCP), 43.9 Ci/mmole), Kd = 25 Ỉ 3, Bmax = 796 Ỉ 49) Each value is the mean Ỉ S.E.M of experiments ND = not determined, DM = dextromethorphan, DX = dextrorphan Fig Effects of BD 1047, a s1 receptor antagonist, or ifenprodil, a s2 receptor antagonist, on the pharmacological action of DM in TMT-induced convulsive behavior in rats Values are expressed as mean convulsive impulse counts (Â1000/20 min) Ỉ S.E.M (Shin et al., 2005a) for six animals DM 12.5 or 25 = DM 12.5 or 25 mg/kg, s.c.; BD or = BD 1047 or mg/kg, i.p.; Ifen or 10 = ifenprodil or 10 mg/kg, i.p Convulsive behavior was not induced in the absence of TMT *P < 0.05 vs Saline + Saline + Saline, #P < 0.05 vs Saline + Saline + TMT, &P < 0.05 vs DM 25 + Saline + TMT, $P < 0.05 vs DM 25 + BD + TMT (two-way ANOVA for repeated measures, followed by Bonferroni’s test) 794 E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 Fig Photomicrographs of fluorescent Nissl-stained tissue sections at 26 days after trimethyltin (TMT) administration Hippocampal tissues from rats treated with saline (A) or dextromethorphan (DM) alone have clearly visible neuronal layers, whereas there is a significant loss of neurons from the pyramidal layer after TMT administration (B) Repeated treatment with DM (25 mg/kg, s.c.) significantly attenuates the loss of neurons from the CA1 and CA3/CA4 regions of the hippocampus (C) Treatment with BD 1047 (2 mg/kg, i.p.) significantly counteracts the neuroprotective effects of DM (25 mg/kg, s.c.) (D) Treatment with ifenprodil (10 mg/kg, i.p.) does not affect the pharmacological action of DM (E) Scale bars = 100 mm The percentage of neurons surviving within the stratum pyramidale was calculated using an image analysis system (Shin et al., 2005a) Sal = saline; DM = dextromethorphan; TMT = trimethyltin; BD = BD 1047; Ifen = ifenprodil Values are means Ỉ S.E.M for six rats *P < 0.01 vs saline + saline; #P < 0.05, ##P < 0.01 vs saline + saline + TMT (8 mg/kg); $P < 0.02, $$P < 0.01 vs saline + DM 25 mg/ kg + TMT (8 mg/kg) (ANOVA with DMR test) E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 the pyramidal layer of the hippocampus compared to controls At 26 days after TMT administration, a significant loss of neurons occurred within the CA1, CA3, and CA4 regions of the hippocampus (saline + saline + saline versus saline + saline + TMT, P < 0.01) Treatment with DM significantly prevented the TMT-induced loss of neurons within the CA1 (P < 0.01), CA3 (P < 0.05), and CA4 (P < 0.05) regions This neuroprotective effect of DM was reversed by BD 1047 (CA1, P < 0.05; CA3 and CA4, P < 0.001) but not by ifenprodil (Fig 2) 3.4 Role of s receptors in the pharmacological action of DM on TMT-induced cognitive dysfunction A two-way ANOVA revealed statistically significant differences among the groups (F (5, 798) = 4.52, P < 0.05), trials (F (19, 798) = 57.58, P < 0.05), and group-by-trial interactions (F (95, 798) = 1.60, P < 0.05) in the hidden platform task Rats in the presence of DM, DM + BD 1047 (data not shown), or DM + ifenprodil (data not shown) showed no significant changes in the escape latencies with the hidden platform test A post hoc test showed significantly impaired performances weeks after TMT injection (P < 0.05) Treatment with DM significantly attenuated the TMT-induced impairment of spatial 795 reference memory (P < 0.05), and this effect induced by DM was reversed by BD 1047 (P < 0.05) but not by ifenprodil (Fig 3A) Treatment with DM, DM + BD 1047 (data not shown), or DM + ifenprodil (data not shown) also did not affect step-through latencies for the retention trial in the passive avoidance task Similar to the hidden platform task, the stepthrough latency was reduced significantly by TMT administration (P < 0.01) Treatment with DM significantly ameliorated the impairments in associative memory induced by TMT (P < 0.01), and this effect induced by DM was also reversed by BD 1047 (P < 0.05) but not by ifenprodil (Fig 3B) 3.5 Effect of DM on the reduction in s1 receptor-like immunoreactivity (s1-IR) induced by TMT In saline-, DM-, DM + BD 1047-, or DM + ifenprodiltreated animals, moderate s1 receptor-like immunoreactivity (s1-IR) was observed in the pyramidal cells and granule cells of the dentate gyrus in the hippocampus (Fig 4, Table 2) In contrast, at 26 days after TMT administration, s1-IR was significantly reduced in the stratum pyramidale (sp) of the CA1 (P < 0.01), CA3 (P < 0.05) and CA4 (P < 0.05) regions Treatment with DM significantly attenuated the TMT-induced reduction in s1-IR (CA1 and CA2 regions; P < 0.01 versus saline + saline + TMT) BD 1047 reversed the DM-mediated attenuation, mainly in the CA1 region (P < 0.01) However, ifenprodil did not significantly affect DM’s pharmacological actions in response to TMT (Fig 4) Discussion Fig Pretreatment with DM significantly attenuates TMT-induced cognitive dysfunction, as evaluated by a hidden platform test (A) and a passive avoidance task (B) Sal = saline; DM = dextromethorphan; TMT = trimethyltin; BD = BD 1047; Ifen = ifenprodil Values are means Æ S.E.M for (A) or 10 (B) animals * P < 0.05, **P < 0.01 vs saline + saline + saline; #P < 0.05, ##P < 0.01 vs saline + saline + TMT; &P < 0.05 vs DM + saline + TMT (two-way ANOVA for repeated measures followed by Bonferroni’s test for the hidden platform test or ANOVA with a DMR test for the passive avoidance task) The main finding of this study was that DM can prevent TMT-induced convulsions, hippocampal neuronal degeneration, and spatial memory impairment in aged rats, apparently by activating the s1 receptor In addition, we showed that DM is a selective s1 ligand with relatively modest affinity for the s2 and NMDA-linked phencyclidine (PCP) sites and that this was more pronounced in Fischer 344 rats than in the Sprague– Dawley rats studied previously (Shin et al., 2005b) These results suggest that PCP sites are not always required for the neuroprotective effects of DM (Kim et al., 2003a; Shin et al., 2005b), although DM is metabolized primarily to the phenolic PCP-like compound dextrorphan (DX), which has prominent anticonvulsant/neuroprotective effects in rodent models of experimental epilepsy (Kim et al., 2003b; Tortella et al., 1988, 1994) Interestingly, other high-affinity DM ligands such as carbetapentane also exhibit relatively high-affinity for the s1 site We have demonstrated previously that the neuroprotective action of carbetapentane is at least partly mediated by s1 receptor modulation (Kim et al., 2001c) The results of these binding studies are consistent with the idea that the brain sites to which radiolabeled DM binds with high-affinity have binding characteristics and regional distributions remarkably similar to those of s1 receptors (Tortella et al., 1989) Thus, a functional relationship may exist between neuroprotective activity and high-affinity DM-binding/s1 sites 796 E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 Fig Representative photomicrographs of rat hippocampus sections immunostained for the s1 receptor after TMT treatment Hippocampal sections from control (saline + saline + saline or DM + saline + saline) animals show moderate s1-like immunoreactivity (s1-IR) in the pyramidal neurons and dentate gyrus The s1-IR is apparently reduced in the pyramidal neurons at 26 days after TMT DM prevented this reduction in s1-IR primarily in the CA1 and CA2 regions BD 1047 counteracted the DM-mediated attenuating effects, mainly in the CA1 region However, ifenprodil did not affect DM’s protective effects Scale bars = 300 mm BD 1047 is an antagonist of both s1 and s2 receptors, although it has a 51-fold greater affinity for s1-binding sites than for s2 sites (Matsumoto et al., 1995) Ifenprodil selectively binds s2 sites at 37 8C (Hashimoto and London, 1993; Hashimoto et al., 1994), but is selective for the N-methyl-Daspartate (NMDA)-polyamine site at 8C in rat brain (Hashimoto et al., 1994) Ifenprodil also binds to the dopamine transporter (DAT) and blocks dopamine uptake (Witkin and Acri, 1995) Previous demonstrations have suggested that s2 receptors regulate DAT activity through protein kinase C (PKC) modulation (Derbez et al., 2002; Nuwayhid