Original article 181

The effect of ketamine on N-methyl-D-aspartate receptor subunit expression in neonatal rats Li-Chun Hana, Li-nong Yaob, Sheng-xi Wuc, Yong-hui Yangb, Li-Xian Xua,M and Wei Chaib,M

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Background and objective Ketamine has been widely used in paediatric anaesthesia but its inﬂuence on development in infants and toddlers still remains unclear. In order to elucidate the inﬂuence of ketamine on brain development in neonatal rats, semiquantitative reverse transcriptase PCR, quantitative reverse transcriptase PCR and immunohistochemistry assays were performed to detect the expression of N-methyl-D-aspartate receptor subtypes expression.

Methods Seven-day-old rats were divided into two random groups. All of them were injected with ketamine intraperitoneally at postnatal day (PND) 7; one group was sacriﬁced at PND 7, but the other group was sacriﬁced at PND 28. Each group was divided into ﬁve random subgroups.

Results In the semiquantitative reverse transcriptase PCR and quantitative reverse transcriptase PCR experiments, ketamine treatment caused a marked increase in mRNA expression in all subtypes at PND 7 and in NR2A subtypes at PND 28. Immunohistochemistry results indicated that NR2A, 2B and 2C receptor protein increased

signiﬁcantly at PND 7, and NR2A receptor protein increased at PND 28.

Conclusions Exposure to ketamine resulted in an increase in N-methyl-D-aspartate receptor subunits at PND 7, and this increase persisted to PND 28 in NR2A. Eur J Anaesthesiol 27:181–186 Q 2010 European Society of Anaesthesiology.

European Journal of Anaesthesiology 2010, 27:181–186

Keywords: hippocampus, immunohistochemistry, ketamine, N-methyl-D- aspartate, quantitative reverse transcriptase PCR, semiquantitative reverse transcriptase PCR

aDepartment of Anesthesiology, School of Stomatology, bDepartment of Anesthesiology, Tangdu Hospital and cDepartment of Anatomy, Histology and Embryology, K. K. Leung Brain Research Centre, Fourth Military Medical University, Xi’an, PR China

Correspondence to Dr Li-Xian Xu, Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, PR China E-mail: kqmzk@fmmu.edu.cn

Correspondence to Dr Wei Chai, Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, 710038, PR China E-mail: tdmzka@fmmu.edu.cn

Received 12 February 2009 Revised 14 July 2009 Accepted 14 July 2009

Introduction Ketamine is used as a general paediatric anaesthetic for surgical procedures in infants and toddlers. It has been reported that it blocks excitatory synaptic transmission by acting as a noncompetitive N-methyl-D-aspartate (NMDA) receptor ion channel blocker [1].

NMDA receptors play important roles in excitatory synap- tic transmission, in brain cell migration, differentiation, survival and activity-dependent synaptic plasticity under- lying learning and memory [2]. Ikonomidou et al. [3] was the ﬁrst to demonstrate that the NMDA receptor antagonists MK-801 and ketamine induce neuroapoptosis in several encephalic regions in rats at postnatal day (PND) 7 after treatment for 8 h. Since then, groups have veriﬁed that NMDA receptor antagonists can provoke neuroapop- tosis in many encephalic regions [4–6].

Many studies have shown that NMDA receptor anta- gonists can change the expression of NMDA receptor subtypes. In the present study, in order to investigate the inﬂuence of ketamine on brain development in neonatal

rats, we aimed to show that ketamine, administered as a classic general anaesthetic agent for short-term anaesthe- sia caused changes in the expression of NMDA receptor subtypes NR1, NR2A, NR2B and NR2C at the mRNA level using quantitative reverse transcriptase (qRT) PCR and semiquantitative reverse transcriptase (sqRT) PCR techniques and at the protein level using immuno- histochemistry.

Materials and methods Animals Seven-day-old male and female Sprague Dawley rats (body weight 11.1–17.5 g) were housed in plastic cages with their mothers and maintained on a 12 : 12 h light/ dark cycle at 22–258C ambient temperature with food and water available ad libitum for the mothers. All of the experimental procedures were approved by the Animal Use and Care Committee for Research and followed the ethical guidelines for investigation of experimental pain in conscious animals [7].

M Dr Li-Xian Xu and Dr Wei Chai contributed equally to the writing of this article.

