Neonatal ketamine exposure results in changes in biochemical substrates of neuronal growth and synaptogenesis, and alters adult behavior irreversibly

Toxicology 249 (2008) 153–159

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Neonatal ketamine exposure results in changes in biochemical substrates of neuronal growth and synaptogenesis, and alters adult behavior irreversibly ´ c , Per Eriksson a , Torsten Gordh c , Anders Fredriksson b Henrik Viberg a,∗ , Emma Ponten a

Department of Environmental Toxicology, Uppsala University, Norbyv¨ agen 18A, 75236 Uppsala, Sweden Department of Neuroscience, Psychiatry Uller˚ aker, Uppsala University, Sweden c Department of Surgical Sciences, Anaesthesiology and Intensive Care, Uppsala University, Sweden b

a r t i c l e

i n f o

Article history: Received 13 March 2008 Received in revised form 25 April 2008 Accepted 25 April 2008 Available online 4 May 2008 Keywords: Ketamine Neonatal Neurotoxicity CaMKII GAP-43 Behavior

a b s t r a c t Ketamine, an anaesthetic agent used in newborns and toddlers, has been shown to induce neurodegeneration and alter adult behavior in mice, when administered during the neonatal period. Mammals have a marked period of rapid brain growth and development (BGS), which is postnatal in mice and rats, spanning the first 3–4 weeks of life and reaching its peak around postnatal day 10. CaMKII and GAP-43 play important roles during the BGS in mammals. In the present study, 10 days old mice were exposed to 5–25 mg ketamine/kg bw and 24 h later brains were analyzed for calcium/calmodulin-dependent protein kinase II (CaMKII) and growth associated protein-43 (GAP-43) and at an age of 2 and 4 months the animals were tested for spontaneous behavior. The protein analysis showed that CaMKII increased significantly in hippocampus, but not in cortex, in animals 24 h after exposure to ketamine. GAP-43 showed a significant increase in hippocampus, but a significant decrease in cortex for the highest ketamine dose. When looking at the adult behavior it was clear that neonatal ketamine exposure affected spontaneous behavior and habituation in a dose–response-related manner and that these behavioral disturbances were not transient but still persisted 2 months later. Taken together, this shows that ketamine affects important proteins involved in normal maturation of the brain and induce functional deficits in the adult individual, which further strengthen our findings concerning ketamine as a developmental neurotoxicological agent. Published by Elsevier Ireland Ltd.

1. Introduction In surgical procedures in newborns and toddlers the drug ketamine is sometimes used as a general paediatric anaesthetic (Miller, 2004), because it produces what is referred to as dissociative anaesthesia, a state of unconsciousness and analgesia. The dissociative anaesthesia is dose-related and short with rapid recovery (Kohrs and Durieux, 1998). For clinically relevant doses ketamine has been shown to act as a non-competitive ion channel blocker of the N-methyl-d-aspartate (NMDA) receptor (Anis et al., 1983; Harrison and Simmonds, 1985). The NMDA receptor is widely distributed throughout the CNS. It is a subtype of the glutamate receptor, which is involved in a variety of processes, including development and differentiation of the nervous system, learning and memory, and synaptic plasticity (Collingridge et al., 1983; D’Souza et al., 1993; Meldrum and Garthwaite, 1990). Exposure to ketamine can coincide in time with an important period of brain development—the brain growth spurt (BGS)

∗ Corresponding author. Tel.: +46 18 4717695; fax: +46 18 518843. E-mail address: [email protected] (H. Viberg). 0300-483X/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.tox.2008.04.019

(Davison and Dobbing, 1968). The BGS is characterized by a series of rapid fundamental developmental changes, for example maturation of dendritic and axonal outgrowth, the establishment of neural connections, and synaptogenesis and proliferation of glia cells with accompanying myelinization (Davison and Dobbing, 1968; Kolb and Whishaw, 1989). This is also the period when animals acquire many new motor and sensory abilities (Bolles and Woods, 1964) and when spontaneous motor behavior peaks (Campbell et al., 1969). Most of the different transmitter systems undergo qualitative and quantitative changes during this developmental period. For example, the glutamatergic system transforms during the BGS and the numbers of NMDA receptors increase during postnatal brain development (D’Souza et al., 1993). Many behavioral characteristics (Karzmar, 1975) and cognitive functions (Bartus et al., 1982; Drachman, 1977) are closely linked to the cholinergic and glutamatergic transmitter systems. In mammals, the period of BGS in terms of onset and duration varies from species to species. In humans, it begins during the third trimester of pregnancy and continues throughout the first 2 years of life, whereas in rodents, such as mice and rats, the BGS is neonatal, spanning the first 3–4 weeks of life, reaching its peak around postnatal day 10.

