Creatine prevents behavioral alterations caused by methylmalonic acid administration into the hippocampus of rats in the open field task

Journal of the Neurological Sciences 244 (2006) 23 – 29 www.elsevier.com/locate/jns

Creatine prevents behavioral alterations caused by methylmalonic acid administration into the hippocampus of rats in the open field task V. Vasques, F. Brinco, C.M. Viegas, M. Wajner a,b,c,* a

Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b Servic¸o de Gene´tica Me´dica, Hospital de Clı´nicas de Porto Alegre, RS, Brazil c Universidade Luterana do Brasil, Canoas, RS, Brazil Received 26 April 2005; received in revised form 30 November 2005; accepted 12 December 2005 Available online 7 February 2006

Abstract Although a variable degree of psychomotor delay/mental retardation is found in a considerable number of patients affected by methylmalonic acidemia, the mechanisms underlying the neuropathology of this disorder are still poorly defined. The present study investigated the effect of acute intrahippocampal administration of methylmalonic acid (MMA), the biochemical hallmark of this disease, on rat behavior in the open field task. Cannulated 60-day-old male Wistar rats received bilateral intrahippocampal injection of MMA (0.1 – 1.0 Amol) 10 min before training. Controls received 0.1 – 1.0 Amol NaCl. Testing session was performed 24 h later. We observed that rats administered with 1.0 Amol MMA, but not with lower doses, did not habituate in the open field task, reflecting a deficit of performance. Motor activity, assessed by the number of crossing responses, was the same at training for the groups infused with MMA or NaCl. The effect of MK-801 (15 nmol) and succinate (1.5 Amol) administered 30 min before MMA injection, and of creatine (50 mg/kg, i.p.) administered twice a day for 3 days on the behavioral alterations provoked by MMA in the open field task revealed that only the energetic substrate creatine prevented these effects, reflecting a possible compromise of brain energy production by MMA. The results indicate that high intrahippocampal concentrations of the major metabolite accumulating in methylmalonic acidemia compromises brain functioning, causing deficit of performance in the open field task that may be related to the psychomotor delay/mental retardation observed in the affected patients. D 2005 Elsevier B.V. All rights reserved. Keywords: Methylmalonic acidemia; Methylmalonic acid; Open field; Inhibitory avoidance; Rat behavior

1. Introduction Methylmalonic acidemia (MMAemia) comprehends a group of relatively frequent organic acidemias caused by the deficient activity of l-methylmalonil-CoA mutase (E.C. 5.4.99.2), primarily leading to tissue accumulation of lAbbreviations: MMA, methylmalonic acid; MMAemia, methylmalonic acidemia; NMDA, N-methyl-d-aspartate; MK-801, dizocilpine maleate; ANOVA, analysis of variance; S.E.M., standard error of the mean; SPSS, Statistical Package of the Social Sciences; NaCl, sodium chloride. * Corresponding author. Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, UFRGS Rua Ramiro Barcelos, 2600 Anexo, CEP 90035-003, Porto Alegre, RS, Brazil. Tel.: +55 51 33165571; fax: +55 51 21018010. E-mail address: [email protected] (M. Wajner). 0022-510X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2005.12.005

methylmalonic acid (MMA) [1]. The levels of MMA in blood and cerebrospinal fluid can be as high as 2.5 –5 mM during metabolic crises and these levels may be even higher in the neuronal cells [2]. Neurological dysfunction is one of the most striking features in patients with methylmalonic acidemia. A large percentage of affected infants dies in the first few weeks or months of life after encephalopathic crises. Those who survive longer are usually under treatment, which basically consists of protein restriction and a commercial formula lacking the precursor amino acids forming methylmalonic acid, supplemented by l-carnitine and also by cobalamin to the vitamin B12 responsive variants [1]. However, despite treatment, a considerable number of ‘‘well-treated’’ patients present a variable degree of physical and psychomotor delay/mental retardation.