Rats (n ¼ 40) were divided into two random groups. In one group (n ¼ 20) the rats were injected with ketamine

0265-0215 (cid:1) 2010 Copyright European Society of Anaesthesiology

DOI:10.1097/EJA.0b013e328330d453

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182 European Journal of Anaesthesiology 2010, Vol 27 No 2

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Table 1 Summary of experimental protocol

PND 7

C K1 K2

Control group 100 mg kg(cid:1)1 ketamine persistently for 6 h 100 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day

K3 K4

50 mg kg(cid:1)1 ketamine persistently for 6 h 50 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day

PND 28

C K1 K2

Control group 100 mg kg(cid:1)1 ketamine persistently for 6 h 100 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day

K3 K4

50 mg kg(cid:1)1 ketamine persistently for 6 h 50 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day

intraperitoneally (i.p.) at PND 7 [8] and sacriﬁced within 24 h. In the other group (n ¼ 20) the rats were also injected with ketamine at PND 7 and were sacriﬁced at PND 28. Each group was divided into ﬁve random subgroups (n ¼ 4 per subgroup). The control group received 0.9% physio- logical saline. The other four groups received i.p. injec- tions of ketamine (K1–K4) [9] (see Table 1).

Semiquantitative reverse-transcriptase PCR and quantitative real-time reverse-transcriptase PCR All animals from the different groups (n ¼ 4) were killed by decapitation under ether anaesthesia. sqRT-PCR was used to qualitatively assess the effect of NMDA subtype receptor expression. qRT-PCR was then applied in order to further quantify the observed effects. Table 2 sum- marizes information about the oligonucleotide primers used in this study. All primer sequences were checked in GenBank (National Center for Biotechnology Infor- mation, Bethesda, Maryland, USA) to avoid inadvertent sequence homologies. b-actin was used as an internal control. Animals were decapitated under ether anaesthe- sia, and the hippocampus was quickly dissected out and frozen at (cid:1)808C until use. Total RNA was isolated using Trizol reagent (Invitrogen, Virginia, USA), according to the manufacturer’s instructions, and then reverse transcribed with Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Invitrogen) and oligo(dT)12–18 primers.

For sqRT-PCR, a PCR reaction mixture containing 10 mmol l(cid:1)1 Tris (pH 8.3), 50 mmol l(cid:1)1 KCI, 1.5 mmol l(cid:1)1 MgCI2, 100 ml of deoxyribonucleotide triphosphate

(dNTP), 2.5 units of Taq DNA polymerase (Takara, Kyoto, Japan), 0.5 ml of synthesized cDNA and 20 pmol of each sense and antisense primer pair. The PCR reac- tion was performed for 30 cycles using a PTC-100 Pro- grammed Thermal Controller (MJ Research, Watertown, Massachusetts, USA) as follows: 1 min at 938C, 30 s at appropriate annealing temperature (Table 2) and 1 min at 728C, with 1 min of 938C treatment before starting the thermal cycles, and, ﬁnally, an 8 min extension at 728C was conducted. PCR was performed simultaneously on control and experimental rat samples, with the internal controls (b-actin) running in parallel with the examined mRNAs. In all reverse transcriptase PCR experiments including negative controls, in which template RNA or reverse transcriptase was omitted, no PCR product was detected. Ten microlitres of each PCR product was electrophoresed on a 3% agarose gel containing ethidium bromide. Resulting gel bands were visualized in an ultraviolet (UV) transilluminator, and images were cap- tured with an eight-bit charge coupled device (CCD) camera (Ultra-Violet Products, Upland, California, USA).

Quantitative PCR was set up using SYBR Green- containing premix from Takara. The reverse transcrip- tase reaction product (100 ng) was ampliﬁed in a 25 ml reaction with 12.5 SYBR Premix EX Taq (Takara, Shiga, Japan). Samples were heated to 908C for 30 s, and then ampliﬁed for 40 cycles consisting of 958C for 15 s and 608C for 15 s. Relative quantiﬁcation of NMDA subtype receptors was performed by a comparative threshold cycle method. All data are expressed as mean (cid:2) SEM. Experimental groups were compared by analysis of var- iance. P values of less than 0.05 were considered to be statistically signiﬁcant.