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In several studies we have shown that the period of rapid brain development, BGS, is vulnerable to insults from different kinds of xenobiotics (Ankarberg et al., 1998; Eriksson, 1992; Eriksson et al., 2000, 2002; Viberg et al., 2003). We have also shown that exposure to ketamine during this critical period of rapid brain development can give rise to functional effects on the behavior and learning and memory in the adult mouse (Fredriksson and Archer, 2004; Fredriksson et al., 2007) and other research groups have also demonstrated that ketamine can alter brain development if exposure occurs during BGS (Ikonomidou et al., 1999; Olney et al., 2004; Riley and McGee, 2005; Scallet et al., 2004). The above presented data shows that ketamine interfere with the critical developmental processes occurring during the brain growth spurt, and it is possible that neonatal exposure to ketamine results in changes in permanent brain function. We hypothesized that the effects of ketamine on critical developmental processes would be reflected by changes in the biochemical substrates underlying them. Therefore, the levels of two important proteins involved in neuronal survival, growth, and synaptogenesis were examined. The proteins assayed were growth associated protein-43 (GAP43), and calcium/calmodulin-dependent protein kinase II (CaMKII). GAP-43 is a phosphoprotein localized to axonal growth cones. It is frequently used as a marker for axonal sprouting and growth and is maximally expressed during nervous system development (Oestreicher et al., 1997) and shows a bell-shaped expression curve during the first four neonatal weeks in mice, with a peak around postnatal day 10 (Viberg et al., 2008). CaMKII is one of the most abundant protein kinases in the mammalian brain (Erondu and Kennedy, 1985). CaMKII exhibits a striking increase in expression during brain development, and is involved in regulation of both synaptogenesis and synaptic plasticity (Frankland et al., 2001; Rongo and Kaplan, 1999; Viberg et al., 2008). Despite the mounting evidences for ketamine as a developmental neurotoxicant there is an ongoing debate on how to interpret the above-mentioned findings and how to apply them in clinical practice (Olney et al., 2005; Soriano and Anand, 2005). Therefore the aim of the present study was to further evaluate how neonatal ketamine exposure affects important proteins involved in neuronal growth, survival, and synaptogenesis, and also investigate if the functional effects, manifested in deranged adult behavior, are dose–responserelated. 2. Materials and methods 2.1. Animals Pregnant NMRI mice were purchased from B&K, Sollentuna, Sweden. Each litter was adjusted within 48 h to 10–12 mice and to contain offspring of both sexes in about equal numbers. The litters were kept together with its respective mother in a plastic cage in a room at temperature of 22 ± 1 ◦ C and at a 12/12 h constant light/dark cycle (lights on between 06.00 a.m. and 18.00 p.m.). The animals were supplied with standardised pellet food and tap water ad libitum. Only the male offspring were used in this study. At the age of 4 weeks the mice were weaned and the males were raised in groups of four to seven animals in a room with male mice only. They were kept as outlined above until behavioral testing. Mice were randomly picked from each treatment group for each of the different experiments (see further in Section 2.2). Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) after approval from the local ethical committee (Uppsala University and Agricultural Research Council), and by the Swedish Committee for Ethical Experiments on Laboratory Animals. 2.2. Treatment and drugs On postnatal day 10 male mouse pups were administered 5, 10 or 25 mg ketamine/kg bw (Ketalar, 10 mg/ml Pfizer Inc., New York, USA) or vehicle (0.9% NaCl) in a volume of 5 ml/kg, by subcutaneous injection in the neck. The highest dose (25 mg/kg bw) was chosen in order to repeat the behavioral results from an earlier study (Fredriksson et al., 1993), and the two lower doses were chosen to be close to doses that are used clinically (FASS, 2007). Each treatment group were derived from four to five different litters. Ten animals from each treatment group were randomly

assigned to spontaneous behavior testing at an age of 55 and 115 days. From the control group and the groups administered 5 or 25 mg ketamine/kg bw, seven to eight animals were randomly assigned to protein analysis and were sacrificed by cervical dislocation 24 h after treatment. For protein analysis the brains were dissected on an ice-cold glass-plate and cortex and hippocampus were collected and stored at −80 ◦ C until analysis. 2.3. Protein analysis: slot-blot analysis of CaMKII and GAP-43 Cortex and hippocampus were homogenized in a RIPA cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% sodium deoxycholate) with the addition of 0.5% protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem). The homogenate was then centrifuged at 14,000 × g for 15 min at 4 ◦ C, and the protein content of the supernatant was measured using the BCA method (Pierce). Subsequently, the supernatant was stored at −80 ◦ C until use. As described in an a recent study (Viberg et al., 2008) we evaluated the status of the antibodies in use by running the GAP-43 (Chemicon AB5220) and CaMKII (Chemicon MAB8699) antibodies in a western blot procedure. In both cases the antibodies turned out to be specific for the protein intended to, which was shown by the presence of only one band at the appropriate molecular weight. We concluded that these particular antibodies were suitable for use in the slot-blotting procedure. In the slot-blotting procedure 4 ␮g of protein was diluted to a final volume of 200 ␮l with sample buffer (120 mM KCl, 20 mM NaCl, 2 mM NaHCO3 , 2 mM MgCl2 , 5 mM HEPES, pH 7.4, 0.05% Tween20, 0.2% NaN3 ) and applied in duplicate to a nitrocellulose membrane (0.45 ␮m, Bio-Rad) using a Bio-Dot SF microfiltration apparatus (Bio-Rad). The membranes were fixed in 25% isopropanol and 10% acetic acid solution, washed, and blocked for 1 h at room temperature in 5% non-fat dry milk containing 0.03% Tween-20. The membranes were then incubated overnight at 4 ◦ C with a mouse monoclonal CaMKII antibody (Chemicon MAB8699, 1:10,000) or a rabbit polyclonal GAP-43 antibody (Chemicon AB5220, 1:5000). Immunoreactivity was detected using a horseradish peroxidase-conjugated secondary antibody against mouse (074-1806, 1:20,000) or rabbit (KPL 074-1506, 1:20,000). Immunoreactive bands were detected using an enhanced chemiluminescent substrate (Pierce, Super Signal West Dura) with imaging on a LAS-1000 (Fuji Film, Tokyo, Japan). The intensity of bands was quantified using IR-LAS 1000 Pro (Fuji Film). 2.4. Spontaneous behavior Mice exposed to 5, 10 or 25 mg ketamine/kg bw on postnatal day 10 were observed for spontaneous behavior at an age of 55 and 115 days as earlier described (Eriksson et al., 1992; Fredriksson et al., 2007). The experimenter was blinded to the different treatments of the mice. The animals were tested between 8 a.m. and 12 p.m. under the same ambient light and temperature conditions as their housing conditions. A total of 10 mice were randomly picked from the three to five different litters in each treatment group. Motor activity was measured for a 60-min period, divided into 3× 20-min spells, in an automated device consisting of cages (40 cm × 25 cm × 15 cm) placed within two series of infrared beams (low and high level) (Rat-O-Matic, ADEA Elektronik AB, Uppsala, Sweden) (Fredriksson, 1994). 2.4.1. Locomotion Counting took place when the mouse moved horizontally through the low-level grid of infrared beams. 2.4.2. Rearing Movement in the vertical plane was registered at a rate of 4 counts per second, when a single high level beam was interrupted, i.e., the number of counts obtained was proportional to time spent rearing. 2.4.3. Total activity All types of vibration within the cage, i.e., those caused by mouse movements, shaking (tremors), and grooming, were registered by a pick-up (mounted on a lever with a counterweight), connected to the test cage. 2.5. Statistical analysis 2.5.1. Protein analysis The data from the CaMKII and GAP-43 analysis were subjected to one-way ANOVA and Newman–Keuls post hoc test (GraphPad Prism 3.03). 2.5.2. Spontaneous behavior The data from locomotion, rearing and total activity data over three consecutive 20-min periods in the activity test chambers were submitted to a split-plot ANOVA design (Kirk, 1968). Pairwise testing between the different treatment groups was performed with Tukey’s HSD test at the 1 and 5% levels of significance.