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Although neurological symptoms are common, the mechanisms underlying the pathophysiology of brain damage in MMAemia are not yet fully established. However, there is compelling evidence showing that mitochondrial dysfunction exerts a relevant role in the neuropathology of this disease [3– 9]. Besides, we have recently shown that in vivo intracerebral administration of MMA provokes a reduction of brain phosphocreatine levels [10]. The mechanisms underlying the reduction of brain phosphocreatine content caused by the metabolite are not yet established, but might be attributed to less available ATP due to inhibition of the respiratory chain caused by MMA [9] or, alternatively, to a reduction of mitochondrial creatine kinase activity, which is responsible for phosphocreatine synthesis [11]. In the present study we investigated the influence of intrahippocampal MMA administration on rat behavior in the open field task. We also tested the involvement of the NMDA-glutamate receptor subtype and energy depletion on MMA-induced behavioral alterations by the administration of the NMDA antagonist MK-801 and the energetic substrates creatine and succinate, respectively.

the dorsal hippocampus (A: 4.2, L: T 3.0, V: + 1.3 mm, according to Paxinos and Watson [12]) (Fig. 1A). The cannulae were fixed to the skull with dental cement [13]. All procedures were performed under aseptic conditions. The animals were allowed to recover unrestrained for 3 days after surgery until the day of the experiment. 2.4. Intrahippocampal drug administration and control of cannula placement Ten minutes before training, a 30-gauge needle was fit into the guide cannula [14] protruding 1 mm from the tip of the cannula in order to administer a bilateral infusion of 1.0 Al of MMA (0.1, 0.5 or 1.0 Amol), or NaCl (0.1, 0.5 or 1 Amol) into the dorsal hippocampus (CA1 area). The pH of each solution was previously adjusted to 7.4 with 0.1 N

2. Materials and methods 2.1. Reagents All reagents were purchased from Sigma Chem. Co. (St. Louis, MO, USA), except MK-801 (dizocilpine maleate) which was purchased from RBI (Natick, MA, USA). 2.2. Animals A total of 104 sixty-day-old male Wistar rats (180 – 230 g) from our own breeding stock of the Central Animal House of the Departamento de Bioquı´mica, ICBS, UFRGS were used for the behavioral experiments. The animals were housed five per cage with food and water freely available under a 12 h light/12 h darkness cycle (lights on at 7:00 AM) at a constant temperature of 22 T 1 -C. The experimental protocol was approved by the Ethics Committee for Animal Research of the Federal University of Rio Grande do Sul, Porto Alegre, Brazil, and followed the ‘‘Principles of laboratory animal care’’ (NIH publication no. 85-23, revised 1985). All experimental protocols were designed carefully in order to keep the number of animals used to a minimum, as well as their suffering. 2.3. Surgical procedure Rats were bilaterally implanted under deep anaesthesia (a mixture of ketamine and xilazine i.p.; 75 and 10 mg/kg, respectively) with 27-gauge stainless cannulae (0.6 mm O.D.) with an inner needle guide (diameter, 0.3 mm) aimed 1.0 mm above the pyramidal cell layer of the CA1 region of

Fig. 1. Schematic drawing (A) and actual picture after methylene blue infusion (B) of a rat brain section at plane 4.20 mm [28] showing the extension of the dorsal hippocampus reached by drug infusion which appears as a filled area. It can be seen in the figure (B) that diffusion of the stain is restricted to the hippocampus.