Immunohistochemistry Rats in the control group and those in the K1 and K3 subgroups (n ¼ 4), which were sacriﬁced on PND 7 or PND 28, were perfused transcardially with 100 ml of 0.01 mol l(cid:1)1 PBS (pH 7.4), followed by 100 ml of 4% (w/v) paraformal- dehyde and 75% (v/v) saturated picric acid in 0.1 mol l(cid:1)1 phosphate buffer (pH 7.4). The brains were then removed immediately and placed into the same fresh ﬁxative for an additional 2 h at 48C. Subsequently, the brains were

Table 2 Oligonucleotide primers used in the quantitative reverse transcriptase PCR and semiquantitative reverse transcriptase PCR experiments

Subunits

Subunits primer sequences

Expected size (bp)

Annealing temperature (8C)

R1

NR2A

NR2B

NR2C

b-actin

50-ATGGCATCATCGGACTTCAG-30 50-GGGCTCTTGGTGGATTGTCA-30 50-ATTCATCCCTTCGTTGGTTG-30 50-GCTATGGGCAGGCAGAGAAG-30 50-GTGGGCACTGAGGACTTGTT-30 50-TGTACGACATCAGCGAGGAC-30 50-TCGTATTCCTCCAGCACCTT-30 50-GATCCAGCCACTCACCGTAG-30 50-TGGTGGGTATGGGTCAGAAGGACTC-30 50-CATGGCTGGGGTGTTGAAGGTCTCA-30

431

395

319

300

265

58

56

56.3

55

57.3

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Effect of ketamine on NMDA receptor subunit expression Han et al. 183

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placed into 30% (w/v) sucrose solution in 0.1 mol l(cid:1)1 phos- phate buffer (pH 7.4) overnight at 48C (the sucrose solution contained 0.02% NaN3), and then cut serially into 30 mm thick coronal sections by the use of a freezing microtome (Kryostat 1720; Leitz, Mannheim, Germany). The sections were placed into ﬁve different dishes accord- ing to their numerical order while cutting (e.g. sections 1 and 7 in dish 1; sections 2 and 8 in dish 2; sections 3 and 9 in dish 3; sections 4 and 10 in dish 4; and sections 5 and 11 in dish 5). Each dish usually contained 28–32 sections. All sections were washed carefully with 0.01 mol l(cid:1)1 PBS. The sections in the ﬁrst three dishes were used for immu- nohistochemistry for NR2A–2C. Brieﬂy, the sections were incubated at 48C sequentially with: a mixture of rabbit anti-NR2A, 2B and 2C serum (1 : 200 dilution; Elek mol- nar) for 24 h; biotinylated goat antirabbit immunoglobulin G (1 : 200 dilution; Vector) for 2 h; and avidin-labelled horseradish peroxidase compound (1 : 100 dilution; Vector) for 1 h. The diluent used for all antibodies was 0.05 mol l(cid:1)1 PBS containing 5% (v/v) normal donkey serum, 0.5% (v/v) Triton X-100, 0.05% (w/v) sodium azide (NaN3) and 0.25% (w/v) carrageenan (pH 7.3). In the fourth dish, normal rabbit serum was used instead of rabbit anti-NR2A, 2B and 2C serum, and the following steps were the same as mentioned above. The ﬁfth dish was used for Nissl stain- ing in order to locate a positive construction. The sections were rinsed at least three times in 0.01 mol l(cid:1)1 PBS (pH 7.4) after each incubation, for at least 10 min. The sections were coloured with diaminobenzidine (DAB) and H2O2, then sections were mounted onto clean glass slides, air dried and cover-slipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine (antifading agent) in 0.01 mol l(cid:1)1 PBS. Finally, the sections were studied under a microscope.

Fig. 1

) n

i t c a - β / ( s l e v e l

A N R m e v i t a l e R

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0

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K4


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Results Semiquantitative reverse-transcriptase PCR and quantitative real-time reverse-transcriptase PCR Reverse transcriptase PCR revealed mRNA expression of NR1, NR2A, NR2B and NR2C receptor subtypes as well as the b-actin in the rat hippocampus. The size of the bands for each receptor corresponded to the expected cDNA fragment size based on the choice of oligonucleo- tide primers (Table 2).