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Fig. 1. Protein levels of CaMKII (a) and GAP-43 (b) in hippocampus and cortex from neonatal mice exposed to either 0.9% NaCl (Control), 5 or 25 mg ketamine/kg bw on postnatal day 10. The statistical differences are indicated as (A) significantly different vs. controls p < 0.01; (a) significantly different vs. controls p < 0.05. The height of the bars represents the mean value ± S.D.

3. Results There were no visual signs of toxicity in the ketamine-treated mice at any given time during the experimental period, nor were there any significant differences in the body weights in the ketamine-treated mice, compared with the vehicle-treated mice (data not shown). 3.1. Effects of ketamine on CaMKII protein levels in neonatal hippocampus and cortex Protein levels of CaMKII in cortex and hippocampus of mice, 24 h after treatment with 5 or 25 mg ketamine/kg bw on postnatal day 10 are presented in Fig. 1a. CaMKII levels were significantly increased (p < 0.01) by 30% in hippocampus in mice exposed to 5 mg ketamine/kg bw, compared to the control group. Furthermore, CaMKII levels were significantly increased (p < 0.001) by 46% in hippocampus in mice exposed to 25 mg ketamine/kg bw, compared to the control group. There was no significant difference between the ketamine-treated groups. In cortex there were no differences in CaMKII levels between the different treatment groups, compared to the control group. 3.2. Effects of ketamine on GAP-43 protein levels in neonatal hippocampus and cortex Protein levels of GAP-43 in cortex and hippocampus of mice, 24 h after treatment with 5 or 25 mg ketamine/kg bw on postnatal day 10 are presented in Fig. 1b. GAP-43 levels were significantly increased (p < 0.05) by 14% in hippocampus in mice exposed to 5 mg ketamine/kg bw, compared to the control group. Furthermore, GAP-43 levels were significantly increased (p < 0.05) by 16% in hippocampus in mice exposed to 25 mg ketamine/kg bw, compared to the control group. In cortex there was a significant decrease (15%, p < 0.01) in the level of GAP-43 in mice exposed to 25 mg

ketamine/kg bw on postnatal day 10, compared to the control group. 3.3. Dose-dependent effects of neonatal ketamine exposure on adult spontaneous behavior The results from the spontaneous behavioral variables locomotion, rearing, and total activity in 55 and 115 days old male mice, after exposure to a single subcutaneous dose of 5, 10 or 25 mg ketamine/kg bw at an age of 10 days, are shown in Figs. 2 and 3, respectively. When tested for spontaneous behavior around 2 months of age (age 55 days), there were significant group × period interactions [F6,88 = 156.51; F6,88 = 213.38; F6,88 = 134.23] for the locomotion, rearing, and total activity variables, respectively (Fig. 2). Pairwise testing between ketamine and control groups showed a significant dose-related change in all three test variables. In control mice, there was a distinct decrease in activity in all three behavioral variables over the 60-min period. Mice exposed neonatally to the highest dose of ketamine (25 mg/kg bw) displayed significantly less activity, for all three behavioral variables, during the first 20-min period (0–20 min) compared with the controls, while during the third 20-min period (40–60 min), they were significantly more active than the control animals in relation to all three behavioral variables. These mice also showed significantly higher activity, for all three variables, during the third period (40–60 min), compared with the mice receiving the middle dose of ketamine (10 mg/kg bw). Mice receiving the middle dose of ketamine (10 mg/kg bw) showed significantly lower activity during the first 20-min period (0–20 min) for all three variables locomotion, rearing and total activity, compared with the controls, but during the third 20-min period (40–60 min), they showed a significantly higher activity compared with the control animals regarding all three behavioral variables locomotion rearing and total activity. These mice also showed significantly lower activity for the locomotion rearing and

