Dietary restriction protects against chronic-ethanol-induced changes in exploratory behavior in Wistar rats

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Dietary restriction protects against chronic-ethanol-induced changes in exploratory behavior in Wistar rats Lucas S.N.M. Pinto a,d , Felipe A.S. Gualberto a,d , Silvia R.C. Pereira c,d , Paula A. Barros b , Glaura C. Franco b , Angela M. Ribeiro a,d,⁎ a

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas-Universidade Federal de Minas Gerais, Brazil Departamento de Estatística-Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Brazil c Departamento de Psicologia-Faculdade de Filosofia e Ciências Humanas, Universidade Federal de Minas Gerais, Brazil d Laboratório de Neurociência Comportamental e Molecular-(LaNeC) Universidade Federal de Minas Gerais, AV Antônio Carlos, 6627, 30270-110-Belo Horizonte, Minas Gerais, Brazil b

A R T I C LE I N FO

AB S T R A C T

Article history:

Chronic ethanol intake causes various types of neural damage and behavioral impairments,

Accepted 21 December 2005

probably acting through oxidative stress and excitotoxicity, while dietary restriction is

Available online 28 February 2006

considered by some authors to protect the central nervous system from these kinds of damage. In the present study, a factorial experimental design was used to investigate the

Keywords:

effects of chronic ethanol and dietary restriction treatments, associated or not, on Wistar

Dietary restriction

rats' exploratory behavior, spatial memory aspects and cortical and hippocampal

Chronic ethanol

acetylcholinesterase (AChE) activity. Dietary restriction lasted for the whole experiment,

Exploratory behavior

while ethanol treatment lasted for only 3 weeks. Despite the short ethanol treatment

Acetylcholinesterase

duration, for two behavior categories assessed, moving and rearing, an interaction was

Nitric oxide

observed between the effects of chronic ethanol and dietary restriction. There were no

Wistar rat

significant differences in AChE activities among the groups. Cerebellar neural nitric oxide synthase (nNOs) activity was measured as a first step to assess oxidative stress. Dietary

Abbreviations:

restriction significantly reduced NO formation. The present results indicate that dietary

AChE, acetylcholinesterase

restriction might exert a protective effect against chronic-ethanol-induced changes in

CNS, central nervous system

exploratory behavior. It is hypothesized that the mechanisms underlying this protection

DR, dietary restriction

can involve prevention of oxidative stress.

DTNB, dithiobisnitrobenzoate

© 2006 Elsevier B.V. All rights reserved.

NMDA, N-methyl-D-aspartate NO, nitric oxide nNOS, neural nitric oxide synthase R, restricted RE, restricted ethanol RW, restricted water C, control CE, control ethanol CW, control water ⁎ Corresponding author. Laboratório de Neurociência Comportamental e Molecular-(LaNeC) Universidade Federal de Minas Gerais, AV Antônio Carlos, 6627, 30270-110-Belo Horizonte, Minas Gerais, Brazil. Fax: +55 31 34995963. E-mail address: [email protected] (A.M. Ribeiro). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.092

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1.

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Introduction

Chronic ethanol intake causes changes in various neurotransmitter systems, white matter loss and neuronal death in many structures of both humans and rodents (Fadda and Rossetti, 1998; Harper, 1998). Some authors consider that ethanolinduced neurotoxicity is probably mediated through oxidative stress and excitotoxic mechanisms (Arendt et al., 1989; Dodd et al., 2000; Fadda and Rossetti, 1998; Harkany et al., 1997; Muscoli et al., 2002; Tsai, 1998). In particular, central nervous system (CNS) levels of nitric oxide (NO), a free radical, seem to be increased in chronic ethanol consumption, as demonstrated both in vitro and in vivo, apparently through overactivation of N-methyl-D-aspartate (NMDA) receptors, and this increase seems to be involved in the pathophysiology of ethanolinduced neuronal damage (for a review, see Syapin, 1998). NO S can react with the super-oxide radical (O2−), generating peroxinitrite, a compound with a high oxidizing capacity, being able to attack and modify protein, lipids and cellular DNA, causing neuronal damage (Torreilles et al., 1999), which may be related to neurodegenerative syndromes and with premature aging (Molina et al., 1998; Ozdemir et al., 2002; Siles et al., 2002). In the behavioral realm, ethanol can produce cognitive deficits, such as impairments in spatial and non-spatial reference and working memory (Arendt et al., 1989; Beracochea et al., 1986, 1992; Melis et al., 1996; Pereira et al., 1998; Santín et al., 2000) and alterations in exploratory and other open-field behaviors (Harkany et al., 1997). Some authors showed that rats treated with NOS inhibitor increase (Prickaerts et al., 1998) or decrease (Sandi et al., 1995) the behavioral activity in the open field. Experimental data obtained with rodents suggest that chronic ethanol consumption induces hypofunction in several cholinergic parameters (Arendt, 1994; Arendt et al., 1989; Beracochea et al., 1986, 1992; Floyd et al., 1997; Melis et al., 1996; Miller and Rieck, 1993), including brain acetylcholinesterase (AChE) activity (Arendt et al., 1989; Floyd et al., 1997), and that a dysfunction in the activity of this enzyme seems to be related to cognitive deficits (Arendt et al., 1989; Das et al., 2005). There is wide evidence that shows a close relation between cognitive impairments and lesions in the cholinergic basal forebrain projection system induced by chronic ethanol intake (Arendt, 1994; Arendt et al., 1989; Beracochea et al., 1986, 1992; Cadete-Leite et al., 1995; Melis et al., 1996). The hypothesis of cholinergic mechanisms underlying the cognitive deficits induced by prolonged ethanol ingestion is supported by findings that fetal brain transplants rich in cholinergic neurons improve spatial memory performance as measured by eight-arm radial maze and increase cholinergic activity in rats treated chronically with ethanol (Arendt et al., 1989) and that administration of physostigmine, a cholinesterase inhibitor, to ethanol-treated mice improves their performance in cognitive tests (Beracochea et al., 1986, 1992). Our group observed that chronic ethanol intake affected remote spatial memory but did not find a correlation between ethanol-induced changes in the cholinergic system and memory deficits, as measured by the eight-arm radial maze. The 32-week ethanol treatment did not cause any alterations