120

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C

#

K1


K2

NR2B


K3


K4


20

Ketamine treatment caused a marked increase in NR1, NR2A, NR2B and NR2C mRNA expressions at PND 7 when compared with the control (P < 0.05; Figs 1 and 2). At PND 28, ketamine treatment resulted in a different pattern of NMDA receptor mRNA expression from that at PND 7. Moreover, we also observed that the expres- sion of NR2A mRNA was signiﬁcantly increased not only at PND 7 but also at PND 28 (P < 0.05; Figs 1 and 2), but no signiﬁcant change was observed in NR1, NR2B or NR2C mRNA expression at PND 28 when compared with the control group (P > 0.05; Figs 1 and 2). More- over, no change was found among groups K1–K4, so we

0

C

K1

K2

K3

K4

NR2C

Histogram summary for relative expression levels of NR1, NR2A, NR2B and NR2C mRNA in the hippocampus of PND 7 and PND 28 rats after administration of ketamine (n ¼ 16, mean (cid:2) SE). C, control group; K1– K4, ketamine-treated groups. (cid:3)P < 0.05, compared with control group at PND 7; $P < 0.05, compared with control group at PND 28; and #P < 0.05, control group at PND 7 compared with the control group at PND 28. PND, postnatal day.

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Electrophoresis strip of NR1, NR2A, NR2B and NR2C mRNA expression in the hippocampus of PND 7 and PND 28 rats after administration of ketamine. The expression of b-actin mRNA was used as an internal control. C, control group; K1–K4, ketamine-treated groups (see Materials and methods). PND, postnatal day.

concluded that the dosage and schedule of exposure might not inﬂuence the effects of ketamine.

In addition, NR1, NR2A and NR2C subtype receptor mRNA expression at PND 28 exhibited a signiﬁcant increase when compared with PND 7 in the control group, but NR2B exhibited a reverse trend (P < 0.05; Figs 1 and 2).

subgroups was higher than that in the control group: NR2A (control, 1.7 (cid:2) 0.1; K1, 7.2 (cid:2) 3.5; K3, 9.9 (cid:2) 4.3), NR2B (control, 1.2 (cid:2) 0.1; K1, 8.8 (cid:2) 3.4; K3, 9.5 (cid:2) 4.5) and NR2C (control, 3.8 (cid:2) 1.5; K1, 16.2 (cid:2) 6.2; K3, 16.8 (cid:2) 6.8). At PND 28, only the NR2A subtype receptor expression was signiﬁcantly increased in the K1 and K3 groups com- pared with the control group: NR2A (control, 10.8 (cid:2) 3.2; K1, 28.5 (cid:2) 4.5; K3, 32.2 (cid:2) 5.4). There was no signiﬁcant difference between the K1 and K3 subgroups.

Discussion In the present study, we observed that neonatal rats receiving ketamine, administered for short periods and at moderate doses, could upregulate the NR1, NR2A, 2B and 2C receptor subtype mRNA and NR2A, 2B and 2C receptor subtype protein at PND 7, and the NR2A recep- tor mRNA and protein expression increase persisted to PND 28. It is well accepted that NMDA receptor antagonists can induce an increase in some NMDA recep- tor subtype mRNA expression during critical periods of [10,11]. Chronic treatment of cultured development neurons from neonatal rat brains with amino-phosphono- pentanoate 5 (AP-5), an NMDA receptor antagonist, increased NR2B mRNA expression, as well as NR1 and NR2A/B polypeptides [12]. Also, increased expression of excitatory amino acid receptor subunit mRNA may con- tribute to the enhanced vulnerability to excitotoxic injury that has been observed after MK-801 treatment [13].

Previous studies [14,15] showed that NMDA receptors participated in central nervous system (CNS) regulation and appeared to regulate the excitatory synaptic trans- mission and synaptic plasticity underlying learning and in glutamate transmission, memory. Abnormalities particularly involving overstimulation of NMDA recep- tors, have been implicated in apoptosis, abnormal axonal arborization and aberrant CNS development [16]. In 2002, Olney [17] observed that NMDA receptor anta- gonists interfered with CNS development and caused abnormalities in morphology and function. Neonatal rats receiving AP-5 or MK-801 during the ﬁrst 2 weeks of life developed abnormal axonal arborizations in the retinal connections to the superior colliculus, interfering with normal visual responses [18]. Neurons with NMDA receptors are exquisitely sensitive to overstimulation, and they are similarly sensitive to understimulation during synaptogenesis. Too much NMDA receptor stimulation triggers excitotoxic neurodegeneration, but too little triggers apoptotic neurodegeneration [17].