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total activity variables during the first 20-min period (0–20 min), compared with the mice receiving the lowest dose of ketamine (5 mg/kg bw). In addition, these mice showed significantly higher activity for all three behavioral variables locomotion, rearing and total activity during the third 20-min period (40–60 min), compared with the mice receiving the lowest dose of ketamine. Mice receiving the lowest dose of ketamine (5 mg/kg bw) displayed significantly less activity, for all three behavioral variables, during the first 20-min period (0–20 min) compared with the controls, while during the third 20-min period (40–60 min), they were significantly more active than the control animals concerning the rearing variable only. When tested for spontaneous behavior around 4 months of age (age 115 days), there were significant group × period interactions [F6,88 = 159.99; F6,88 = 115.66; F6,88 = 78.72] for the locomotion, rearing, and total activity variables, respectively (Fig. 3). Pairwise testing between ketamine and control groups showed a significant doserelated change in all three test variables. In control mice, there was a distinct decrease in activity in all three behavioral variables over the 60-min period. Mice exposed neonatally to the highest dose of ketamine (25 mg/kg bw) displayed significantly less activity, for all three behavioral variables, during the first 20-min period (0–20 min) compared with the controls, while during the third 20min period (40–60 min), they were significantly more active than the control animals in relation to all three behavioral variables. These mice also showed significantly lower activity for all three variables during the first 20-min period and higher activity, for

Fig. 3. Spontaneous motor activity of adult mice exposed to either 0.9% NaCl (Control), 5, 10, or 25 mg ketamine/kg bw, on postnatal day 10 and tested in the activity test chamber at 4 months age (115 days). Mean locomotion, rearing and the total activity counts of three consecutive 20-min periods. The statistical differences are indicated as: (A) significantly different vs. controls, p < 0.01; (B) significantly different vs. 5 mg ketamine/kg bw, p < 0.01; (C) significantly different vs. 10 mg ketamine/kg bw, p < 0.01; (c) significantly different vs. 10 mg ketamine/kg bw, p < 0.05. The height of the bars represents the mean value ± S.D.

Fig. 2. Spontaneous motor activity of adult mice exposed to either 0.9% NaCl (Control), 5, 10, or 25 mg ketamine/kg bw, on postnatal day 10 and tested in the activity test chamber at 2 months age (55 days). Mean locomotion, rearing and the total activity counts of three consecutive 20-min periods. The statistical differences are indicated as: (A) significantly different vs. controls, p < 0.01; (a) significantly different vs. controls, p < 0.05; (B) significantly different vs. 5 mg ketamine/kg bw, p < 0.01; (C) significantly different vs. 10 mg ketamine/kg bw, p < 0.01; (c) significantly different vs. 10 mg ketamine/kg bw, p < 0.05. The height of the bars represents the mean value ± S.D.

all three variables, during the third period (40–60 min), compared with the mice receiving the middle dose of ketamine (10 mg/kg bw). Mice receiving the middle dose of ketamine (10 mg/kg bw) showed significantly lower activity during the first 20-min period (0–20 min) for all three variables locomotion, rearing and total activity, compared with the controls, but during the third 20-min period (40–60 min), they showed a significantly higher activity compared with the control animals regarding all three behavioral variables locomotion rearing and total activity. These mice also showed significantly lower activity for the locomotion and rearing variables during the first 20-min period (0–20 min), compared with the mice receiving the lowest dose of ketamine (5 mg/kg bw). In addition, these mice showed significantly higher activity for all three behavioral variables locomotion, rearing and total activity during the third 20-min period (40–60 min), compared with the mice receiving the lowest dose of ketamine. Mice receiving the lowest dose of ketamine (5 mg/kg bw) displayed significantly less activity, for the rearing variable, during the first 20-min period (0–20 min) compared with the controls, while during the third 20min period (40–60 min), they did not differ in activity for any of the three variables locomotion, rearing and total activity, compared with the control animals. 4. Discussion We have previously shown that exposure to ketamine during the brain growth spurt results in increased neurodegeneration/apoptosis and changes in levels of BDNF in the maturing brain