in cortical cholinergic parameters (Pereira et al., 1998). In other studies, our group (Fernandes et al., 2002; Pires et al., 2001) and other authors (Calingasan and Gibson, 2000) found similar results for different ethanol treatment durations. However, we did find a chronic-ethanol-induced reduction in the activity of AChE in the hippocampus (Pires et al., 2001). There is evidence that AChE is involved in open-field behavior and spatial memory performance (Das et al., 2005; Mach et al., 2004; Takahata et al., 2005) and that this enzyme activity can be changed by chronic ethanol consumption (Arendt et al., 1989). Lamprea et al. (2003) showed that AChE is related to exploratory behavior as measured in the open field. This behavioral category is also affected by chronic ethanol intake (Harkany et al., 1997). Particularly, the cholinergic system seems to be related both to exploration of novel environments and habituation to them (Thiel et al., 1998). Other authors assessed more specific cholinergic parameters, for instance, extracellular ACh, and found that the cholinergic system seems to be related to exploratory behavior (Thiel et al., 1998; Torres et al., 1994). Dietary restriction (DR), on the other hand, is known to increase lifespan (Sohal et al., 1994) and to have several protective effects in the CNS, not only retarding and/or preventing age-related effects (Dubey et al., 1996; EcklesSmith et al., 2000; Idrobo et al., 1987; Ingram et al., 1987; Mattson et al., 2003; Prolla and Mattson, 2001; Stewart et al., 1989), but also reducing and/or preventing several types of damage to the CNS of adult rodents (Bruce-Keller et al., 1999; Calingasan and Gibson, 2000; Duan et al., 2001; Prolla and Mattson, 2001; Zhu et al., 1999), in part through reduction of oxidative stress (Duan et al., 2001; Kim et al., 2002; Lee et al., 2000b,c). Besides, DR was shown to enhance neurogenesis and neurotrophins expression (Duan et al., 2001; Lee et al., 2000b,c). In short: (i) chronic ethanol consumption causes a wide range of biochemical, morphological and behavioral deficits; (ii) ethanol-induced neurotoxicity probably involves changes in cerebellar NO production; (iii) both central AChE and cerebellar NO activities seem to be important in cognitive processes and exploratory behavior; (iv) at least part of ethanol-induced deficits seem to be a consequence of damages to activities of these enzymes; and (v) DR could prevent neural damage induced by a number of factors, including oxidative stress and excitotoxic damage. Hence, we decided to investigate the effects of restricted diet and chronic ethanol treatment, associated or not, on: (i) cortical and hippocampal AChE activity; (ii) cerebellar nitric oxide synthase activity; (iii) exploratory behavior; (iv) reference and working spatial memory; and (v) the correlation between these biochemical and behavioral parameters. Cerebellar nNOs activity was measured as a first step to verify whether oxidative stress is involved in the effects caused by chronic ethanol and dietary restriction.

2.

Results

2.1.

Animals and treatment

2.1.1.

Food intake

Data are shown in Fig. 1A. Animals from group CW consumed significantly more chow than animals from group CE

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2.1.2.