Immunohistochemistry of NR2A, 2B and 2C subtype protein Representative photomicrographs of NR2A, 2B and 2C subtype receptor staining and statistical analysis with the Student’s t-test among the groups are presented in Fig. 3. At PND 7, NR2A, 2B and 2C subtype receptor expression was observed mainly in the hippocampus, and the corresponding receptor expression in the K1 and K3

Ketamine treatment may alter glutamatergic synaptic transmission through NMDA receptors and contributes to the upregulation of NMDA receptor mRNA expres- sion, but how increased expression of excitatory amino acid receptor subunit mRNA contributes to excitotoxic injury and neuroapoptosis remains controversial. Some researchers have found that glutamate binding to NMDA

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Effect of ketamine on NMDA receptor subunit expression Han et al. 185

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Representative photomicrographs of brain sections showing N-methyl-D-aspartate receptor subtype neurons in the CA1 region of the hippocampus in rat brains in the control (a, d, g), K1 (b, e, h) and K3 (c, f, i) groups. (m and n) The statistical analysis of NMDA receptor subtype neurons in different groups on PND 7 and PND 28. Ketamine (K1 and K3) produced much higher expressions of NR2A, 2B and 2C on PND 7, and NR2A on PND 28 than the control groups; scale bar ¼ 100 mm in (l) for (a)–(l). (cid:3)Groups K1 and K3 are statistically significantly different (P < 0.05) from group C in the CA1 region on PND 7 and PND 28. NMDA, N-methyl-D-aspartate; PND, postnatal day.

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186 European Journal of Anaesthesiology 2010, Vol 27 No 2

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receptors caused Ca2þ inﬂux that activates second mes- sengers, thus regulating neuronal migration, differen- tiation and synaptic plasticity [19–22]. High concen- trations of glutamate resulted in too much Ca2þ inﬂux, causing excitotoxicity. Here, we showed that ketamine could increase mRNA expression of NMDA receptor subtypes. However, if this block is eliminated, the increased expression of NMDA receptors might result in increased glutamate binding, thus inducing excessive Ca2þ inﬂux. This altered level of Ca2þ inﬂux could lead to neuroapoptosis. Further studies on ketamine-induced changes in apoptosis and Ca2þ-binding proteins are necessary to elucidate this possibility.

Glutamate and NMDA receptors mediate a variety of complicated biological processes such as induction, generation, differentiation, apoptosis, migration, synaptic formation and neural network establishment [2,14– 15,17,23]. Synaptic and extrasynaptic NMDA receptors have fundamentally different effects on the fate of neurons. Synaptic NMDA receptors promote survival, whereas extrasynaptic NMDA receptors trigger neuronal degeneration and cell death [24,25]. Therefore, an increase in the expression of some NMDA receptor subunits might result in changes in subunit composition and possibly inﬂuence all of these developmental pro- cesses. Although the mechanisms underlying upregula- tion of expression were not clear, Wang et al. [26] suggested that increased NMDA receptor expression might be due to an increased rate of transcription or decreased rate of degradation.

Taken together, these studies suggest that neonatal animals receiving chronic NMDA receptor antagonists developed abnormal neuronal structure and altered CNS function. Our present study demonstrated that ketamine, administered for short periods and at clinical application doses, induced changes in NMDA receptor subunit com- position and increased some NMDA receptor subtype expressions in neonatal rats, and this effect persisted to PND 28. The expressions of NMDA receptor subunits showed a period speciﬁcity at both the transcriptional and translational levels. Ketamine as a kind of NMDA recep- tor antagonist might change the period-speciﬁc expres- sion of some NMDA receptor subunits.

3 Ikonomidou C, Olney JW, Bosch F, et al. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 1999; 283:70–74.

4 Jevtovic-Todorovic V, Hartman RE, Izumi Y, et al. Early exposure to common

anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deﬁcits. J Neurosci 2003; 23:876–882.