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as well as changes in adult spontaneous behavior and learning ´ et al., and memory in mice (Fredriksson et al., 2004, 2007; Ponten submitted for publication). The present study further explores possible mechanisms of the developmental neurotoxicity of ketamine and we examined the effects of ketamine on two proteins that are critically involved in the processes underlying brain development. The data shows that administration of ketamine to mice on PND 10 altered the amount of CaMKII and GAP-43, 24 h after exposure, during the peak of the brain growth spurt, which indicates that these two proteins may serve as markers of neuronal development. Furthermore, neonatal exposure to ketamine gave rise to dose–response-related changes in adult spontaneous behavior. These changes persisted from 2 months of age to 4 months of age, indicating that the effects seen are not transient or reversible. To detect and explain the mechanisms by which chemicals can damage the CNS, assay of proteins involved in brain development may be useful. Traditionally measures of morphology has been used as indicators of neuronal damage, but in some cases alterations in brain proteins may be more sensitive indicators of damage (O’Callaghan and Miller, 1988a,b). CaMKII, GAP-43 and BDNF are proteins highly enriched in the nervous system and are signaling proteins that regulate neuronal processes (survival, growth, and synapatogenesis) which peak during the brain growth spurt. A recent study has shown that the levels of BDNF are affected in ´ et the neonatal brain due to neonatal ketamine exposure (Ponten al., submitted for publication). CaMKII is one of the most abundant protein kinases in neuronal tissues (Erondu and Kennedy, 1985) and plays a significant role in a number of processes such as long-term potentiation (LTP) (Lisman and Goldring, 1988), apoptosis (Heist and Schulman, 1998), axonal and dendritic arborization (Zou and Cline, 1999) and synaptogenesis (Kazama et al., 2003, 2007; Shi and Ethell, 2006; Yamauchi, 2005). In the present study neonatal ketamine exposure significantly increased CaMKII levels in the hippocampus for both ketamine doses (5 and 25 mg/kg bw), but had no effect in the cortex. The effect in hippocampus occurred at a time (PND 10) when levels of CaMKII are rapidly increasing (Viberg et al., 2008). To date there is little information on the effects of developmental neurotoxicants on CaMKII, but a recent study from our research group has shown the exact same changes after neonatal exposure to the environmental pollutant brominated flame retardant PBDE 209 (Viberg et al., 2008). In a study from another research group it was concluded that neonatal exposure to another brominated flame retardant, PBDE 47, affected hippocampal LTP and the phosphorylated (active) form of CaMKII (Dingemans et al., 2007). While the effects of increased levels of CaMKII are not clear, increased expression of CaMKII could impair normal axonal and dendritic outgrowth and thereby negatively affect the normal development of communications between neurons (Zou and Cline, 1999). GAP-43 is a phosphoprotein that shows in vivo expression that is almost exclusively neuronal in presynaptic terminals (Skene, 1989). Convergent evidence from many labs indicates that GAP-43 plays a key role in guiding the growth of axons and modulating the formation of new connections, both during the development in the CNS and during regeneration in the PNS. Due to its characteristics and pattern of expression GAP-43 is frequently used as a marker for axonal sprouting and growth (Oestreicher et al., 1997). In the present study neonatal ketamine exposure significantly increased the level of GAP-43 in the hippocampus for both ketamine doses (5 and 25 mg/kg bw). In contrast the highest dose of ketamine (25 mg/kg bw) decreased GAP-43 levels in the cortex. This pattern of change has earlier been seen after neonatal exposure to the flame retardant PBDE 209, and this effect at PND 10, a time when GAP-43 is decreasing in the hippocampus and increasing in the cortex (Viberg et al., 2008), is consistent with a ketamine-induced delay in mat-

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uration (i.e., a change to levels observed on PND 7). Other studies have reported changes in GAP-43 after developmental exposure. Kobayashi and co-workers found that fetal and lactational exposure to PTU (propylthiouracil) induced the expression of GAP-43 in cerebral cortex but not in hippocampus, demonstrating diverging results between brain areas during the postnatal brain developmental period (Kobayashi et al., 2005). The changes in CaMKII and GAP-43 were induced at doses, which also result in persistent changes in spontaneous behavior. Although small in magnitude (between 14 and 46%) the changes in CaMKII and GAP-43 are consistent with previous studies of neonatal exposure to neurotoxicants. For example, Viberg et al. (2008) found that neonatal exposure to the brominated flame retardant PBDE 209 resulted in CaMKII and GAP-43 protein changes in the magnitude of 13–38%, and O’Callaghan and Miller (1988a,b) found changes in neurotypic and gliotypic proteins of similar magnitude after neonatal exposure to tributyltin and triethyltin. The observed changes in protein expression could result from (1) a direct effect of the toxicant on protein expression, (2) an up- or down-regulation of protein expression as a compensatory response to toxicant-induced changes in developmental processes, or (3) the gain or loss of the anatomical substrate in which the protein is predominantly localized. Currently it is not clear how exposure to ketamine leads to the observed effects. However, because the changes in CaMKII and GAP43 proceeded the behavioral effects observed in adult animals after neonatal exposure, developmentally regulated proteins that are associated with critical cellular processes (e.g. neurite outgrowth and synaptogenesis) could serve as biomarkers for developmental neurotoxicity resulting from different substances such as ketamine and the recently investigated PBDE 209 (Viberg et al., 2008). Regional differences in the effects of neonatal ketamine exposure could result from both the developmental processes occurring at the time of the toxic insult and/or the amount of ketamine or its metabolites reaching the two brain areas. In the present study the changes in CaMKII and GAP-43 were most pronounced in hippocampus. A recent study by Dingemans and co-workers showed changes in LTP in hippocampus after neonatal exposure to the brominated flame retardant PBDE 47 (Dingemans et al., 2007). In our earlier studies we have shown that neonatal exposure to ketamine can increase the neurodegeneration/apoptosis in hippocampus as well as decrease the levels of BDNF in hippocampus ´ et al., submitted for publication) and other researchers (Ponten have reported that general anaesthesia on postnatal day 7 can induce both increases and decreases in the levels of BDNF, depending on the area of the brain (Lu et al., 2006; Yon et al., 2005). The ontogeny of different variables of the glutamatergic system (D’Souza et al., 1993) parallels the development of CaMKII, GAP-43 and BDNF, as do the ontogeny of different variables of the cholinergic system (Coyle and Yamamura, 1976; Fiedler et al., 1987; Kuhar et al., 1980). At the same time the development of spontaneous behavior peaks (Campbell et al., 1969). The interactions between the studied protein markers and the development of the glutamatergic system in the hippocampus might be a possible mechanism behind the effects on behavior, habituation, learning and memory seen after neonatal ketamine exposure in the present study and recent studies (Fredriksson and Archer, 2004; Fredriksson et al., ´ et al., submitted for publication). 2004, 2007; Ponten The changes in the developmentally important proteins CaMKII and GAP-43 are not the only effects seen after exposure to ketamine on postnatal day 10. These neonatal effects may contribute and be coupled to behavioral changes in the adult animals. Spontaneous behavior is dependent on the integration of sensory input into motor output, and the ability of animals to habituate to a new environment and integrate new information with previously attained information, and thereby be a measure of cognitive function. In the

