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Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: Role of NMDA receptors
Neurochemistry International 53 (2008) 33–37
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Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: Role of NMDA receptors Luiz Fernando Freire Royes a,b, Michele Rechia Fighera b,c, Ana Fla´via Furian b,d, Mauro Schneider Oliveira b,d, Nata´lia Gindri Fiorenza b, Juliano Ferreira e, Andre´ Cesar da Silva f, Margareth Rose Priel f, E´rika Sayuri Ueda f, Joa˜o Batista Calixto g, Esper Abra˜o Cavalheiro f, Carlos Fernando Mello b,* a
Centro de Educac¸a˜o Fı´sica e Desportos, Departamento de Me´todos e Te´cnicas Desportivas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Centro de Cieˆncias da Sau´de, Laborato´rio de Neurotoxicidade e Psicofarmacologia (LABNEURO), Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil c Centro de Cieˆncias da Sau´de, Departamento de Pediatria, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Programa de Po´s-Graduac¸a˜o em Cieˆncias Biolo´gicas: Bioquı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil e Centro de Cieˆncias Naturais e Exatas, Laborato´rio de Neurotoxicidade e Psicofarmacologia (LABNEURO), Departamento de Quı´mica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil f Laborato´rio de Neurologia Experimental, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil g Centro de Cieˆncias Biolo´gicas, Departamento de Farmacologia, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2008 Accepted 25 April 2008 Available online 2 May 2008
The creatine (Cr) and phosphocreatine (PCr) system is essential for the buffering and transport of highenergy phosphates. Although achievements made over the last years have highlighted the important role of creatine in several neurological diseases, the adaptive processes elicited by this guanidino compound in hippocampus are poorly understood. In the present study, we showed that creatine (0.5–25 mM) gradually increases the amplitude of first population spike (PS) and elicits secondary PS in stratum radiatum of the CA1 region, in hippocampal slices. Creatine also decreased the intensity of the stimulus to induce PS, when compared with hippocampal slices perfused with artificial cerebrospinal fluid (ACSF). The competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 100 mM) attenuated creatine-induced increase of amplitude of PS and appearance of secondary PS, providing pharmacological evidence of the involvement of NMDA receptors in the electrophysiological effects of creatine. Accordingly, creatine (0.01–1 mM) increased [3H]MK-801 binding to hippocampal membranes by 55%, further indicating that this compound modulates NMDA receptor function. These results implicate the NMDA receptor in amplitude and population spike increase elicited by creatine in hippocampus. Furthermore, these data suggest that this guanidino compound may also play a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by an increase in glutamatergic function. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Creatine Electrophysiology Hippocampus NMDA [3H]MK-801
1. Introduction Creatine (N-[aminoiminomethyl]-N-methyl glycine) is a guanidino compound endogenously synthesized from glycine, arginine and S-adenosylmethionine in the kidneys, liver and pancreas or ingested in small quantities (the amount can vary from 0 to 7 g of more, depending on the diet), especially with fresh fish (up to 10 g/kg) and meat (approximately 5 g/kg) (Wyss and Kaddurah-
* Corresponding author. Fax: +55 55 3220 8241. E-mail addresses: [email protected], furian.anafl[email protected], [email protected] (C.F. Mello). 0197-0186/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2008.04.008
Daouk, 2000). Creatine is the substrate for cytosolic and mitochondrial creatine kinases (CK). These isoenzymes modulate ATP metabolism by buffering fluctuations in ATP and ADP concentrations in the cell through CK reaction, and by facilitating intracellular transport of high-energy phosphates (Wyss and Schulze, 2002). This high-energy phosphate is utilized as an energy buffer, as well as an energy transport molecule (Wallimann et al., 1992), preventing ATP depletion caused by several conditions and agents, such as 3-hydroxyglutaric (Das et al., 2003) and methylmalonic acid (Royes et al., 2003, 2006) exposure, brain trauma, Parkinson and Huntington diseases, anoxia, hypoxia and ischemia (Sullivan et al., 2000; Sakellaris et al., 2006; Hass et al., 2007; Bender et al., 2005; Ferrante et al., 2000; Wilken et al., 1998;
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Baker-Fulco et al., 2006; Balestrino et al., 2002). Furthermore, it has been demonstrated that long-term creatine supplementation leads to an increase in health life span in mice accompanied by favorable effects on neurobehavioral functioning, especially memory skills (Bender et al., 2007). In agreement with this view, the intrahippocampal administration of creatine leads to spatial memory improvement (Oliveira et al., 2008). Although it is believed that mechanisms underlying creatineinduced neuronal function improvement and neuroprotection involve enhanced energy storage (Brewer and Wallimann, 2000), a direct neuromodulatory role for creatine has also been proposed (Persky and Brazeau, 2001). In this context, it has been shown that creatine is not only synthesized and taken up by neurons, but also released in an action potential-dependent manner, suggesting a neuromodulatory role for this guanidino compound in the brain (Almeida et al., 2006). Therefore, considering that creatine has been considered as prototype drug to sustain brain function (Wyss and KaddurahDaouk, 2000), which could potentially alleviate the functional deficits associated with selected neurodegenerative diseases, and its putative neuromodulatory role, we investigated whether creatine alters hippocampal excitability in vitro, and the involvement of NMDA receptors in the currently reported excitatory effects of this guanidine compound. 2. Materials and methods 2.1. Animals and reagents Adult male Wistar rats (270–300 g) maintained under controlled light and environment (12:12 h light–dark cycle, 24 1 8C, 55% relative humidity) with free access to food (Guabi, Santa Maria, Brazil) and water were used. All experimental protocols were designed aiming to keep the number of animals used to a minimum, as well as their suffering. All experimental protocols were conducted in accordance with national and international legislation (guidelines of Brazilian College of Animal Experimentation (COBEA) and of U.S. Public Health Service’s Policy on Humane Care and Use of Laboratory Animals - PHS Policy), and with the approval of the Ethics Committee for animal research of the Federal University of Santa Maria. All reagents were purchased from Sigma (St. Louis, USA). 