and Werling, 2006) or in a calcium-dependent manner (Derbez et al., 2002) 797 E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 Table Effects of DM on the reduction in s1 receptor-like immunoreactivity induced by TMT in the rat hippocampus Treatments Sal + Sal + Sal DM + Sal + Sal Sal + Sal + TMT DM + Sal + TMT DM + BD + TMT DM + Ifen + TMT Neurons (Stratum pyramidale: sp) DG CA1 CA2 CA3 CA4 1.6 Ỉ 0.2 1.4 Ỉ 0.3 0.2 Ỉ 0.1** 1.8 Ỉ 0.2# 0.3 Ỉ 0.1$ 1.6 Ỉ 0.2 1.0 Ỉ 0.2 1.2 Æ 0.3 0.8 Æ 0.2 1.8 Æ 0.3# 1.7 Æ 0.3 1.5 Ỉ 0.2 0.6 Ỉ 0.1 0.5 Ỉ 0.2 0.2 Ỉ 0.1 * 0.5 Ỉ 0.2 0.2 Ỉ 0.1 0.5 Ỉ 0.1 0.6 Ỉ 0.1 0.5 Ỉ 0.1 0.1 Æ 0.1* 0.3 Æ 0.1 0.1 Æ 0.1 0.2 Æ 0.2 1.0 Ỉ 0.2 1.2 Ỉ 0.2 1.3 Ỉ 0.3 1.3 Ỉ 0.1 1.4 Ỉ 0.3 1.2 Ỉ 0.2 Each value is the mean Ỉ S.E.M of animals Sal = saline, DM = DM 25 mg/kg, s.c., BD = mg/kg, i.p of BD 1047 Ifen = 10 mg/kg, i.p of ifenprodil *P < 0.05, P < 0.01 vs Sal + Sal + Sal, #P < 0.01 vs Sal + Sal + TMT, $P < 0.01 vs DM + Sal + TMT (one-way ANOVA with DMR) ** Ifenprodil also produces an antagonistic effect against the a1adrenergic receptor (Gotti et al., 1988) The release of dopamine (Earley et al., 1992) or norepinephrine (Andersson et al., 1995; Earley et al., 1992) does not seem to change after TMT administration in rat hippocampus (Nittykoski et al., 1998) Although ifenprodil possesses multifunctional effects, we used ifenprodil as a s2 receptor antagonist because a highly selective s2 receptor antagonists is not available The behavioral toxicities we observed are consistent with a previous study showing that the administration of this neurotoxin produced convulsions followed by impaired performance on spatial memory tests (Dyer et al., 1982; Hagan et al., 1988) TMT-induced behavioral effects were accompanied by a significant reduction in the binding of [3H] (+)-pentazocine to s sites, affecting mainly hippocampal pyramidal neurons (O’Connell et al., 1994) The immunodistribution profile of the s1 receptor observed in the present study is consistent with the autoradiographic distribution of [3H]-(+)pentazocine binding sites in the rodent brain (Walker et al., 1992) As [3H]-pentazocine has been shown to bind primarily to s1 receptor sites (Quirion et al., 1992; Kitaichi et al., 2000), we raise the possibility that the TMT-induced reduction in s1-IR may be attributable, in part, to s1 receptor loss Although DM treatment significantly attenuated the TMT-induced reduction of s1-IR in the CA1 and CA2 regions, and it appeared to prevent the reduction in the CA3 region without reaching statistical significance, BD 1047 counteracted this protective effect only in the CA1 region, which possesses a high density of NMDA receptors A marked reduction in PCP receptor density following TMT treatment was also observed The PCP receptor consists of an allosteric site located on the NMDA receptor ion channel (Loo et al., 1986) NMDA/PCP receptors have been shown to exist on intrinsic neurons in the cortex (Greenamyre et al., 1985); however, unlike the cortex, studies on hippocampal formation have suggested that NMDA/PCP receptors exist as presynaptic autoreceptors regulating the release of glutamate for excitatory amino acid nerve terminals PCP receptor density was found to be the highest in the hippocampal CA1 region (pyramidal and molecular layers), olfactory tubercle, and amygdala The density of PCP receptors was relatively low in other brain regions PCP receptors appear to be more sensitive to the effects of TMT than muscarinic cholinergic receptors, with losses in the hippocampus (CA1 and CA4) of more than 50% (O’Connell et al., 1994) NMDA/PCP receptors are believed to play a role in regulating the cholinergic system, and PCP receptors have been shown to modulate the release of cortical (Lodge and Johnson, 1985) and striatal acetylcholine (Snell and Johnson, 1986) Cortical acetylcholine is released from nerve terminals that project from the nucleus basalis of Meynert (Nbm) (Johnson et al., 1979), and NMDA/PCP receptors located in the Nbm may be involved in acetylcholine release from these terminals (Maragos et al., 1991) Interestingly, it