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present study neonatal exposure to different doses of ketamine also produces changes in the adult spontaneous behavior. At an age of 2 months animals exposed to 25 mg ketamine/kg bw on postnatal day 10 showed a significantly hypoactive state in the beginning of the 60-min test period, while they were significantly hyperactive towards the end of the 60-min test period, compared to the control animals. The same was true for the animals exposed to 10 mg ketamine/kg bw. These changes in spontaneous behavior were seen for all three behavioral variables, locomotion rearing and total activity. In addition, these animals did not show normal habituation to the new environment, which is the normal pattern of behavior during this particular behavior test (Eriksson et al., 1992; Fredriksson et al., 1993). When looking at the lowest dose of ketamine treatment (5 mg/kg bw) it is clear that the spontaneous behavior is disturbed, but the effect is not as pronounced as for the higher treatment groups. This marks a clear dose–response-dependent action of ketamine on spontaneous behavior. Recent studies from our research group (Fredriksson and Archer, 2004; Fredriksson et al., ´ et al., submitted for publication) where expo2004, 2007; Ponten sure to 25 or 50 mg ketamine/kg bw on postnatal day 10 has given a similar outcome in the spontaneous behavior test, support the present findings. This type of dose–response-dependent effect on spontaneous behavior has earlier been seen for a number of compounds administered during the brain growth spurt, for example brominated flame retardants (Viberg et al., 2003, 2004). When looking at the response in spontaneous behavior 2 months later, i.e. age 115 days, it is clear that the behavioral disturbances have persisted and are still dose–response-related. The effect of the lowest dose of ketamine (5 mg/kg bw) seems to be some what less pronounced than at the earlier testing occasion (age 55 days). This indicates that this dose might be close to the so-called threshold dose, leading to disturbances in some animals but not all animals, which by chance may occur by selecting the animals randomly at each testing occasion. The behavioral disturbances mean that the neonatal exposure to one single injection of the anaesthetic drug ketamine can change the spontaneous behavior and habituation in the adult animal, changes that are not transient or reversible, but in fact persistent throughout adult life. Human neonates exposed to alcohol develop FAS, foetal alcohol syndrome, or the less severe foetal alcohol spectrum disorders (FASD) (Riley and McGee, 2005). Children with FAS/FASD demonstrate altered cognitive functions such as reduced attention span, less social skills and learning difficulties, which is another example of perinatal exposure to xenobiotics that can result in effects still seen in adolescence and adulthood. The effect of ketamine on habituation, which is a form of learning and memory, may be coupled to the ability of ketamine to act as a NMDA receptor antagonist and the NMDA system is important learning. Animals neonatally exposed to ketamine and other NMDA-antagonists show spatial learning deficits in adulthood (Gorter and de Bruin, 1992) and can demonstrate taste aversion learning and taste recognition memory (Mickley et al., 2001), which further supports our present findings. In conclusion, the present results show that biochemical assessment of proteins involved in normal brain development may be useful biomarkers for developmental neurotoxicity. Although it is not clear how ketamine exposure alters the levels of CaMKII and GAP-43, significant changes in these proteins may alter the normal ontogeny of regional brain development, including neurite outgrowth and synaptogenesis. The relationship between chemical-induced changes in these biochemical substrates of growth and plasticity during the brain growth spurt and possible morphological and/or functional consequences remains to be further determined. One step in that direction is the results presented here showing that neonatal ketamine exposure change adult spon-