2.2. Hippocampal slice preparations and electrophysiology The animals were anaesthetized with ketamine (50 mg/kg) and decapitated. The brains were rapidly removed and placed in cold (4 8C) sucrose-artificial cerebrospinal fluid (ACSF) containing (in mM): 75 sucrose, 3.5 KCl, 2 CaCl2, 5 MgSO4, 0.15 NaH2PO4, 22 NaHCO3, 25 D-glucose, and were aerated with 95% O2 and 5% CO2 (pH 7.4; 303 mOsm). Transverse 400 mm-thick hippocampal slices were cut with a Campden vibroslicer and immediately transferred to a holding recovery chamber, filled with ‘‘normal’’ ACSF containing (in mM): 125 NaCl, 3.5 KCl, 2 CaCl2, 2 MgSO4, 15 D-glucose, and aerated with 95% O2, 5% CO2, pH 7.4, at room temperature. After >1 h of recovery, the slice was transferred to a temperature-controlled (32 0.5 8C) interface recording chamber warmed and humidified with 95% O2–5% CO2 vapor, that was maintained over the exposed surface of the slice. Slices were continuously perfused (at 1.5 ml/min) with ‘‘normal’’ ACSF. The creatine was diluted in ACSF just before use and applied via bath superfusion with a peristaltic pump. Extracellular field potential recordings were made from the stratum pyramidale of the CA1 region of the hippocampus (population spike recordings). Recording pipettes pulled from borosilicate glass had a resistance of 3–5 MV when filled with 1 M NaCl. To elicit opulation spike (PS) responses, electrical stimuli were delivered using Tefloncoated bipolar iridium–platinum electrodes (10–20 mm tips) positioned in the Schaffer collaterals in the stratum radiatum. Constant square-current pulses (100–150 ms, 0.05– 0.03 Hz) were delivered through a constant-current isolation unit (Isoflex, AMPI, Jerusalem, Israel). Stimulus strengths were adjusted to evoke reproducible response amplitudes (70% of the maximal amplitude). Control traces were recorded at test stimuli for at least 30 min in order to establish baseline responses. Input–output curves were constructed to select optimal stimulation intensities and compare the effect of creatine exposure. Stimulation intensities ranged from 5 to 50 mA. In order to test the effect of creatine and participation of NMDA receptor in PS responses, slices were bathed for 60 min with a different concentration of creatine ranging from 0.5 to 25 mM (Balestrino et al., 2002) and AP-5 (100 mM) (Igartua et al., 2007). The amplitude of primary and secondary population spikes was measured from the peak negativity to the next positive peak. The maximum negative signal was measured relative to the mean and the maximum positive signal is measured relative the maximum negative signal (the peak-to-peak
amplitude) and then divided by two. In addition, input–output curves were elaborated by plotting the population spike response amplitudes (mV) versus different stimuli intensities (V). Signals were amplified and recorded with an Axoclamp-2B amplifier, digitized at 10 kHz with a DIGIDATA 1200A/D board (Axon Instruments, Molecular Devices, CA, USA), and responses were analyzed off-line using the Clampfit-6 software (Axon Instruments, Inc.). 2.3. Cell membrane fraction preparation and [3H]MK-801 binding assay The effect of creatine (5–1000 mM) on the NMDA receptor antagonist [3H]MK801 binding to hippocampal washed cell membrane fraction was determined by method described by Bailey et al. (2001). The animals were killed by decapitation under halothane anesthesia. The right and the left hippocampus from each rat was isolated and homogenized in 10 volumes (w/v) of 10 mM Tris–HCl, 300 mM sucrose, and 2 mM EDTA (pH 7.4). This homogenate was centrifuged at 1000 g for 10 min at 4 8C. The resulting supernatant was centrifuged at 20,000 g for 30 min at 4 8C. The resulting pellet was then resuspended in 1 ml of homogenization buffer and frozen at 70 8C until analyzed. Homogenates were incubated for 30 min at 37 8C and diluted with five volumes of wash buffer (50 mM Tris–HCl and 2 mM EDTA, pH 7.4), mixed, and centrifuged at 13,000 g for 10 min at 4 8C. This washing procedure was repeated twice, and the final pellet was resuspended in binding assay buffer (20 mM HEPES and 1 mM EDTA, pH 7.4). Protein content was measured colorimetrically by the method of Bradford (1976) and bovine serum albumin (1 mg/ml) was used as standard. [3H]MK-801 binding was determined in reaction vessels containing 200 mg of the washed cell membrane fraction, 5 nM [3H]MK-801 (21.7 Ci/mmol; PerkinElmer), 100 mM glutamate, 100 mM glycine, and binding assay buffer to give a total volume of 500 ml. Nonspecific binding of [3H]MK-801 was determined in the presence of 100 mM MK-801. Samples were incubated for 3 h at 37 8C and the reaction was terminated by rapid vacuum filtration through Whatman GF/B glass fiber filters that were pre-wet with 5 ml of ice-cold binding assay buffer. The filters were washed twice with 5 ml of ice-cold binding assay buffer, and the radioactivity remaining on the filters was quantified by liquid scintillation spectrometry (Packard Model 1409). 2.4. Statistical analysis Neurochemical data were analyzed by one or two-way analysis of variance (ANOVA), depending on the experimental design. Post hoc analyses were carried out by the F test for simple effect or the Student–Newman–Keuls test, when appropriate. Effective concentration 50 (EC50) values for [3H]MK801 binding were obtained by nonlinear regression analysis using a sigmoidal dose–response model (Prism 4.0; GraphPad Software, San Diego, CA). All data were expressed as mean S.E.M. Statistical analyses were performed utilizing the SPSS (Statistical Package for the social Sciences) software in a PC-compatible computer. P < 0.05 was considered significant.
3. Results The effect of creatine on extracellular field potential in the stratum pyramidale of the CA1 region of the hippocampus is shown in Fig. 1. Extracellular responses in control tissue were characterized by a simple population spike at different stimuli intensities (Doller and Weight, 1982; Arida et al., 2004). The mean amplitude of the baseline responses was 10.73 1.5 mV compared to the postcreatine (0.5–5 mM) amplitude of 17.00 1.61 mV and 17.20 1.59, respectively), representing a significant enhancement of 50–60% after creatine treatment. Statistical analysis showed that slices perfused with creatine (0.5–5 mM) resulted in gradual amplitude increase of the first population spikes [F(1,39) = 4.57; P < 0.05, Fig. 1A] followed by appearance of secondary PS (25 mM) [F(1,39) = 36.98; P < 0.05, Fig. 1B], also demonstrated by representative experiments in Fig. 2. The effect of creatine on stimulus intensity that evoked initial PS in hippocampal slices is shown in Fig. 3. Statistical analysis revealed that hippocampal slices perfused with creatine (0.5– 25 mM) decreased the stimulus threshold (in V) for obtaining PS when compared with hippocampal slices perfused with ACSF [F(1,39) = 15.07; P < 0.05]. In order to study the role of NMDA receptor on creatine-induced gradual amplitude increase of population spikes, the slices were perfused with AP-5, a competitive NMDA receptor antagonist. Post hoc analysis showed that slices perfused with AP-5 (100 mM)
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Fig. 3. Effect of creatine on stimulus threshold (in V) that evoked initial population spikes in hippocampal slices. Slices perfused with creatine (0.5–25 mM) decreased the stimulus intensity necessary for initial PS when compared with hippocampal slices perfused with ACSF. *P < 0.05 compared with basal ACSF group. Data are the means S.E.M. for n = 8 animals in each group.