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Fluid intake

Data are presented in Fig. 1B. Statistical analysis showed significant main effect of dietary restriction (F1,44 = 42.07; P b 0.001); restricted groups consumed more fluid when compared to standard groups. There was a significant interaction between the effects of the two treatments (F1,44 = 28.70; P b 0.001). During treatment with ethanol, animals from group CW had a significantly higher water intake (P b 0.05) than animals from group RW, while animals from group CE had a significantly lower ethanol intake (P b 0.05) than animals from group RE. After treatment with ethanol, rats from control groups (CW and CE) had a significantly higher fluid intake than rats from restricted groups (RW and RE) (P b 0.05). The animals from the ethanoltreated groups consumed an average of 11.9 to 13.1 g/kg/day.

2.1.3.

Body weight

Data are shown in Fig. 1C. Statistical analysis revealed a significant main effect of dietary restriction (F1,44 = 80.00; P b 0.001). Average body weight of the animals receiving ad libitum diet was significantly higher than that of those receiving restricted diet. At the end of the experiment, on day 39, mean body weight of restricted rats corresponded to 66.8% of the body weight of the control animals.

2.1.4.

Serum albumin

There were no significant main effects for dietary restriction (F1,23 = 4.12; P N 0.05), ethanol (F1,23 = 0.02; P N 0.05) and no significant interaction (F1,23 = 0.01; P N 0.05) on albumin blood serum concentrations among the groups (data not shown).

Fig. 1 – Animals and treatment (means ± SE). Striped bars at the bottom of the graphics represent duration of ethanol treatment. (A) Food intake (average intake in g/day). Since all restricted rats consumed a fixed amount of food (15 g per day), data were not included in the graphic. (B) Fluid intake (average intake in ml/day). (C) Body weight averages (g). Statistical differences are detailed in the Results section.

(F1,22 = 14.57; P b 0.01). So, although the restricted groups were offered a fixed amount of food (15 g/day), the magnitude of dietary restriction was different when groups CW × RW and CE × RE were compared: on day 27 (at the end of treatment with ethanol), the average food intake of the animals from group CW was 31.3 g, 52.12% greater than group RW; animals from group CE had an average intake of 24.5 g, 38.84% greater than group RE, or, if 15 g is taken as 100% of chow intake, group CW average intake was 208.66% and that of group CE was 163.33%.

2.2.

Behavioral studies

2.2.1.

Open-field test

Data collected in the third session were analyzed for the first and second half separately. Data for the second half of this session are not shown, except for the category touching. For the other behavioral categories, we will refer only to the first half of session 3 as session 3a. Since a new category, namely touching, was introduced in the second half of the third session, it had more categories than the first half of this session and than the two others. So, unlike the first half, the second half of the third session could not be compared to the other sessions as the relative frequencies of the categories would be different. For rearing (Fig. 2A), 2 × 2 × 3 ANOVA showed a significant main effect for dietary restriction (F1,40 = 7.81; P b 0.01) and significant interaction between the treatments (F1,40 = 4.70; P b 0.05). When separate 2 × 2 ANOVA was done for each session, there was significant dietary restriction main effect (F1,47 = 4.67; P b 0.05 for the first session and F1,47 = 10.92; P b 0.01 for the third session). For moving (Fig. 2B), there was a significant dietary effect (F1,44 = 4.03; P b 0.05) and an interaction between dietary restriction and ethanol treatment (F1,44 = 4.50; P b 0.05). When separate 2 × 2 ANOVA was done for each session, there was significant dietary restriction main effect (F1,47 = 4.10; P b 0.05 for the first session) and a significant interaction between treatments (F1,47 = 4.97; P b 0.05). For grooming (Fig. 2C), statistical analysis showed significant time effect (F1,80 = 4.63; P b 0.025). Post hoc analysis showed significant difference between sessions 1 and 2 (P b 0.01), with frequency increasing

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Fig. 2 – Open-field behavioral categories (means ± SE): rearing (A); moving (B); grooming (C); and touching (D). Statistical differences are detailed in the Results section.

along sessions. There was no significant interaction between dietary restriction and ethanol treatment for this behavior (F1,40 = 1.74; P N 0.05). Groups presented no significant main effect for dietary restriction (F1,43 = 0.14; P N 0.05) and ethanol (F1,43 = 2.63; P N 0.05) in touching, with no significant interaction (F1,43 = 1.16; P N 0.05). (Fig. 2D).

2.2.2.

Water maze test

2.2.2.1. Spatial reference memory. There were no significant main effects (dietary restriction: F1,44 = 0.90; P N 0.05; ethanol: F1,44 = 0.15; P N 0.05) on animals' performances in the reference memory task. There was, however, a significant time effect (F3,132 = 97.72; P b 0.01) indicating that all animals learned the task equally well. For probe trial, ANOVA 2 × 2 showed no significant ethanol (F1,44 = 0.68; P N 0.05) or dietary restriction (F1,44 = 0.07; P N 0.05) main effect nor significant interaction (F1,44 = 1.21; P N 0.05) (data not shown). 2.2.2.2. Spatial working memory. There were no significant main effects (dietary restriction: F1,44 = 0.44; P N 0.05; ethanol: F1,44 = 0.04; P N 0.05) on animals' performances in the working

memory task. There was, however, a significant time effect (F3,176 = 5.33; P b 0.025) indicating that the animals improved their performance along sessions.