V. Vasques et al. / Journal of the Neurological Sciences 244 (2006) 23 – 29

NaOH. Infusions were performed at a rate of 1 Al/min. After the infusions were completed, the cannula was left in place for an additional 30 s. Thus, the entire bilateral infusion procedure took 150 s. At the end of behavioral evaluation, the animals were killed by decapitation and infused with 1 Al of 4% methylene blue through the cannula. The brains were removed and stored in 3.7% formalin for histological evaluation of cannula placement [15]. Fig. 1B, which is an actual picture after methylene blue infusion, shows that diffusion was restricted to the hippocampus. In case the cannula was not in the right place or when the infusion surpassed the limits of the hippocampus, the rats were discarded. In the experiments designed to evaluate the participation of glutamatergic mechanisms mediated by NMDA-glutamate receptors on the MMA-induced behavioral effects, the animals were intrahippocampally preinjected with 1.0 Al NaCl (1.0 Amol) or MK-801 (15 nmol) 30 min before they were injected with 1.0 Al MMA (1.0 Amol) or NaCl (1.0 Amol). A similar approach was used to test the influence of succinate (1.5 Amol) on the behavioral alterations provoked by MMA (1.0 Amol). The effect of creatine administration on MMA-induced behavioral alterations was also evaluated by preinjecting the animals for 3 days, two injections per day, with creatine (50 mg/kg, i.p.) or NaCl (0.9% 10 ml/kg, i.p.), after which the animals were intrahippocampally injected with 1.0 Al MMA (1.0 Amol) or NaCl (1.0 Amol). 2.5. Behavioral procedure 2.5.1. Open-field habituation The rats’ ability to habituate to a new environment was assessed by subjecting the animals to two consecutive 3-min sessions (training and testing) spaced 24 h in an open field. Animals were tested only once, so that different treatments required a new set of rats. The rats were injected with 1.0 Al MMA (0.1 –1.0 Amol) or NaCl (0.1 – 1.0 Amol) into the hippocampus 10 min before (16 animals) training session, as described above. The apparatus consisted of a wooden box measuring 60  40  50 cm with a frontal glass wall. The floor was divided into 12 equal squares by black lines. The animals were placed gently on the left rear quadrant of the open field, and the number of squares crossed with the four paws, number of rearing responses and number of fecal boli recorded by an observer who was not aware of the subject condition [16]. The number of fecal boli (defecation) at training session was considered as a measure of emotionality [17,18]. The number of squares crossed at training session was indicative of motor activity and the reduction in the number of rearing responses along the sessions was considered as a measure of habituation [19 – 21]. For the experiments designed to evaluate the influence of NMDA-receptor antagonist MK-801, succinate and creatine on the behavioral effects caused by MMA in the open field task, 88 animals were used.

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2.6. Statistical analysis Data from the open field experiments were analyzed by Student’s t test and by one-way analysis of variance (ANOVA), and are expressed as mean and standard error of the mean (S.E.M.). The statistical analyses were performed using the SPSS (Statistical Package for the Social Sciences) software in a PC-compatible computer. p < 0.05 was considered significant.

3. Results Fig. 2 shows the effect of pretreatment with 1.0 Amol MMA on the number of rearing responses in the open field task. Statistical analysis of rearing scores revealed a deficit in habituation to a novel environment in rats injected with MMA since this animal group did not reduce the number of rearing responses at testing session [t(8) = 0.373, p > 0.05]. In contrast, animals receiving 1.0 Amol NaCl before training significantly reduced the number of rearing responses [t(8) = 3.353, p < 0.05], indicating habituation to the task. Animals receiving 1.0 Amol MMA also showed more rearing responses than NaCl infused animals at testing session, which reinforces a deficit of habituation in MMAtreated animals [t(14) = 2.932, p < 0.05)]. In contrast, rats receiving 0.1 or 0.5 Amol MMA did not present any behavioral alteration as compared to NaCl-treated animals (results not shown). The effect of pre-training intrahippocampal injection of MMA on the number of crossing responses (motor activity) in the open field task during training is depicted in Table 1. Statistical analysis showed no significant differences between NaCl- and MMA-treated animals [t(16) = 0.998,

Fig. 2. Effect of intrahippocampal administration of methylmalonic acid (MMA) on the number of rearing responses of adult rats submitted to the open field task. The rats were injected 10 min before training with NaCl (1.0 Amol) or MMA (1.0 Amol). Data are mean T S.E.M., for n = 8 rats per group. Asterisks indicate significant differences between training and testing sessions, p < 0.05 (Student’s t test for paired samples). #Different from NaCl group at testing session, p < 0.05 (Student’s t test for unpaired samples).