Fig. 1. (A) Effect of creatine and AP-5 on population spikes amplitude from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (0.5 and 5 mM) induced an increase in the amplitude of primary PS. Slices perfused with creatine (5 mM) plus AP-5 (0.1 mM) decreased of creatine-induced amplitude of primary PS. *P < 0.05 compared with basal ACSF group. Data are the means S.E.M. for n = 8 animals in each group. (B) Effect of creatine (0.5–25 mM) and AP-5 (0.1 mM) on number of spikes from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (25 mM) induced the appearance of secondary PS. Slices perfused with creatine (25 mM) plus AP-5 (0.1 mM) decreased of spikes. *P < 0.05 compared with basal ACSF group. Data are the means S.E.M. for n = 8 animals in each group.
attenuated the creatine-induced gradual amplitude increase of the first population spikes (Fig. 1A) and appearance of secondary PS (Fig. 1B), also shown by representative experiments in Fig. 4. These
results suggest that creatine exerts its effects by interacting with NMDA receptor and/or events that follow its activation. In order to investigate whether creatine modulates NMDA receptor function, we carried out a [3H]MK801 binding assay, which reflects functional activity of the NMDA receptor (Gibson et al., 2002). Fig. 5 shows the effect of creatine on hippocampal [3H]MK-801 binding assay. Statistical analysis revealed that creatine (5–1000 mM) addition to membrane preparation enhanced the binding of [3H] MK-801 [F(4,11) = 5.28; P < 0.001] indicating not only a direct interaction of creatine with NMDA receptor but also a stimulatory effect of creatine on NMDA receptor. The EC50 value of creatine was 67 mM and the maximal effect was 158 16% at 1000 mM of creatine. These results appear to provide a neurochemical correlate of its ability to enhance neuronal firing evidenced in electrophysiological recordings. 4. Discussion Historically, over the last century, the creatine kinase and creatine field went through alternating periods of excitement and depression, concerning new ideas and concepts of creatine
Fig. 2. Representative experiments demonstrating the effect of creatine (0.5–25 mM) on population spikes from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (0.5 and 5 mM) induced an increase in the amplitude of primary population spike followed by appearance of secondary population spikes in slices perfused with creatine (25 mM). Calibrations bars, 2 mV and 5 ms.
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Fig. 4. Representative experiments demonstrating the effect of creatine (25 mM) and AP-5 (0.1 mM) on population spikes from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (25 mM) plus AP-5 (0.1 mM) antagonized the creatine-induced appearance of secondary population spikes. Calibrations bars, 2 mV and 5 ms.
function, particularly in the area of brain and muscle biochemistry (Salomons and Wyss, 2007). In this context, it has been proposed that creatine provides a buffer for the synthesis of ATP, enhancing the performance of high-intensity exercise (Bemben and Lamont, 2005). Moreover, creatine supplementation protects against ATP depletion and neuronal injury induced by several conditions, such as brain trauma, Parkinson and Huntington diseases, anoxia, hypoxia and ischemia (Sullivan et al., 2000; Sakellaris et al., 2006; Hass et al., 2007; Bender et al., 2005; Ferrante et al., 2000; Wilken et al., 1998; Baker-Fulco et al., 2006; Balestrino et al., 2002). Here, we showed that creatine facilitates hippocampal synaptic transmission in the hippocampus, an effect which is attenuated by NMDA receptor antagonist AP5. We also shown that creatine increases the binding of [3H]MK-801 to membranes, further supporting a functional modulatory role for this amino acid on NMDA receptors. Synaptic transmission in cerebral tissue can fail rapidly in some conditions, such as hypoxia or anoxia (Kono et al., 2007; Nieber et al., 1999). It has been demonstrated that the transmission failure in guinea pig hippocampal slices can be delayed by exposing the tissue to extracellular creatine (Cr), an effect that is associated with an increase of tissue phosphocreatine (PCr) concentration (Whittingham and Lipton, 1981). Of note, the levels of PCr and free creatine largely increase after creatine (25 mM) incubation in hippocampal slices during normoxia and protects against PCr depletion induced by hypoxia (Whittingham and Lipton, 1981; Lipton and Whittingham, 1982; Carter, 1994). Moreover, PCr levels increase after 3-h incubation with 1 mM of creatine (Balestrino et al., 1999) in hippocampal slices. In the current study, we showed that perfusion with creatine (0.5–25 mM) for 60 min facilitates neural transmission in the stratum pyramidale of the CA1 region, measured by the gradual amplitude increase of PS and the decrease of stimulus intensity to evoke them. The currently reported increase in hippocampal excitability by creatine is in agreement with the results from Cox and Bachelard (1985), who reported that 25 mM creatine increases neuronal excitability in hippocampal slices from
Fig. 5. Effect of creatine on hippocampal [3H] MK-801 binding assay. Creatine (5– 1000 mM) addition to membrane preparation enhanced the binding of [3H]MK-801. Data are the means S.E.M., n = 6–8 animals in each group.
