Effects of l-arginine and creatine administration on spatial memory in rats subjected to a chronic variable stress model

Abstract

Context: Chronic stress results from repeated exposure to one or more types of stressors over a period, ranging from days to months, and can be associated with physical, behavioral, and neuropsychiatric manifestations. Some physiological alterations resulting from chronic stress can potentially cause deficits on spatial learning and memory.

Objective: This study investigated the effects of chronic variable stress (CVS) and administration of l-arginine and creatine on spatial memory in rats. Furthermore, body, heart, adrenal weight, and plasma glucose and corticosterone levels were analyzed.

Material and methods: Male Wistar rats were subjected to a CVS model for 40 days and evaluated for spatial memory after the stress period. Chronically stressed animals were treated daily by gavage with: 0.5% carboxymethylcellulose (Group Cs), 500 mg/kg l-arginine (Group Cs/La), 300 mg/kg creatine (Group Cs/Cr); and 500 mg/kg l-arginine and 300 mg/kg creatine (Group Cs/La + Cr) during the entire experimental period.

Results: Our results showed that animals in the Cs/Cr and Cs/La + Cr groups presented significantly decreased corticosterone levels compared to group Cs (p < 0.05); animals in group Cs/Cr were more efficient in finding the platform, in the working memory task, compared to all other groups (p < 0.01); and animals in group Cs/La + Cr significantly improved in reference memory retention compared to controls (p < 0.05).

Discussion and conclusion: Overall, these results demonstrated that a single administration of creatine improves working memory efficiency, and, when co-administrated with l-arginine, improves reference memory retention, a phenomenon that is possibly associated with increased creatine/phosphocreatine levels and l-arginine-derived NO synthesis.

• Chronic stress
• creatine
• l-arginine
• memory
• nitric oxide.

Introduction

Chronic stress results from continuous or repeated exposure to one or more types of stressors over a period ranging from days to months (Ulrich-Lai & Herman, 2009). It is a condition associable to several physical, behavioral and neuropsychiatric manifestations (Chrousos, 2009). Studies have demonstrated that chronic stress is associated with Na⁺, K⁺, ATPases activity reduction, impairment of energy metabolism and oxidative stress in the hippocampus (Vasconcellos et al., 2005; Tagliari et al., 2010). These alterations can potentially cause deficits on spatial learning and memory observed in different tasks (Kleen et al., 2006; Mclay et al., 1998; Vasconcellos et al., 2005).

l-Arginine is a non-essential amino acid involved in several metabolic pathways that produce a variety of biologically active compounds such as agmatine, polyamines, ornithine, citrulline and nitric oxide (NO) (Huynh & Chin-Dusting, 2006). An important role in memory formation has been proposed to the l-arginine-NO pathway because NO is synthesized and released from post-synaptic neuron, and acts as a retrograde messenger directly in pre-synaptic neurons. Moreover, it coordinates the enhancement of pre- and post-synaptic mechanisms involved in synaptic plasticity such as the long-term potentiation (LTP) (a synaptic correlate for learning and memory mostly pronounced in the cerebral cortex and hippocampus) (Esplugues, 2002; Paul & Ekambaram, 2011).

l-Arginine also participates in the synthesis of creatine, which is a guanidinic compound that is considered the most popular nutritional supplement consumed in muscular strengthening, fitness, and bodybuilding. Moreover, this substance also presents antioxidant properties (Lawler et al., 2002), has a neuroprotective role in various models of neurological disorders involving impairment of energy metabolism (Klein & Ferrante, 2007), and is suggested to play a critical role in cognitive functions such as memory (Benton & Donohoe, 2011; Oliveira et al., 2008; Rae et al., 2003).

Several studies on cognitive process involve the single administration of l-arginine or creatine, however, the co-administration of these compounds was used only in studies on vascular function (Jahangir et al., 2009) and muscular system (Little et al., 2008). Data from the literature indicate that NO synthesis reflects only a minimum portion of the l-arginine flux. A significantly greater proportion of this amino acid flux participates in creatine formation. However, long-term creatine supplementation can reduce the activity of glycine amidinotransferase (AGAT) by feedback inhibition, and thereby increase the availability of l-arginine to other metabolic pathways, such as NO synthesis (Derave et al., 2004; Jahangir et al., 2009). Thus, our hypothesis is that the combined use of l-arginine and creatine could enhance the bioavailability of creatine and NO. This phenomenon could be used as an interesting therapeutic strategy for various pathological--physiological states involving NO and creatine depletion in the brain, such as psychiatric and neurocognitive disorders induced by chronic stress.

Therefore, this study aimed to analyze the effects of chronic variable stress (CVS) and administration of l-arginine and creatine on spatial memory and other morphologic and biochemical parameters such as body, heart, and adrenal weight, and plasma glucose and corticosterone levels in rats.