has been suggested that NMDA/PCP receptor loss, which occurs in Alzheimer’s disease (Greenamyre et al., 1987), may be secondary to degeneration of the cholinergic system, suggesting a similarity to the TMT case The selective protection of neurons in the CA1 field by NMDA receptor antagonists (dizocilpine, ketamine, and PCP) may be related to the major distribution of NMDA receptors in the CA1 field (Tortella et al., 1989) Thus, one of the neuroprotective mechanisms of DM, acting as a s1 ligand, may be at least partly related to the blockade of NMDA receptorrelated signal transduction pathways in rats However, as DM possesses mild NMDA receptor antagonistic properties, its actions at s1 receptors may not fully account for DM’s pharmacological effects In this study, the disorder of pyramidal cells in the CA2 region induced by TMT was weaker than that in the CA1 and CA3/CA4 regions It is known that the pyramidal cells in CA2 are relatively resistant to glutamate-induced neurodegeneration (Mattson and Kaster, 1989; Onley et al., 1979), whereas the pyramidal cells in CA1 and CA3/CA4 are more vulnerable (Mattson and Kaster, 1989; Onley et al., 1979) We observed that DM attenuated the loss of neurons from CA1 and CA3/ CA4 after TMT administration This protection appeared to be most pronounced in the CA1 region Similarly, DM has been shown to protect against brain damage associated with seizures, especially damage in the CA1 field of the hippocampus (Kim et al., 1999) DM binding sites may represent functional receptors responsible for mediating the anticonvulsant/neuroprotective effects of this drug (Tortella et al., 1989) The localization of [3H]-DM to the hippocampal formation suggests that CA1 is a primary site for the neuroprotective actions of DM (Tortella et al., 1989) Interestingly, a variety of calcium channel antagonists also compete for the high-affinity DM (presumably s1) site (Tortella et al., 1989) We previously demonstrated that DM affects the 798 E.-J Shin et al / Neurochemistry International 50 (2007) 791–799 dihydropyridine-binding site of the L-type voltage-sensitive calcium channel (L-VSCC), by modulating the expression of the AP-1 transcription factor (Shin et al., 2004) DM has been shown to block voltage-gated calcium currents in neurons (Netzer et al., 1993) The L-VSCC antagonist nimodipine enhanced hippocampal-dependent learning in aged rabbits at a dose that also increases the excitability of aged CA1 neurons (Straube et al., 1990) Moreover, several investigators have reported that nimodipine enhances hippocampal-dependent memory in a variety of animals and tasks (Deyo et al., 1989; Levere and Walker, 1992; Sandin et al., 1990) Similar to the novel calcium channel blocker levemopamil, DM attenuated the spatial learning deficit and neuronal damage induced by global ischemia in rats (Block and Schwarz, 1998) We also previously demonstrated that DM directly prevents the behavioral effects induced by the L-type calcium channel activator BAY k-8644, in a dose-dependent manner (Shin et al., 2004) We cannot rule out the possibility that DM attenuates neuronal degeneration as well as learning impairment via inhibition of the L-VSCC Thus, the neuroprotective action mediated by DM might be implicated in complex mechanisms regulated by s1 and NMDA receptors, and the voltage-gated calcium channels, although more evidence is needed In conclusion, although systemic DM administration at relatively high doses produces behavioral side effects (Holtzman, 1994; Jhoo et al., 2000; Shin et al., 2005b), the application of the optimal dose of DM is beneficial in blocking TMTinduced neurotoxicity, apparently by activating the s1 receptor Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project (A020007), Ministry of Health & welfare, Republic of Korea, by a grant (M103KV01001306K220201310) from the Brain Research Center from the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea, and by Brain Korea 21 project Equipments at the Institute of Pharmaceutical Science 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