guinea pig (Cox and Bachelard, 1985). On the other hand, our results are in disagreement with previous studies that have not found increase in the amplitude of PS in the stratum pyramidale of the CA1 region of the hippocampus when perfused with 10 mM creatine (Parodi et al., 2003). In this context, it is interesting that Parodi et al. (2003) have identified a nonsignificant increase in the amplitude of PS in the presence of 10 mM of creatine which, according to the authors, did not achieve statistical significance due to the high variability of the recorded responses. The determining factor for this discrepancy is not known, but we may speculate that this phenomenon may be a related to the model itself. While the period of incubation with creatine (3 h) descrited by Parodi et al. (2003) is the time required by creatine to be transformed in neuroprotectant derived PCr (Reith et al., 1997), the creatine-induced amplitude increase evidenced 60 min after incubation suggest a possible pharmacological effect of this compound on neural transmission. In agreement with our results, it has been described that the addition of 25 mM creatine to guinea pig hippocampal slices superfusate increases neuronal excitability (Cox and Bachelard, 1985). Despite the apparent conflict between our data and data from Parodi et al. (2003), in the present study we show, for the first time, pharmacological evidence for the involvement of NMDA receptors in the effects of creatine, since it revealed a significant facilitation of [3H]MK-801 binding to hippocampal membrane preparations. In this context, although the main known effect of creatine is to increase high-energy phosphate levels, this guanidino compound seems also to play a modulatory role in the central transmission process (Persky and Brazeau, 2001). In fact, recent evidence suggests a neuromodulatory role for this guanidino compound (Almeida et al., 2006), since it accumulates in neocortex slices in a Na+dependent manner, and is electrically released from neocortex, caudate putamen and hippocampus slices, an effect that is dependent of Ca2+ influx and Na+ channel stimulation. These in vitro data indicate that creatine is not only synthesized and taken up by central neurons, but also released in exocytotic manner (Almeida et al., 2006). In this study we found that AP-5 reduces creatineinduced amplitude increase of the first PS and number of spikes in slices, suggesting the involvement of NMDA receptors in the excitatory effect of this compound. Another remarkable finding of the current study that further supports the involvement of NMDA receptors in the effects of creatine is the facilitation of [3H]MK-801 binding to hippocampal membrane preparations. Considering that [3H]MK-801 binding reflects functional activity of the NMDA receptor (Gibson et al., 2002), our results demonstrate not only a direct interaction of creatine with the NMDA receptor, but also suggests that this interaction is stimulatory. Importantly, these results may be of physiological relevance, since the EC50 value obtained for creatine (67 mM) is similar to the cerebrospinal fluid (34–42 mM) concentration (Perasso et al., 2003; Sto¨ckler et al., 1997), and constitute circumstantial evidence that creatine is a neuromodulator in the central nervous system. In this context, results from single cell recording studies in the hippocampus have demonstrated that neurons encode spatial,
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temporal, and goal-related information by activation of several neuronal receptors, such as NMDA (Ho¨lscher et al., 2003; Axmacher et al., 2006). Regarding this point, previous results from our group demonstrated the involvement of NMDA receptors in spatial memory improvement induced by bilateral microinjection of creatine into hippocampus (Oliveira et al., 2008). For instance, Schulze (2003) reported that creatine deficiency syndromes are characterized by development delay or regression and mental retardation, suggesting that creatine is important for mental activities in brain. In summary, while in this study we report that creatine facilitates hippocampal excitatory postsynaptic potentials, we also present exciting novel data about the role of NMDA receptors in this effect of creatine. Regarding this point, we show electrophysiological and neurochemical evidence suggesting that creatine-induced increase of neuronal firing in the hippocampus involves NMDA receptors. Therefore, in addition to its role as a precursor of energy-rich compounds that maintain nearly all cellular functions, including neurotransmission (Liu and Xie, 2002; Wu et al., 2004; Hoyer et al., 2004), our results suggest a putative role for this amino acid in neuronal plasticity. Acknowledgements Work supported by CNPq (grant: 500120/2003-0), CAPES, FAPERGS and FAPESP. C.F. Mello, A.F. Furian and J. Ferreira are the recipients of CNPq fellowships. M.S Oliveira is the recipient of CAPES fellowships. References Almeida, L.S., Salomons, G.S., Hogenboom, F., Jakobs, C., Schoffelmeer, A.N.M., 2006. Exocytotic release of creatine in rat brain. Synapse 60, 118–123. Arida, R.M., Sanabria, E.R., da Silva, A.C., Faria, L.C., Scorza, F.A., Cavalheiro, E.A., 2004. Physical training reverts hippocampal electrophysiological changes in rats submitted to the pilocarpine model of epilepsy. Physiol. Behav. 83, 165– 171. Axmacher, N., Mormann, F., Ferna´ndez, G., Elger, C.E., Fell, J., 2006. Memory formation by neuronal synchronization. Brain Res. Rev. 52 (1), 170–182. Bailey, C.D., Brien, J.F., Reynolds, J.N., 2001. Chronic prenatal ethanol exposure increases GABA(a) receptor subunit protein expression in the adult guinea pig cerebral cortex. J. Neurosci. 21, 4381–4389. Baker-Fulco, C.J., Fulco, C.S., Kellogg, M.D., Glickman, E., Young, A.J., 2006. Voluntary muscle function after creatine supplementation in acute hypobaric hypoxia. Med. Sci. Sports Exerc. 38 (8), 1418–1424. Balestrino, M., Rebaudo, R., Lunardi, G., 1999. Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose–effect relationship. Brain Res. 816, 124–130. Balestrino, M., Lensman, M., Parodi, M., Perasso, L., Rebaudo, R., Melani, R., Polenov, S., Cupello, A., 2002. Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage. Amino Acids 23, 221–229. Bemben, M.G., Lamont, H.S., 2005. Creatine supplementation and exercise performance. Sports Med. 35, 107–125. Bender, A., Beckers, J., Schneider, I., Ho¨lter, S.M., Haack, T., Ruthsatz, T., VogtWeisenhorn, D.M., Becker, L., Genius, J., Rujescu, D., Irmler, M., Mijalski, T., Mader, M., Quintanilla-Martinez, L., Fuchs, H., Gailus-Durner, V., de Angelis, M.H., Wurst, W., Schmidt, J., Klopstock, T., 2007. Creatine improves health and survival of mice. Neurobiol. Aging, doi:10.1016/j.neurobiolaging.2007.03.001 Bender, A., Auer, D.P., Merl, T., Reilmann, R., Saemann, P., Yassouridis, A., Bender, J., Weindl, A., Dose, M., Gasser, T., Klopstock, T., 2005. Creatine supplementation lowers brain glutamate levels in Huntington’s disease. J. Neurol. 252 (1), 36–40. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248–254. Brewer, G.J., Wallimann, T.W., 2000. Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons. J. Neurochem. 74 (5), 1968–1978. Carter, C., 1994. The Neuropharmacology of Polyamines. Academic, London. Cox, D.W., Bachelard, H.S., 1985. Effect of creatine on granule cell evoked activity in the presence of normal and decreased levels of glucose. Biomed. Biochim. Acta 44 (10), 1483–1490. Das, A.M., Lu¨cke, T., Ullrich, K., 2003. Glutaric aciduria I: creatine supplementation restores creatinephosphate levels in mixed cortex cells from rat incubated with 3-hydroxyglutarate. Mol. Genet. Metab. 78 (2), 108–111.