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Table 1 Effect of intrahippocampal administration of MMA on the number of crossing responses and fecal boli in the open field task of adult rats at training session

Control MMA

Number of crossings

Number of fecal boli

67.2 T 3.5 68.4 T 5.0

3.1 T1.1 2.0 T 0.7

Data are mean T S.E.M. for n = 8 rats per group. No significant differences between controls and MMA-treated group were detected at training session (Student’s t test for unpaired samples).

p > 0.05], indicating that the administered drugs did not influence motor activity of the animals. Table 1 also shows the effect of MMA on the number of fecal boli. Defecation of rats submitted to the open field was similar in NaCl- and MMA-infused groups at training [t(16) = 0.871, p > 0.05], suggesting a normal emotionality of the animals in the open field task. Deficit of brain energy metabolism [6] caused by pathophysiological concentrations of MMA and convulsions induced by MMA mediated by NMDA receptors have been previously reported [22]. Therefore, we also tested the effect of pre-administration of the NMDA antagonist MK801 and the energetic substrates succinic acid and creatine on the MMA-induced behavioral alterations in the open field task in order to elucidate possible mechanisms interfering with the lack of habituation observed in this task. The effect of MK-801 on rat performance in the open field task was first evaluated. Fig. 3 shows that the control group (NaCl-infused animals) reduced the number of rearing responses along the sessions [t (5) = 3.008, p < 0.05], whereas the groups administered with MK-801 [t(6) = 0.553, p > 0.05], MMA [ F(5) = 0.733, p > 0.05] or MK-801 plus MMA [t(8) = 0.533, p > 0.05] did not decrease the number of rearing responses along the sessions.

Fig. 3. Influence of pre-administration of MK-801 on the number of rearing responses of rats infused with methylmalonic acid (MMA) and submitted to the open field task. The pre-treatment protocol is described in the text. Rats were injected 10 min before training with 1 Al of MMA (1.0 Amol). Data are mean T S.E.M., for n = 6 – 9 rats per group. Asterisks indicate significant differences between training and testing sessions within each group, p < 0.05 (Student’s t test for paired samples). #Different from NaCl group at testing session, p < 0.05 (Student’s t test for unpaired samples).

Fig. 4. Influence of pre-administration of succinate on the number of rearing responses of rats infused with methylmalonic acid (MMA) and submitted to the open field task. The pre-treatment protocol is described in the text. Rats were injected 10 min before training with 1 Al of MMA (1.0 Amol). Data are mean T S.E.M., for n = 7 rats per group. Asterisks indicate significant differences between training and testing sessions within each group, p < 0.05 (Student’s t test for paired samples). #Different from NaCl group at testing session, p < 0.05 (one-way ANOVA).

In addition, regarding the number of rearing responses, no differences between NaCl and MK801 groups were found at testing session, whereas MMA [t(11) = 3.151, p < 0.05] and MK801 + MMA [t(13) = 2.964; p < 0.05] groups performed differently than NaCl-injected animals. These data indicate that overstimulation of NMDA receptors was not responsible for the lack of habituation produced by MMA. Furthermore, the lack of habituation provoked by the NMDA receptor antagonist per se is expected since MK801 may cause amnesia. We then tested the effect of succinic acid pre-treatment on the number of rearing responses of rats submitted to the open field habituation task since it has been showed that MMA is a competitive inhibitor of succinate dehydrogenase [4] (Fig. 4). Controls and succinate-infused groups showed a significant decrease of rearing responses along the sessions

Fig. 5. Influence of pre-administration of creatine on the number of rearing responses of rats infused with methylmalonic acid (MMA) and submitted to the open field task. The pre-treatment protocol is described in the text. Rats were injected 10 min before training with 1 Al of MMA (1.0 Amol). Data are mean T S.E.M., for n = 8 rats per group. Asterisks indicate significant differences between training and testing sessions within each group, * p < 0.05 (Student’s t test for paired samples). #Different from NaCl group at testing session, p < 0.05 (one-way ANOVA).