taneous behavior, changes that are dose–response-related and not transient, but seem to be persistent throughout adulthood. Acknowledgements Supported by grants from the Foundation for Strategic Environment Research, Stockholm, Sweden and the Swedish Research Council for Environmental, Agricultural Sciences and Spatial Planning, Stockholm, Sweden. References Anis, N.A., Berry, S.C., Burton, N.R., Lodge, D., 1983. The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methyl-aspartate. Br. J. Pharmacol. 79, 565–575. Ankarberg, E., Fredriksson, A., Eriksson, P., 1998. Interactive effects of PCB and nicotine administered during the neonatal brain development. Organohalogen Compd. 37, 93–96. Bartus, R.T., Dean 3rd, R.L., Beer, B., Lippa, A.S., 1982. The cholinergic hypothesis of geriatric memory dysfunction. Science 217, 408–414. Bolles, R.C., Woods, P.J., 1964. The ontogeny of behaviour in the albino rat. Anim. Behav. 12, 427–441. Campbell, B.A., Lytle, L.D., Fibiger, H.C., 1969. Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science 166, 635–637. Collingridge, G.L., Kehl, S.J., McLennan, H., 1983. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334, 33–46. Coyle, J.T., Yamamura, H.I., 1976. Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain. Brain Res. 118, 429–440. Davison, A.N., Dobbing, J., 1968. Applied Neurochemistry. Blackwell, Oxford, pp. 178–221, 253–316. Dingemans, M.M., Ramakers, G.M., Gardoni, F., van Kleef, R.G., Bergman, A., Di Luca, M., van den Berg, M., Westerink, R.H., Vijverberg, H.P., 2007. Neonatal exposure to brominated flame retardant BDE-47 reduces long-term potentiation and postsynaptic protein levels in mouse hippocampus. Environ. Health Perspect. 115, 865–870. Drachman, D.A., 1977. Memory and cognitive function in man: does the cholinergic system have a specific role? Neurology 27, 783–790. D’Souza, S.W., McConnell, S.E., Slater, P., Barson, A.J., 1993. Glycine site of the excitatory amino acid N-methyl-d-aspartate receptor in neonatal and adult brain. Arch. Dis. Child. 69, 212–215. Eriksson, P., 1992. Neuroreceptor and behavioural effects of DDT and pyrethroids in immature and adult mammals. In: Isaacson, R.L., Jensen, K.F. (Eds.), The Vulnerable Brain and Environmental Risks. Plenum Press, New York, pp. 235–251. Eriksson, P., Ahlbom, J., Fredriksson, A., 1992. Exposure to DDT during a defined period in neonatal life induces permanent changes in brain muscarinic receptors and behaviour in adult mice. Brain Res. 582, 277–281. Eriksson, P., Ankarberg, E., Fredriksson, A., 2000. Exposure to nicotine during a defined period in neonatal life induces permanent changes in brain nicotinic receptors and in behaviour of adult mice. Brain Res. 853, 41–48. Eriksson, P., Viberg, H., Jakobsson, E., Orn, U., Fredriksson, A., 2002. A brominated flame retardant, 2,2 ,4,4 ,5-pentabromodiphenyl ether: uptake, retention, and induction of neurobehavioral alterations in mice during a critical phase of neonatal brain development. Toxicol. Sci. 67, 98–103. Erondu, N.E., Kennedy, M.B., 1985. Regional distribution of type II Ca2+ /calmodulindependent protein kinase in rat brain. J. Neurosci. 5, 3270–3277. ¨ ¨ ¨ ¨ veterinarmedicinskt ¨ FASS, V., 2007. Fass vet:forteckning over lakemedel for bruk, ¨ ¨ Lakemedelsindustrif oreningen LIF, Stockholm. Fiedler, E.P., Marks, M.J., Collins, A.C., 1987. Postnatal development of cholinergic enzymes and receptors in mouse brain. J. Neurochem. 49, 983–990. Frankland, P.W., O’Brien, C., Ohno, M., Kirkwood, A., Silva, A.J., 2001. Alpha-CaMKIIdependent plasticity in the cortex is required for permanent memory. Nature 411, 309–313. Fredriksson, A., 1994. MPTP-induced Behavioural Deficits in Mice: Validity and Utility of a Model of Parkinsonism. Uppsala University, Uppsala. Fredriksson, A., Archer, T., 2004. Neurobehavioural deficits associated with apoptotic neurodegeneration and vulnerability for ADHD. Neurotox. Res. 6, 435–456. Fredriksson, A., Fredriksson, M., Eriksson, P., 1993. Neonatal exposure to paraquat or MPTP induces permanent changes in striatum dopamine and behavior in adult mice. Toxicol. Appl. Pharmacol. 122, 258–264. Fredriksson, A., Archer, T., Alm, H., Gordh, T., Eriksson, P., 2004. Neurofunctional deficits and potentiated apoptosis by neonatal NMDA antagonist administration. Behav. Brain Res. 153, 367–376. Fredriksson, A., Ponten, E., Gordh, T., Eriksson, P., 2007. Neonatal exposure to a combination of N-methyl-d-aspartate and gamma-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107, 427–436. Gorter, J.A., de Bruin, J.P., 1992. Chronic neonatal MK-801 treatment results in an impairment of spatial learning in the adult rat. Brain Res. 580, 12–17. Harrison, N.L., Simmonds, M.A., 1985. Quantitative studies on some antagonists of Nmethyl-d-aspartate in slices of rat cerebral cortex. Br. J. Pharmacol. 84, 381–391.