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Clinical pharmacology of the dietary supplement creatine monohydrate. Pharmacol. Rev. 53, 161–176. Reith, J., Jorgensen, H.S., Nakayama, H., Raaschou, H.O., Olsen, T.S., 1997. Seizures in acute stroke: predictors and prognostic significance—the Copenhagen Stroke Study. Stroke 28, 1585–1589. Royes, L.F., Fighera, M.R., Furian, A.F., Oliveira, M.S., da Silva, L.G., Malfatti, C.R., Schneider, P.H., Braga, A.L., Wajner, M., Mello, C.F., 2003. Creatine protects against the convulsive behavior and lactate production elicited by the intrastriatal injection of methilmalonate. Neuroscience 118, 1079–1090. Royes, L.F., Fighera, M.R., Furian, A.F., Oliveira, M.S., Myskiw, J.C., Fiorenza, N.G., Petry, J.C., Coelho, R.C., Mello, C.F., 2006. Effectiveness of creatine monohydrate on seizures and oxidative damage induced by methylmalonate. Pharmacol. Biochem. Behav. 83, 136–144. 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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: Role of NMDA receptors Luiz Fernando Freire Royes a,b, Michele Rechia Fighera b,c, Ana Fla´via Furian b,d, Mauro Schneider Oliveira b,d, Nata´lia Gindri Fiorenza b, Juliano Ferreira e, Andre´ Cesar da Silva f, Margareth Rose Priel f, E´rika Sayuri Ueda f, Joa˜o Batista Calixto g, Esper Abra˜o Cavalheiro f, Carlos Fernando Mello b,* a
Centro de Educac¸a˜o Fı´sica e Desportos, Departamento de Me´todos e Te´cnicas Desportivas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Centro de Cieˆncias da Sau´de, Laborato´rio de Neurotoxicidade e Psicofarmacologia (LABNEURO), Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil c Centro de Cieˆncias da Sau´de, Departamento de Pediatria, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Programa de Po´s-Graduac¸a˜o em Cieˆncias Biolo´gicas: Bioquı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil e Centro de Cieˆncias Naturais e Exatas, Laborato´rio de Neurotoxicidade e Psicofarmacologia (LABNEURO), Departamento de Quı´mica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil f Laborato´rio de Neurologia Experimental, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil g Centro de Cieˆncias Biolo´gicas, Departamento de Farmacologia, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2008 Accepted 25 April 2008 Available online 2 May 2008
The creatine (Cr) and phosphocreatine (PCr) system is essential for the buffering and transport of highenergy phosphates. Although achievements made over the last years have highlighted the important role of creatine in several neurological diseases, the adaptive processes elicited by this guanidino compound in hippocampus are poorly understood. In the present study, we showed that creatine (0.5–25 mM) gradually increases the amplitude of first population spike (PS) and elicits secondary PS in stratum radiatum of the CA1 region, in hippocampal slices. Creatine also decreased the intensity of the stimulus to induce PS, when compared with hippocampal slices perfused with artificial cerebrospinal fluid (ACSF). The competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 100 mM) attenuated creatine-induced increase of amplitude of PS and appearance of secondary PS, providing pharmacological evidence of the involvement of NMDA receptors in the electrophysiological effects of creatine. Accordingly, creatine (0.01–1 mM) increased [3H]MK-801 binding to hippocampal membranes by 55%, further indicating that this compound modulates NMDA receptor function. These results implicate the NMDA receptor in amplitude and population spike increase elicited by creatine in hippocampus. Furthermore, these data suggest that this guanidino compound may also play a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by an increase in glutamatergic function. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Creatine Electrophysiology Hippocampus NMDA [3H]MK-801
1. Introduction Creatine (N-[aminoiminomethyl]-N-methyl glycine) is a guanidino compound endogenously synthesized from glycine, arginine and S-adenosylmethionine in the kidneys, liver and pancreas or ingested in small quantities (the amount can vary from 0 to 7 g of more, depending on the diet), especially with fresh fish (up to 10 g/kg) and meat (approximately 5 g/kg) (Wyss and Kaddurah-
* Corresponding author. Fax: +55 55 3220 8241. E-mail addresses: [email protected], furian.anafl[email protected], [email protected] (C.F. Mello). 0197-0186/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2008.04.008
Daouk, 2000). Creatine is the substrate for cytosolic and mitochondrial creatine kinases (CK). These isoenzymes modulate ATP metabolism by buffering fluctuations in ATP and ADP concentrations in the cell through CK reaction, and by facilitating intracellular transport of high-energy phosphates (Wyss and Schulze, 2002). This high-energy phosphate is utilized as an energy buffer, as well as an energy transport molecule (Wallimann et al., 1992), preventing ATP depletion caused by several conditions and agents, such as 3-hydroxyglutaric (Das et al., 2003) and methylmalonic acid (Royes et al., 2003, 2006) exposure, brain trauma, Parkinson and Huntington diseases, anoxia, hypoxia and ischemia (Sullivan et al., 2000; Sakellaris et al., 2006; Hass et al., 2007; Bender et al., 2005; Ferrante et al., 2000; Wilken et al., 1998;
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Baker-Fulco et al., 2006; Balestrino et al., 2002). Furthermore, it has been demonstrated that long-term creatine supplementation leads to an increase in health life span in mice accompanied by favorable effects on neurobehavioral functioning, especially memory skills (Bender et al., 2007). In agreement with this view, the intrahippocampal administration of creatine leads to spatial memory improvement (Oliveira et al., 2008). Although it is believed that mechanisms underlying creatineinduced neuronal function improvement and neuroprotection involve enhanced energy storage (Brewer and Wallimann, 2000), a direct neuromodulatory role for creatine has also been proposed (Persky and Brazeau, 2001). In this context, it has been shown that creatine is not only synthesized and taken up by neurons, but also released in an action potential-dependent manner, suggesting a neuromodulatory role for this guanidino compound in the brain (Almeida et al., 2006). Therefore, considering that creatine has been considered as prototype drug to sustain brain function (Wyss and KaddurahDaouk, 2000), which could potentially alleviate the functional deficits associated with selected neurodegenerative diseases, and its putative neuromodulatory role, we investigated whether creatine alters hippocampal excitability in vitro, and the involvement of NMDA receptors in the currently reported excitatory effects of this guanidine compound. 2. Materials and methods 2.1. Animals and reagents Adult male Wistar rats (270–300 g) maintained under controlled light and environment (12:12 h light–dark cycle, 24 1 8C, 55% relative humidity) with free access to food (Guabi, Santa Maria, Brazil) and water were used. All experimental protocols were designed aiming to keep the number of animals used to a minimum, as well as their suffering. All experimental protocols were conducted in accordance with national and international legislation (guidelines of Brazilian College of Animal Experimentation (COBEA) and of U.S. Public Health Service’s Policy on Humane Care and Use of Laboratory Animals - PHS Policy), and with the approval of the Ethics Committee for animal research of the Federal University of Santa Maria. All reagents were purchased from Sigma (St. Louis, USA). 