Materials and methods

Animals

The study included 34 male Wistar rats (120 days old) weighing an average of 374 ± 3.12 g. The animals were maintained in cages (3–4 animals per cage) with controlled temperature (26 ± 1 °C), light–dark cycle of 12/12 h, and access to water and feed (PURINA®) ad libitum. All experimental procedures were approved by the Ethics Committee on Animal Use (protocol n.015/2011); efforts were made to minimize animal suffering.

Experimental design

Rats were randomly assigned into five groups: control (Ct, n = 7), chronic stress (Cs, n = 7), chronic stress + l-arginine (Cs/La, n = 7), chronic stress + creatine (Cs/Cr, n = 7), and chronic stress + l-arginine + creatine (Cs/La + Cr, n = 6).

The treatments were performed through daily gavages (always around 12 pm) during the entire experimental period. To avoid unnecessary stress in the Ct group, gavages were not performed in these animals. The substances were administered at the following dosages.

Group Cs: 1.0 ml vehicle solution containing 0.5% carboxymethylcellulose (CMC); Group Cs/La: 1.0 ml of l-arginine solution (500 mg/kg body weight); Group Cs/Cr: 1.0 ml of creatine solution (300 mg/kg body weight) suspended in 0.5% CMC; and Group Cs/La + Cr: 1.5 ml of l-arginine solution (500 mg/kg body weight) + creatine solution (300 mg/kg body weight) suspended in 0.5% CMC. The selected doses of l-arginine (Heinzen & Pollack, 2003) and creatine (Magni et al., 2007; Saraiva et al., 2012) were based on previous studies.

Chronic variable stress

A model of chronic variable stress adapted from Vasconcellos et al. (2005) was used with some modifications in intensity and type of stressors. The animals in groups Cs, Cs/La, Cs/Cr, and Cs/La + Cr were exposed to stress for 40 days; the control animals remained undisturbed in their cages during the same period. The following stressors were applied: crowded environment (7 animals per cage for 24 h); 1 h exposure to cold; 1–2 h of restraint; inclination of cages at a 45° angle for 2–4 h; flashing light during 1–2 h; 24 h of food deprivation (once a week); and 15–40 min of noise. These seven stressors were applied daily and randomly at different times in order to minimize stressor predictability by the animal.

Spatial memory evaluation

An adapted version of the Morris water maze was used (Morris, 1984). The water maze was a black circular pool (135  cm in diameter) with controlled water temperature (21 ± 1 °C) containing one escape acrylic platform (10 × 10  cm) that was centered in a virtual quadrant of the pool and at 1.5 cm below the water surface. Cornstarch (300 g) was added to the water to increase turbidity and hinder platform visualization.

This apparatus was placed in a 7.5 m² room with walls rich in distal visual cues. The reference memory evaluation began after the stress period. This task consisted of five training and one testing sessions. The main objective of the training sessions (acquisition of memory) was for the animals to find the platform (located at the center of one virtual quadrant). Animals were subjected to four trials daily starting from different quadrant locations (N, S, W and E). An interval of 10 min between each trial was applied.

The rats were allowed to search the platform for a maximum of 60 sec in each trial and to remain on it for 10 sec. In the event the rat failed to find the platform, the rat was gently conducted to the platform by the experimenter and allowed to remain on it for 10 sec. The animals were dried and returned to their cages after each trial.

These animals were subsequently subjected to a testing session (retention of memory) at around 24 h after the last training session. This session was carried out for 60 s in the absence of the escape platform. The following parameters were evaluated: latency in reaching the original platform position, percentage (%) of time spent in the target quadrant. The animals’ spontaneous locomotion activity was determined by the total number of crossings between quadrants.

The working memory evaluation began 48 h after the testing session for reference memory. This task used the same apparatus used in the previous task: however, it consisted of four trials per day (5 min inter-trial interval) during four consecutive days. The platform was placed in different positions each day. The working memory performance was evaluated by calculating latencies in finding the platform in every first, second, third, and fourth trial on the four testing days (Netto et al., 1993).

Blood biochemical parameters

Animals of all groups were sacrificed by decapitation 24 h after the last behavioral session and 12 h of fasting. Total blood was collected in heparinized tubes and plasma was separated and used for analyses of corticosterone and glucose levels. Corticosterone levels were determined by the Siemens ADVIA Centaur® Cortisol assay, which is a competitive immunoassay using direct chemiluminescent technology. The results are expressed in µg/dL. Glucose levels were measured using a commercial kit (Labtest®, Lagoa Santa, Minas Gerais, Brazil) with data expressed in mg/dL.

Morphological parameters

Body weight was measured before euthanasia. The heart and left adrenal gland were removed and weighed following euthanasia. The heart weight (mg) was used to calculate the heart weight-to-body weight ratio, and as a marker for the degree of myocardial hypertrophy expressed by the following ratio: heart weight (mg)/body weight (g) (Wallen et al., 2000).