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[NaCl: t(6) = 3.032, p < 0.05; succinate: t(6) = 3.022, p < 0.05]. In contrast, the groups infused with MMA and with succinate plus MMA did not reduce the number of rearing responses at testing session [MMA: t(6) = 0.430, p > 0.05; succinate plus MMA: t(6) = 0.367, p > 0.05]. The effect of treatment on the number of rearing responses at testing session revealed that the group infused with MMA presented more rearing responses than the groups infused with NaCl and with succinate. In addition, the animals infused with succinate plus MMA performed similarly as the animals infused with MMA regarding the number of rearing responses at testing session [ F(3,24) = 3.197, p < 0.05], suggesting that acute succinate (1.5 Amol) pretreatment did not prevent the performance deficit provoked by MMA in this task. Fig. 5 shows the effect of creatine pre-treatment on the number of rearing responses of rats submitted to intrahippocampal infusion of MMA in the open field task. NaCl-, creatine- and creatine plus MMA-infused groups showed a significant decrease of rearing responses along the sessions [NaCl: t(7) = 3.614, p < 0.01; creatine: t(7) = 4.706, p < 0.01; creatine plus MMA: t(7) = 2.686, p < 0.05]. In contrast, the rats infused only with MMA did not habituate [t(7) = 0.826, p > 0.05]. At testing session, the group infused with creatine and MMA performed similarly than controls [ F(3,28) = 7.00, p < 0.01], suggesting that chronic creatine pre-treatment prevented the lack of habituation provoked by MMA. We also investigated whether the tested compounds (MK-801, succinic acid and creatine) could alter motor activity and emotionality by evaluating the number of crossing responses and fecal boli, respectively, at training session in the open field task. The number of crossing responses [MK-801: F(3,27) = 1.303, p > 0.05; succinate: F (3,27) = 0.209, p > 0.05; creatine: F (3,31) = 0.895, p > 0.05] and fecal boli [MK-801: F(3,27) = 0.484, p > 0.05; succinate: F(3,27) = 0.861, p > 0.05; creatine: F(3,31) = 1.793, p > 0.05] at training were not significantly different between the various groups (Table 2). These findings indicate that pre-infusion of the tested drugs did not provoke any alteration on locomotion (crossing response) and emotionality (fecal boli) in the open field task.

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4. Discussion Neurological alterations, including psychomotor delay/ mental retardation, are observed in a considerable number of patients with MMAemia [23]. To our knowledge, the present work describes for the first time that high intrahippocampal concentrations of MMA, the principal metabolite accumulating in MMAemia, cause deficit of performance in a behavioral task. We first observed that animals pretreated with 1.0 Amol MMA, but not with lower doses, 10 min before training presented no habituation as revealed by the lack of reduction of rearing responses along sessions in the open field task, whereas the controls (NaCl-injected animals) showed normal habituation. In the open field, rats present an exploratory behavior consisting of increased motor activity and rearing responses when first exposed to the box (training session). Memory retention or habituation to a novel environment can be measured by reduction of rearing responses along sessions in this task, in which the hippocampus and amygdala plays a crucial role [20,24 – 28]. Since a reduction of number of rearing responses along sessions in the open field habituation can be interpreted to indicate that animals recognize the environment and remember the previous exposure to this environment [20,27,28], and since in the present study MMA-treated animals did not show this pattern, it is assumed that this reflects a behavioral alteration caused by this organic acid. Alterations of motor activity or emotionality probably did not contribute to the deficit of habituation caused by MMA since all groups of animals behaved similarly as regards to the number of crossing responses (locomotor activity) and fecal boli, which seems to be primarily related to the dimension of emotional reactivity, and occur in parallel to serum corticosterone levels [24]. There is considerable evidence indicating that mitochondrial dysfunction occurs in MMAemia [3,5 – 9]. In this context, we also showed here that chronic creatine pretreatment prevented the MMA-induced lack of habituation, a fact that suggests mytochondrial dysfunction in the hippocampus after MMA administration. Indeed, previous studies demonstrated reduced brain phosphocreatine concentrations after in vivo intrastriatal injection of MMA [10]

Table 2 Effect of pre-administration of MK-801 (15 nmol), succinate (1.5 Amol) and creatine (50 mg/kg) on the number of crossing responses and fecal boli of rats infused intrahippocampally with 1.0 Amol of methylmalonic acid (MMA) in the open field task at training session MK-801