2.2. Hippocampal slice preparations and electrophysiology The animals were anaesthetized with ketamine (50 mg/kg) and decapitated. The brains were rapidly removed and placed in cold (4 8C) sucrose-artificial cerebrospinal fluid (ACSF) containing (in mM): 75 sucrose, 3.5 KCl, 2 CaCl2, 5 MgSO4, 0.15 NaH2PO4, 22 NaHCO3, 25 D-glucose, and were aerated with 95% O2 and 5% CO2 (pH 7.4; 303 mOsm). Transverse 400 mm-thick hippocampal slices were cut with a Campden vibroslicer and immediately transferred to a holding recovery chamber, filled with ‘‘normal’’ ACSF containing (in mM): 125 NaCl, 3.5 KCl, 2 CaCl2, 2 MgSO4, 15 D-glucose, and aerated with 95% O2, 5% CO2, pH 7.4, at room temperature. After >1 h of recovery, the slice was transferred to a temperature-controlled (32 0.5 8C) interface recording chamber warmed and humidified with 95% O2–5% CO2 vapor, that was maintained over the exposed surface of the slice. Slices were continuously perfused (at 1.5 ml/min) with ‘‘normal’’ ACSF. The creatine was diluted in ACSF just before use and applied via bath superfusion with a peristaltic pump. Extracellular field potential recordings were made from the stratum pyramidale of the CA1 region of the hippocampus (population spike recordings). Recording pipettes pulled from borosilicate glass had a resistance of 3–5 MV when filled with 1 M NaCl. To elicit opulation spike (PS) responses, electrical stimuli were delivered using Tefloncoated bipolar iridium–platinum electrodes (10–20 mm tips) positioned in the Schaffer collaterals in the stratum radiatum. Constant square-current pulses (100–150 ms, 0.05– 0.03 Hz) were delivered through a constant-current isolation unit (Isoflex, AMPI, Jerusalem, Israel). Stimulus strengths were adjusted to evoke reproducible response amplitudes (70% of the maximal amplitude). Control traces were recorded at test stimuli for at least 30 min in order to establish baseline responses. Input–output curves were constructed to select optimal stimulation intensities and compare the effect of creatine exposure. Stimulation intensities ranged from 5 to 50 mA. In order to test the effect of creatine and participation of NMDA receptor in PS responses, slices were bathed for 60 min with a different concentration of creatine ranging from 0.5 to 25 mM (Balestrino et al., 2002) and AP-5 (100 mM) (Igartua et al., 2007). The amplitude of primary and secondary population spikes was measured from the peak negativity to the next positive peak. The maximum negative signal was measured relative to the mean and the maximum positive signal is measured relative the maximum negative signal (the peak-to-peak
amplitude) and then divided by two. In addition, input–output curves were elaborated by plotting the population spike response amplitudes (mV) versus different stimuli intensities (V). Signals were amplified and recorded with an Axoclamp-2B amplifier, digitized at 10 kHz with a DIGIDATA 1200A/D board (Axon Instruments, Molecular Devices, CA, USA), and responses were analyzed off-line using the Clampfit-6 software (Axon Instruments, Inc.). 2.3. Cell membrane fraction preparation and [3H]MK-801 binding assay The effect of creatine (5–1000 mM) on the NMDA receptor antagonist [3H]MK801 binding to hippocampal washed cell membrane fraction was determined by method described by Bailey et al. (2001). The animals were killed by decapitation under halothane anesthesia. The right and the left hippocampus from each rat was isolated and homogenized in 10 volumes (w/v) of 10 mM Tris–HCl, 300 mM sucrose, and 2 mM EDTA (pH 7.4). This homogenate was centrifuged at 1000 g for 10 min at 4 8C. The resulting supernatant was centrifuged at 20,000 g for 30 min at 4 8C. The resulting pellet was then resuspended in 1 ml of homogenization buffer and frozen at 70 8C until analyzed. Homogenates were incubated for 30 min at 37 8C and diluted with five volumes of wash buffer (50 mM Tris–HCl and 2 mM EDTA, pH 7.4), mixed, and centrifuged at 13,000 g for 10 min at 4 8C. This washing procedure was repeated twice, and the final pellet was resuspended in binding assay buffer (20 mM HEPES and 1 mM EDTA, pH 7.4). Protein content was measured colorimetrically by the method of Bradford (1976) and bovine serum albumin (1 mg/ml) was used as standard. [3H]MK-801 binding was determined in reaction vessels containing 200 mg of the washed cell membrane fraction, 5 nM [3H]MK-801 (21.7 Ci/mmol; PerkinElmer), 100 mM glutamate, 100 mM glycine, and binding assay buffer to give a total volume of 500 ml. Nonspecific binding of [3H]MK-801 was determined in the presence of 100 mM MK-801. Samples were incubated for 3 h at 37 8C and the reaction was terminated by rapid vacuum filtration through Whatman GF/B glass fiber filters that were pre-wet with 5 ml of ice-cold binding assay buffer. The filters were washed twice with 5 ml of ice-cold binding assay buffer, and the radioactivity remaining on the filters was quantified by liquid scintillation spectrometry (Packard Model 1409). 2.4. Statistical analysis Neurochemical data were analyzed by one or two-way analysis of variance (ANOVA), depending on the experimental design. Post hoc analyses were carried out by the F test for simple effect or the Student–Newman–Keuls test, when appropriate. Effective concentration 50 (EC50) values for [3H]MK801 binding were obtained by nonlinear regression analysis using a sigmoidal dose–response model (Prism 4.0; GraphPad Software, San Diego, CA). All data were expressed as mean S.E.M. Statistical analyses were performed utilizing the SPSS (Statistical Package for the social Sciences) software in a PC-compatible computer. P < 0.05 was considered significant.
3. Results The effect of creatine on extracellular field potential in the stratum pyramidale of the CA1 region of the hippocampus is shown in Fig. 1. Extracellular responses in control tissue were characterized by a simple population spike at different stimuli intensities (Doller and Weight, 1982; Arida et al., 2004). The mean amplitude of the baseline responses was 10.73 1.5 mV compared to the postcreatine (0.5–5 mM) amplitude of 17.00 1.61 mV and 17.20 1.59, respectively), representing a significant enhancement of 50–60% after creatine treatment. Statistical analysis showed that slices perfused with creatine (0.5–5 mM) resulted in gradual amplitude increase of the first population spikes [F(1,39) = 4.57; P < 0.05, Fig. 1A] followed by appearance of secondary PS (25 mM) [F(1,39) = 36.98; P < 0.05, Fig. 1B], also demonstrated by representative experiments in Fig. 2. The effect of creatine on stimulus intensity that evoked initial PS in hippocampal slices is shown in Fig. 3. Statistical analysis revealed that hippocampal slices perfused with creatine (0.5– 25 mM) decreased the stimulus threshold (in V) for obtaining PS when compared with hippocampal slices perfused with ACSF [F(1,39) = 15.07; P < 0.05]. In order to study the role of NMDA receptor on creatine-induced gradual amplitude increase of population spikes, the slices were perfused with AP-5, a competitive NMDA receptor antagonist. Post hoc analysis showed that slices perfused with AP-5 (100 mM)
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Fig. 3. Effect of creatine on stimulus threshold (in V) that evoked initial population spikes in hippocampal slices. Slices perfused with creatine (0.5–25 mM) decreased the stimulus intensity necessary for initial PS when compared with hippocampal slices perfused with ACSF. *P < 0.05 compared with basal ACSF group. Data are the means S.E.M. for n = 8 animals in each group.