Statistical analyses

All results are presented as mean ± S.E.M. The statistical analysis was performed using a one-way ANOVA and ANOVA with repeated-measures. Differences were considered statistically significant at p < 0.05. The post-hoc Newman–Keuls test was used to identify differences between groups when appropriate.

Results

Blood biochemical parameters

Results from the blood biochemical parameters are shown in Table 1. No significant difference was observed in plasma glucose levels among the experimental groups [F_(4.29) = 1.47, p > 0.05]. However, Cs/Cr and Cs/La + Cr groups exhibited plasma corticosterone levels significantly lower than the Cs group [F_(4.29) = 2.12, p < 0.05].

Table 1. Effects of chronic variable stress and treatments on blood biochemical parameters.

	Groups
Parameters	Ct	Cs	Cs/La	Cs/Cr	Cs/La + Cr
Corticosterone (μg/dL)	0.91 ± 0.2	1.35 ± 0.3	0.95 ± 0.2	0.65 ± 0.1*	0.67 ± 0.1*
Glucose (mg/dL)	81.1 ± 6.7	85.1 ± 4.2	92.3 ± 3.3	94.4 ± 3.4	88.4 ± 4.1

Data are expressed as mean ± S.E.M.

*Significant differences when compared to the Cs group (One-way ANOVA with the post-hoc Newman–Keuls test, p < 0.05), n = 7–6 animals/group.

Morphological parameters

No significant difference was observed in the body weight among the experimental groups from the beginning of the study to the end. However, animals from the Cs/La + Cr and Cs/Cr groups presented significant reduction in the left adrenal gland weight compared to animals in the Cs group [F_(4.29) = 4.14, p < 0.05].

The heart morphologic parameters showed significant differences between animals from the different experimental groups. Animals in the Cs/La group showed increased heart weight [F_(4.29) = 3.45, p < 0.05] and exhibited greater heart weight-to-body weight ratio than animals in the control and stressed groups [F_(4.29) = 4.00, p < 0.05]. The results from the studied morphological parameters are shown in Table 2.

Table 2. Effects of chronic variable stress and treatments on morphological parameters.

	Groups
Parameters	Ct	Cs	Cs/La	Cs/Cr	Cs/La + Cr
Final body weight (g)	435.3 ± 5.8	425.3 ± 7.8	418.6 ± 11	431 ± 11	438.7 ± 12
Left adrenal weight (mg/g)	0.09 ± 0.0	0.11 ± 0.0†	0.10 ± 0.0	0.08 ± 0.0	0.07 ± 0.0
Heart weight (mg)	1530 ± 95	1446 ± 48	1903 ± 110*	1653 ± 97	1727 ± 123
Heart weight (mg)/body weight (g)	3.52 ± 0.2	3.40 ± 0.1	4.53 ± 0.3*	3.82 ± 0.2	3.94 ± 0.3

Data are expressed as mean ± S.E.M.

†Significantly different from the Cs/Cr and Cs/La + Cr groups.

*Significantly different from the Ct and Cs groups. (One-way ANOVA with the post-hoc Newman–Keuls test, p < 0.05), n = 7–6 animals/group.

Spatial memory evaluation

The results showed that animals from all groups presented spatial learning because of the decreased latency in finding the platform within five days of training for memory acquisition (p < 0.05). No significant difference in latency was observed between the different experimental groups (Figure 1).

Figure 1. Effects of chronic variable stress and treatments on spatial reference memory acquisition. Data are expressed as mean ± S.E.M. These values represent the median latency (s) in finding the platform from four trials during five days of training. No significant differences were observed between groups (One-way ANOVA with the post-hoc Newman–Keuls test, p > 0.05), n = 7–6 animals/group.

No statistical differences were observed between the experimental groups regarding latency in finding the platform during the spatial reference memory retention evaluation [F_(4.29) = 0.69, p > 0.05] (Figure 2A). However, the Cs/La + Cr group spent more time in the target quadrant compared to animals from the Ct, Cs, and Cs/La groups [F_(4.29) = 4.48, p < 0.05) (Figure 2B). The total number of crossings between quadrants did not show statistical difference between the experimental groups [F_(4.29) = 0.25, p > 0.05].

Figure 2. Effects of chronic variable stress and treatments on reference memory retention: (A) latency in finding the platform and (B) mean % time spent in the target quadrant (T.Q.). Data are expressed as mean ± S.E.M. *Significantly different from Ct, Cs, and Cs/La groups (One-way ANOVA with the post-hoc Newman–Keuls test, p < 0.05), n = 7–6 animals/group.