Control Treated MMA Treated + MMA

Succinate

Creatine

Crossings

Fecal boli

Crossings

Fecal boli

Crossings

Fecal boli

102.5 T 11.9 76.0 T 6.0 75.7 T 11.8 87.1 T 6.7

2.5 T 0.6 3.0 T 0.7 3.8 T 1.1 3.2 T 0.6

81.6 T 13.5 89.4 T 18.3 80.0 T 8.4 75.3 T 8.4

3.64 T 0.7 3.0 T 1.7 2.4 T 0.6 2.1 T 0.7

70.0 T 5.3 67.1 T 4.3 78.2 T 6.2 68.9 T 4.9

1.6 T 0.6 2.6 T 0.8 0.8 T 0.5 1.3 T 0.4

Data are mean T S.E.M., for n = 6 – 9 rats per group. There were no significant differences between groups at training session (ANOVA). F values are shown in the text. Control rats received NaCl. Treated animals received MK-801, succinate or creatine. No significant differences between the various groups were detected (ANOVA).

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and inhibition of mitochondrial creatine kinase [11], which is mainly responsible for phosphocreatine synthesis [29,30]. Therefore, it may be presumed that high energy phosphate levels were restored by chronic treatment with creatine, preventing the MMA-induced open-field habituation deficit. Interestingly it has been previously demonstrated that a knockout mice model lacking the mitochondrial isoform of creatine kinase present diminished habituation and other deficit of performance [31]. The investigators also found that these animals had normal sensory and motor functions and postulated a crucial role for mitochondrial dysfunction in the hippocampal-dependent learning circuitry of brain. It should be pointed out that synaptic plasticity in the hippocampus is affected by mitochondrial dysfunction [32,33], creatine supplementation improves brain performance [34], succinic acid may be a psychoenergizer [35] and cognitive deficits are prominent features in models of mitochondrial disease [36] and in mitochondrial encephalopathies [37]. In our present study succinate pre-administration was not able to prevent the behavioral alterations provoked by MMA, in contrast to other studies showing that this energetic substrate did prevent the convulsions elicited by MMA [10,22]. Since methylmalonate is a weak competitive inhibitor of succinate dehydrogenase [4,9], it appears that higher succinate doses should be tested before we can conclude that succinate is not able to prevent the MMAinduced deficit of performance in our model. However, based on the present available data, it appears that convulsive properties caused by MMA are more susceptible to succinate beneficial action. The significance of our present findings and their possible relationship to the human condition are far from clear. Although the actual concentrations of MMA achieved in our model are unknown, we presume that the brain concentrations achieved by this organic acid after its administration may be sufficient to cause metabolic alterations, such as an impairment of energy production, in pathways involved in learning/memory and in other neurological functions. Therefore, it is possible that the neurobehavioral alterations in MMAemia, such as psychomotor delay/mental retardation, may also result from an increase in MMA concentrations in the brain above a certain threshold which may compromise essential pathways necessary for cerebral functioning. It might be interesting to stress that, although plasma and CSF concentrations of MMA can reach approximately 3 mM, these levels may be even higher within neural cells of methylmalonic acidemic patients [2]. Previous studies have demonstrated that chronic subcutaneous administration of methylmalonic acid to rats during early development provokes neuromotor developmental delay and selective compromise of learning abilities in adult rats after a month of recovery when brain MMA levels are normal. These data indicate a long-lasting brain damage caused by MMA administration, but does not rule out that an indirect effect occurred [38,39].

The present work demonstrates that acute high concentrations of MMA in the hippocampus, which probably mimics the episodes of metabolic decompensation in which dramatic brain increases of MMA occur, compromise rat performance in a behavioral task, indicating that this organic acid interferes with the learning/memory processes occurring in this cerebral structure. In conclusion, even though at the present time it is difficult to correlate slower or deficit of performance with biochemical defects, our results allied to a great body of evidence demonstrating that MMA disturbs brain energy metabolism, strongly suggest that energy deficit provoked by MMA may be related to the behavioral changes observed in MMA-treated rats. Therefore, it is presumed that energetic substrates, besides the usual measures, may be useful as an adjuvant therapy for methylmalonic acidemic children, at least during the metabolic crises in which the levels of this organic acid increase dramatically.

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