Fig. 1. (A) Effect of creatine and AP-5 on population spikes amplitude from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (0.5 and 5 mM) induced an increase in the amplitude of primary PS. Slices perfused with creatine (5 mM) plus AP-5 (0.1 mM) decreased of creatine-induced amplitude of primary PS. *P < 0.05 compared with basal ACSF group. Data are the means S.E.M. for n = 8 animals in each group. (B) Effect of creatine (0.5–25 mM) and AP-5 (0.1 mM) on number of spikes from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (25 mM) induced the appearance of secondary PS. Slices perfused with creatine (25 mM) plus AP-5 (0.1 mM) decreased of spikes. *P < 0.05 compared with basal ACSF group. Data are the means S.E.M. for n = 8 animals in each group.
attenuated the creatine-induced gradual amplitude increase of the first population spikes (Fig. 1A) and appearance of secondary PS (Fig. 1B), also shown by representative experiments in Fig. 4. These
results suggest that creatine exerts its effects by interacting with NMDA receptor and/or events that follow its activation. In order to investigate whether creatine modulates NMDA receptor function, we carried out a [3H]MK801 binding assay, which reflects functional activity of the NMDA receptor (Gibson et al., 2002). Fig. 5 shows the effect of creatine on hippocampal [3H]MK-801 binding assay. Statistical analysis revealed that creatine (5–1000 mM) addition to membrane preparation enhanced the binding of [3H] MK-801 [F(4,11) = 5.28; P < 0.001] indicating not only a direct interaction of creatine with NMDA receptor but also a stimulatory effect of creatine on NMDA receptor. The EC50 value of creatine was 67 mM and the maximal effect was 158 16% at 1000 mM of creatine. These results appear to provide a neurochemical correlate of its ability to enhance neuronal firing evidenced in electrophysiological recordings. 4. Discussion Historically, over the last century, the creatine kinase and creatine field went through alternating periods of excitement and depression, concerning new ideas and concepts of creatine
Fig. 2. Representative experiments demonstrating the effect of creatine (0.5–25 mM) on population spikes from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (0.5 and 5 mM) induced an increase in the amplitude of primary population spike followed by appearance of secondary population spikes in slices perfused with creatine (25 mM). Calibrations bars, 2 mV and 5 ms.
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Fig. 4. Representative experiments demonstrating the effect of creatine (25 mM) and AP-5 (0.1 mM) on population spikes from the CA1 area following stimulation of Schaffer collaterals. Slices perfused with creatine (25 mM) plus AP-5 (0.1 mM) antagonized the creatine-induced appearance of secondary population spikes. Calibrations bars, 2 mV and 5 ms.
function, particularly in the area of brain and muscle biochemistry (Salomons and Wyss, 2007). In this context, it has been proposed that creatine provides a buffer for the synthesis of ATP, enhancing the performance of high-intensity exercise (Bemben and Lamont, 2005). Moreover, creatine supplementation protects against ATP depletion and neuronal injury induced by several conditions, such as brain trauma, Parkinson and Huntington diseases, anoxia, hypoxia and ischemia (Sullivan et al., 2000; Sakellaris et al., 2006; Hass et al., 2007; Bender et al., 2005; Ferrante et al., 2000; Wilken et al., 1998; Baker-Fulco et al., 2006; Balestrino et al., 2002). Here, we showed that creatine facilitates hippocampal synaptic transmission in the hippocampus, an effect which is attenuated by NMDA receptor antagonist AP5. We also shown that creatine increases the binding of [3H]MK-801 to membranes, further supporting a functional modulatory role for this amino acid on NMDA receptors. Synaptic transmission in cerebral tissue can fail rapidly in some conditions, such as hypoxia or anoxia (Kono et al., 2007; Nieber et al., 1999). It has been demonstrated that the transmission failure in guinea pig hippocampal slices can be delayed by exposing the tissue to extracellular creatine (Cr), an effect that is associated with an increase of tissue phosphocreatine (PCr) concentration (Whittingham and Lipton, 1981). Of note, the levels of PCr and free creatine largely increase after creatine (25 mM) incubation in hippocampal slices during normoxia and protects against PCr depletion induced by hypoxia (Whittingham and Lipton, 1981; Lipton and Whittingham, 1982; Carter, 1994). Moreover, PCr levels increase after 3-h incubation with 1 mM of creatine (Balestrino et al., 1999) in hippocampal slices. In the current study, we showed that perfusion with creatine (0.5–25 mM) for 60 min facilitates neural transmission in the stratum pyramidale of the CA1 region, measured by the gradual amplitude increase of PS and the decrease of stimulus intensity to evoke them. The currently reported increase in hippocampal excitability by creatine is in agreement with the results from Cox and Bachelard (1985), who reported that 25 mM creatine increases neuronal excitability in hippocampal slices from
Fig. 5. Effect of creatine on hippocampal [3H] MK-801 binding assay. Creatine (5– 1000 mM) addition to membrane preparation enhanced the binding of [3H]MK-801. Data are the means S.E.M., n = 6–8 animals in each group.