5 Luk KC, Kennedy TE, Sadikot AF. Glutamate promotes proliferation of

striatal neuronal progenitors by an NMDA receptor-mediated mechanism. J Neurosci 2003; 23:2239–2250.

6 Mellon RD, Simone AF, Rappaport BA. Use of anesthetic agents in neonates and young children. Anesth Analg 2007; 104:509–520. 7 Zimmermann M. Ethical guidelines for investigations of experimental pain in

conscious animals. Pain 1983; 16:109–110.

8 Mickly GA, Remmers-Roeber DR, Crouse C. Ketamine blocks a taste- mediated conditioned motor response in perinatal rats. Pharmacol Biochem Behav 2000; 66:547–552.

9 Wolfensohn S, Lloyd M. Handbook of laboratory animal management and

welfare. Oxford, UK: Wiley-Blackwell Inc.; 2003; 185–186.

10 Trevisan L, Fitzgerald LW, Brose N, et al. Chronic ingestion of ethanol up- regulates NMDAR1 receptor subunit immunoreactivity in rat hippocampus. J Neurochem 1994; 62:1635–1638.

11 Hinoi E, Fujimori S, Nakamura Y, et al. Constitutive expression of

heterologous N-methyl-D-aspartate receptor subunits in rat adrenal medulla. J Neurosci Res 2002; 68:36–45.

12 Follesa P, Ticku MK. NMDA receptor upregulation: molecular studies in cultured mouse cortical neurons after chronic antagonist exposure. Neuroscience 1996; 16:2172–2178.

13 Wilson MA, Kinsman SL, Johnston MV. Expression of NMDA receptor subunit mRNA after MK-801 treatment in neonatal rats. Dev Brain Res 1998; 109:211–220.

14 Kato K, Li ST, Zorumski CF. Modulation of long-term potentiation induction

in the hippocampus by NMDA-mediated presynaptic inhibition. Neuroscience 1999; 92:1261–1272.

15 Hudspith MJ. Glutamate: a role in normal brain function, anesthesia,

analgesia and CNS injury. Br J Anesth 1997; 78:731–747.

16 Haberny KA, Paule MG, Scallet AC, et al. Ontogeny of the N-methyl-D-

aspartate (NMDA) receptor system and susceptibility to neurotoxicity. Toxicol Sci 2002; 68:9–17.

17 Olney JW. New insights and new issues in developmental neurotoxicology.

Neurotoxicology 2002; 23:659–668.

18 Simon DK. NMDA receptor antagonists disrupt the formation of a

mammalian neural map. Proc Natl Acad Sci U S A 1992; 89:10593– 10597.

19 Akopian A, Witkovsky P. Calcium and retinal function. Mol Neurobiol 2002;

2:113–132.

20 Holt M, Cooke A, Wu MM, et al. Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells. J Neurosci 2003; 23:1329–1339. 21 Weiler R, Janssen BU. Spinule-type neurite outgrowth from horizontal cells

during light adaptation in the carp retina: an actin-dependent process. J Neurocytol 1993; 22:129–139.

22 Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodeling. Nat Rev Mol Cell Biol 2003; 4:517–529.

23 Behar TN, Scott CA, Greene CL, et al. Glutamate acting at NMDA

receptors stimulates embryonic cortical neuronal migration. J Neurosci 1999; 19:4449–4461.

24 Wittmann M, Bengtson CP, Bading H. Extrasynaptic NMDA receptors:

mediators of excitotoxic cell death. Pharmacol Cereb Ischemia 2004;253–266.

25 Jiang Q, Gu Z, Zhang G, et al. NMDA receptor activation results in

regulation of extracellular signal-regulated kinases by protein kinases and phosphatases in glutamate-induced neuronal apoptotic-like death. Brain Res 2000; 887:285–292.

26 Wang C, Sadovova N, Fu X, et al. The role of the N-methyl-D-aspartate

Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (nos 30470556 and 30570683).

receptor in katemine-induced apoptosis in rat forebrain culture. Neuroscience 2005; 132:967–977.

References 1 Yamamura T, Harada K, Okamura A, Kemmotsu O. Is the site of action of ketamine anesthesia the N-methyl-D-aspartate receptor? Anesthesiology 1990; 72:704–710.

2 Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors.

Science 1993; 260:95–97.

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