H. Viberg et al. / Toxicology 249 (2008) 153–159 Heist, E.K., Schulman, H., 1998. The role of Ca2+ /calmodulin-dependent protein kinases within the nucleus. Cell Calcium 23, 103–114. Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., Dikranian, K., Tenkova, T.I., Stefovska, V., Turski, L., Olney, J.W., 1999. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283, 70– 74. Karzmar, A.G., 1975. Cholinergic Influences on Behaviour. Raven Press, New York. Kazama, H., Morimoto-Tanifuji, T., Nose, A., 2003. Postsynaptic activation of calcium/calmodulin-dependent protein kinase II promotes coordinated pre- and postsynaptic maturation of Drosophila neuromuscular junctions. Neuroscience 117, 615–625. Kazama, H., Nose, A., Morimoto-Tanifuji, T., 2007. Synaptic components necessary for retrograde signaling triggered by calcium/calmodulin-dependent protein kinase II during synaptogenesis. Neuroscience 145, 1007–1015. Kirk, R., 1968. Experimental Design: Procedures for the Behavioural Sciences. Brooks/Cole, Belmont, CA. Kobayashi, K., Tsuji, R., Yoshioka, T., Kushida, M., Yabushita, S., Sasaki, M., Mino, T., Seki, T., 2005. Effects of hypothyroidism induced by perinatal exposure to PTU on rat behavior and synaptic gene expression. Toxicology 212, 135–147. Kohrs, R., Durieux, M.E., 1998. Ketamine: teaching an old drug new tricks. Anesth. Analg. 87, 1186–1193. Kolb, B., Whishaw, I.Q., 1989. Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog. Neurobiol. 32, 235–276. Kuhar, M.J., Birdsall, N.J.M., Burgen, A.S.V., Hulme, E.C., 1980. Ontogeny of muscarinic receptors in rat brain. Brain Res. 184, 375–383. Lisman, J.E., Goldring, M.A., 1988. Feasibility of long-term storage of graded information by the Ca2+ /calmodulin-dependent protein kinase molecules of the postsynaptic density. Proc. Natl. Acad. Sci. U.S.A. 85, 5320–5324. Lu, L.X., Yon, J.H., Carter, L.B., Jevtovic-Todorovic, V., 2006. General anesthesia activates BDNF-dependent neuroapoptosis in the developing rat brain. Apoptosis 11, 1603–1615. Meldrum, B., Garthwaite, J., 1990. Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11, 379–387. Mickley, G.A., Remmers-Roeber, D.R., Dengler, C.M., Kenmuir, C.L., Crouse, C., 2001. Paradoxical effects of ketamine on the memory of fetuses of different ages. Brain Res. Dev. Brain Res. 127, 71–76. Miller, R.D. (Ed.), 2004. Miller’s Anesthesia, 6th edition. Churchill Livingstone. O’Callaghan, J.P., Miller, D.B., 1988a. Acute exposure of the neonatal rat to tributyltin results in decreases in biochemical indicators of synaptogenesis and myelinogenesis. J. Pharmacol. Exp. Ther. 246, 394–402. O’Callaghan, J.P., Miller, D.B., 1988b. Acute exposure of the neonatal rat to triethyltin results in persistent changes in neurotypic and gliotypic proteins. J. Pharmacol. Exp. Ther. 244, 368–378. Oestreicher, A.B., De Graan, P.N., Gispen, W.H., Verhaagen, J., Schrama, L.H., 1997. B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system. Prog. Neurobiol. 53, 627– 686.

159

Olney, J.W., Young, C., Wozniak, D.F., Ikonomidou, C., Jevtovic-Todorovic, V., 2004. Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? Anesthesiology 101, 273–275. Olney, J.W., Young, C., Wozniak, D.F., Ikonomidou, C., Jevtovic-Todorovic, V., 2005. Of mice and men: should we extrapolate rodent experimental data to the care of human neonates? Anesthesiology 102, 868–869. ´ E., Viberg, H., Eriksson, P., Gordh, T., Fredriksson, A., submitted for pubPonten, lication. Exposure to ketamine during a defined transient vulnerable period of neonatal life accelerates neurodegeneration and affects neonatal levels of brain derived neurotrophic factor (BDNF) and alters adult behaviour in mice. Riley, E.P., McGee, C.L., 2005. Fetal alcohol spectrum disorders: an overview with emphasis on changes in brain and behavior. Exp. Biol. Med. (Maywood) 230, 357–365. Rongo, C., Kaplan, J.M., 1999. CaMKII regulates the density of central glutamatergic synapses in vivo. Nature 402, 195–199. Scallet, A.C., Schmued, L.C., Slikker Jr., W., Grunberg, N., Faustino, P.J., Davis, H., Lester, D., Pine, P.S., Sistare, F., Hanig, J.P., 2004. Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons. Toxicol. Sci. 81, 364–370. Shi, Y., Ethell, I.M., 2006. Integrins control dendritic spine plasticity in hippocampal neurons through NMDA receptor and Ca2+ /calmodulin-dependent protein kinase II-mediated actin reorganization. J. Neurosci. 26, 1813–1822. Skene, J.H., 1989. Axonal growth-associated proteins. Annu. Rev. Neurosci. 12, 127–156. Soriano, S.G., Anand, K.J., 2005. Anesthetics and brain toxicity. Curr. Opin. Anaesthesiol. 18, 293–297. Viberg, H., Fredriksson, A., Eriksson, P., 2003. Neonatal exposure to polybrominated diphenyl ether (PBDE 153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol. Appl. Pharmacol. 192, 95–106. Viberg, H., Fredriksson, A., Eriksson, P., 2004. Investigations of strain and/or gender differences in developmental neurotoxic effects of polybrominated diphenyl ethers in mice. Toxicol. Sci. 81, 344–353. Viberg, H., Mundy, W., Eriksson, P., 2008. Neonatal exposure to decabrominated diphenyl ether (PBDE 209) results in changes in BDNF, CaMKII and GAP-43, biochemical substrates of neuronal survival, growth, and synaptogenesis. Neurotoxicology 29, 152–159. Yamauchi, T., 2005. Neuronal Ca2+ /calmodulin-dependent protein kinase II—discovery, progress in a quarter of a century, and perspective: implication for learning and memory. Biol. Pharm. Bull. 28, 1342–1354. Yon, J.H., Daniel-Johnson, J., Carter, L.B., Jevtovic-Todorovic, V., 2005. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135, 815–827. Zou, D.J., Cline, H.T., 1999. Postsynaptic calcium/calmodulin-dependent protein kinase II is required to limit elaboration of presynaptic and postsynaptic neuronal arbors. J. Neurosci. 19, 8909–8918.