guinea pig (Cox and Bachelard, 1985). On the other hand, our results are in disagreement with previous studies that have not found increase in the amplitude of PS in the stratum pyramidale of the CA1 region of the hippocampus when perfused with 10 mM creatine (Parodi et al., 2003). In this context, it is interesting that Parodi et al. (2003) have identified a nonsignificant increase in the amplitude of PS in the presence of 10 mM of creatine which, according to the authors, did not achieve statistical significance due to the high variability of the recorded responses. The determining factor for this discrepancy is not known, but we may speculate that this phenomenon may be a related to the model itself. While the period of incubation with creatine (3 h) descrited by Parodi et al. (2003) is the time required by creatine to be transformed in neuroprotectant derived PCr (Reith et al., 1997), the creatine-induced amplitude increase evidenced 60 min after incubation suggest a possible pharmacological effect of this compound on neural transmission. In agreement with our results, it has been described that the addition of 25 mM creatine to guinea pig hippocampal slices superfusate increases neuronal excitability (Cox and Bachelard, 1985). Despite the apparent conflict between our data and data from Parodi et al. (2003), in the present study we show, for the first time, pharmacological evidence for the involvement of NMDA receptors in the effects of creatine, since it revealed a significant facilitation of [3H]MK-801 binding to hippocampal membrane preparations. In this context, although the main known effect of creatine is to increase high-energy phosphate levels, this guanidino compound seems also to play a modulatory role in the central transmission process (Persky and Brazeau, 2001). In fact, recent evidence suggests a neuromodulatory role for this guanidino compound (Almeida et al., 2006), since it accumulates in neocortex slices in a Na+dependent manner, and is electrically released from neocortex, caudate putamen and hippocampus slices, an effect that is dependent of Ca2+ influx and Na+ channel stimulation. These in vitro data indicate that creatine is not only synthesized and taken up by central neurons, but also released in exocytotic manner (Almeida et al., 2006). In this study we found that AP-5 reduces creatineinduced amplitude increase of the first PS and number of spikes in slices, suggesting the involvement of NMDA receptors in the excitatory effect of this compound. Another remarkable finding of the current study that further supports the involvement of NMDA receptors in the effects of creatine is the facilitation of [3H]MK-801 binding to hippocampal membrane preparations. Considering that [3H]MK-801 binding reflects functional activity of the NMDA receptor (Gibson et al., 2002), our results demonstrate not only a direct interaction of creatine with the NMDA receptor, but also suggests that this interaction is stimulatory. Importantly, these results may be of physiological relevance, since the EC50 value obtained for creatine (67 mM) is similar to the cerebrospinal fluid (34–42 mM) concentration (Perasso et al., 2003; Sto¨ckler et al., 1997), and constitute circumstantial evidence that creatine is a neuromodulator in the central nervous system. In this context, results from single cell recording studies in the hippocampus have demonstrated that neurons encode spatial,
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temporal, and goal-related information by activation of several neuronal receptors, such as NMDA (Ho¨lscher et al., 2003; Axmacher et al., 2006). Regarding this point, previous results from our group demonstrated the involvement of NMDA receptors in spatial memory improvement induced by bilateral microinjection of creatine into hippocampus (Oliveira et al., 2008). For instance, Schulze (2003) reported that creatine deficiency syndromes are characterized by development delay or regression and mental retardation, suggesting that creatine is important for mental activities in brain. In summary, while in this study we report that creatine facilitates hippocampal excitatory postsynaptic potentials, we also present exciting novel data about the role of NMDA receptors in this effect of creatine. Regarding this point, we show electrophysiological and neurochemical evidence suggesting that creatine-induced increase of neuronal firing in the hippocampus involves NMDA receptors. Therefore, in addition to its role as a precursor of energy-rich compounds that maintain nearly all cellular functions, including neurotransmission (Liu and Xie, 2002; Wu et al., 2004; Hoyer et al., 2004), our results suggest a putative role for this amino acid in neuronal plasticity. Acknowledgements Work supported by CNPq (grant: 500120/2003-0), CAPES, FAPERGS and FAPESP. C.F. Mello, A.F. Furian and J. Ferreira are the recipients of CNPq fellowships. M.S Oliveira is the recipient of CAPES fellowships. References Almeida, L.S., Salomons, G.S., Hogenboom, F., Jakobs, C., Schoffelmeer, A.N.M., 2006. Exocytotic release of creatine in rat brain. Synapse 60, 118–123. Arida, R.M., Sanabria, E.R., da Silva, A.C., Faria, L.C., Scorza, F.A., Cavalheiro, E.A., 2004. Physical training reverts hippocampal electrophysiological changes in rats submitted to the pilocarpine model of epilepsy. Physiol. Behav. 83, 165– 171. Axmacher, N., Mormann, F., Ferna´ndez, G., Elger, C.E., Fell, J., 2006. Memory formation by neuronal synchronization. Brain Res. Rev. 52 (1), 170–182. Bailey, C.D., Brien, J.F., Reynolds, J.N., 2001. Chronic prenatal ethanol exposure increases GABA(a) receptor subunit protein expression in the adult guinea pig cerebral cortex. J. Neurosci. 21, 4381–4389. Baker-Fulco, C.J., Fulco, C.S., Kellogg, M.D., Glickman, E., Young, A.J., 2006. Voluntary muscle function after creatine supplementation in acute hypobaric hypoxia. Med. Sci. Sports Exerc. 38 (8), 1418–1424. Balestrino, M., Rebaudo, R., Lunardi, G., 1999. Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose–effect relationship. Brain Res. 816, 124–130. Balestrino, M., Lensman, M., Parodi, M., Perasso, L., Rebaudo, R., Melani, R., Polenov, S., Cupello, A., 2002. Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage. Amino Acids 23, 221–229. Bemben, M.G., Lamont, H.S., 2005. Creatine supplementation and exercise performance. Sports Med. 35, 107–125. Bender, A., Beckers, J., Schneider, I., Ho¨lter, S.M., Haack, T., Ruthsatz, T., VogtWeisenhorn, D.M., Becker, L., Genius, J., Rujescu, D., Irmler, M., Mijalski, T., Mader, M., Quintanilla-Martinez, L., Fuchs, H., Gailus-Durner, V., de Angelis, M.H., Wurst, W., Schmidt, J., Klopstock, T., 2007. Creatine improves health and survival of mice. Neurobiol. Aging, doi:10.1016/j.neurobiolaging.2007.03.001 Bender, A., Auer, D.P., Merl, T., Reilmann, R., Saemann, P., Yassouridis, A., Bender, J., Weindl, A., Dose, M., Gasser, T., Klopstock, T., 2005. Creatine supplementation lowers brain glutamate levels in Huntington’s disease. J. Neurol. 252 (1), 36–40. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248–254. Brewer, G.J., Wallimann, T.W., 2000. Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons. J. Neurochem. 74 (5), 1968–1978. Carter, C., 1994. The Neuropharmacology of Polyamines. Academic, London. Cox, D.W., Bachelard, H.S., 1985. Effect of creatine on granule cell evoked activity in the presence of normal and decreased levels of glucose. Biomed. Biochim. Acta 44 (10), 1483–1490. Das, A.M., Lu¨cke, T., Ullrich, K., 2003. Glutaric aciduria I: creatine supplementation restores creatinephosphate levels in mixed cortex cells from rat incubated with 3-hydroxyglutarate. Mol. Genet. Metab. 78 (2), 108–111.
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