The results showed that the time spent in finding the platform over four trials during the working memory evaluation decreased in all the groups (p < 0.05). A significant difference was observed between the second [F_(4.12) = 2.82, p < 0.05] and third trials [F_(4.12) = 6.61, p < 0.001] when comparing the performance among groups; the Cs/Cr group performed better than all other groups (Figure 3).

Figure 3. Effects of chronic variable stress and treatments on spatial working memory. Data are expressed as mean ± S.E.M. These values represent mean latencies in finding the platform in each trial during four testing days. Statistical difference between the Cs/Cr group and other groups in the second (*p < 0.05) and third trials (**p < 0.001) (repeated-measures ANOVA with the post-hoc Newman–Keuls test), n = 7–6 animals/group.

Considering the performance in all trials, the repeated-measures ANOVA showed that the Cs/Cr group presented better efficiency in finding the platform compared to the other groups [F_(4.12) = 6.33, p < 0.01].

Discussion

The results showed that the administration of creatine and creatine associated with l-arginine reduced corticosterone levels that had increased in plasma and also reduced the left adrenal weight compared with chronically stressed animals (group Cs). Creatine supplementation could be decreasing corticosterone levels and other stress responses by acting through brain GABA-A receptors (Koga et al., 2005). The l-arginine-NO pathway is also involved in modulating corticosterone plasma concentrations, and l-arginine-derived NO has been reported as significantly decreasing the production of basal and ACTH-stimulated corticosterone in the zona fasciculata adrenal cells in rats (Cymeryng et al., 1999).

We observed that the animals in the Cs/La group presented a significant increase in heart weight and myocardial hypertrophy compared to animals in the Ct and Cs groups. The overproduction of l-arginine-derived NO may also be detrimental because it generates peroxynitrite-free radicals (Kronon et al., 1999), which are associated with cardiac fibrosis, hypertrophy, and cardiac dilation (Mungrue et al., 2002). Creatine presents an antioxidant property with significant ability to remove peroxynitrite (Lawler et al., 2002), which can be linked to blockade of cardiac hypertrophy mediated by excessive peroxynitrite production that could have occurred in the animals of Cs/La group in this study.

Our results showed that co-administration of l-arginine and creatine improved the reference memory retention. According to the literature, the l-arginine-NO pathway and the creatine-phosphocreatine system are involved in memory formation. NO is synthesized from post-synaptic neurons in response to the activation of NMDA receptors by amino acid glutamate. NO acts as a retrograde messenger directly in pre-synaptic neurons after being released. Moreover, it coordinates the enhancement of pre- and post-synaptic mechanisms involved in synaptic plasticity, namely LTP (a synaptic correlate for learning and memory mostly pronounced in the cerebral cortex and hippocampus) (Esplugues, 2002; Paul & Ekambaram, 2011), and is involved in memory facilitation induced by spermidine in rats (Guerra et al., 2006).

Studies showed that creatine supplementation resulted in better memory in vegetarians and elder individuals, positive effect on intelligence tests, and increased the levels of creatine and phosphocreatine in the hippocampus (Benton et al., 2011; McMorris et al., 2007b; Rae et al., 2003; Valenzuela et al., 2003). In addition to participating in brain energy metabolism, recent data showed that creatine modulates sites of polyamines on NMDA receptors providing improved spatial memory in rats (Oliveira et al., 2008).

It is important to emphasize that altered spontaneous locomotor activity is probably not the cause of the observed effects because the total number of crossings between quadrants did not show differences between animals from different experimental groups.

Decreased latency in finding the platform was observed in all groups throughout four testing days during the working memory evaluation. This indicates that all the animals presented working memory. However, the isolated administration of creatine significantly improved efficiency in finding the platform during the working memory task evaluation. Studies showed that creatine supplementation improves working memory and intelligence in young vegetarians (Rae et al., 2003), and numeric and spatial working memory in elderly individuals (McMorris et al., 2007b); moreover, creatine supplementation enhances the performance in complex central executive and working memory tasks under stress conditions (McMorris et al., 2006, 2007a).

Conclusion

The results in this study indicate that single administration of creatine improves efficiency in finding the platform during a working memory task, and when co-administrated with l-arginine improves reference memory retention, which is a phenomenon possibly associated with increased production of creatine and l-arginine-derived NO. Future studies could corroborate the results observed and extend the contributions from this study. Improved knowledge about biochemical mechanisms underlying the l-arginine and creatine co-administration could help to explain the positive results obtained from administration combined of these substances.

Declaration of interest

The authors report no declarations of interest. The authors are grateful to Coordination for Enhancement of Higher Education Personnel (CAPES) (Brazil), National Council for Scientific and Technological Development (CNPq) (Brazil), and Araucaria Foundation of Parana (Brazil) for financial support of this work. This manuscript has been reviewed by a professional science editor and a native English-speaking science editor.