2.3.

Neocortical and hippocampal AChE activity

The cortex AChE activities (mean ± SE) measured as micromoles of acetyltiocholine hydrolyzed/min/g of tissue were 3.05 ± 0.28, 3.25 ± 0.22, 3.02 ± 0.16 and 2.89 ± 0.29 for groups control water, control ethanol, restricted water and restricted ethanol, respectively. Main effects of dietary restriction (F1,47 = 0.665; P N 0.05), ethanol (F1,47 = 0.016; P N 0.05) were not significant. There was no significant interaction between these effects (F1,47 = 0.696; P N 0.05). The hippocampal AChE activities (mean ± SE) measured as micromoles of acetyltiocholine hydrolyzed/min/g of tissue were 3.45 ± 0.15, 3.63 ± 0.42, 3.40 ± 0.18 and 3.54 ± 0.28 for groups standard water, standard ethanol, restricted water and restricted ethanol, respectively. Main effects of dietary restriction (F1,47 = 0.007; P N 0.05) and ethanol (F1,47 = 0.332; P N 0.05) were not significant. There was no significant interaction between these effects (F1,47 = 0.008; P N 0.05).

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Fig. 3 – Nitric oxide synthase activity (mean ± SE of the percentage of nitrite produced in relation to the average of SW group which was considered 100%) in the cerebellum of animals from the four groups: standard-water (SW); standard-ethanol (SE); restricted-water (RW); and restricted-ethanol (RE). The asterisk indicates that there is a significant main effect for dietary restriction (P b 0.025).

2.4.

Cerebellar nNOS activity

The data referring to cerebellar nNOS activity are shown in Fig. 3. Two-way ANOVA test revealed a significant main effect for dietary restriction (F1,12 = 8.24; P b 0.025), but there was no significant main effect for ethanol (F1,12 = 1.06; P N 0.05) and no significant interaction (F1,12 = 1.24; P N 0.05). Post hoc Newman– Keuls test revealed a significant difference (P b 0.05) between groups SE and RW. The absolute average value of cerebellar nNOS activity for the control condition (SW group) was 0.54 mmol of nitrite produced/mg of protein.

3.

Discussion

In the present study, we show that chronic ethanol intake has a suppressant effect on rats' exploratory behavior and that this effect disappears when chronic ethanol intake is associated with dietary restriction. Rats from group CE had significantly lower frequencies in the exploratory behavioral categories rearing in sessions 1 and 3 and moving in session 1, when compared to all other groups, including group RE. In other words, chronic ethanol has different effects on rats treated with standard versus restricted diet. It is important to note that rats were submitted to ethanol abstinence 13 days before the open-field test, suggesting that the observed alterations were indeed due to chronic ethanol consumption, and not to acute ethanol anxiolytic effects. Although the groups did not differ in the category touching, which is also an exploratory behavior, there was a tendency for rats from group CE to show lower frequencies of touching than all other groups, following the same pattern of both rearing and moving. Harkany et al. (1997) also described a chronic-

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ethanol-induced reduction in rat's exploratory behavior. They also found a significant reduction in moving and rearing and an increase in the latency to start open-field exploration. Likewise, File and Mabbutt (1990) observed a chronic-ethanol-induced disruption in habituation and reduced response to novel objects 3 and 5 months after interruption of ethanol administration. Our finding of no changes in exploratory activity of dietaryrestricted rats consuming water is also in accordance with the literature (Wu et al., 2002). Yet, to our knowledge, this is the first time that a combined effect of both DR and chronic ethanol on the exploratory behavior of rats is described. The mechanism by which chronic ethanol induces these alterations is not well understood. Steps towards an answer come from the studies of Sasvari et al. (1997) and Harkany et al. (1997), which correlate these changes, respectively, to loss of Ca+2 homeostasis and to increased oxidative stress. Caloric restriction seems to act as a protector, not an enhancer; that is, rats receiving water and restricted diet do not perform differently from ad-libitum-fed controls. The differential effect is only observed when there is an agent that causes some type of damage. In this case, chronic ethanol reduced exploratory behavior, while its association with restricted diet maintained exploratory behavior at control levels. Following the same logic, these findings are in accordance with other studies (Dubey et al., 1996; Idrobo et al., 1987; Ingram et al., 1987; Stewart et al., 1989), in which DR either attenuated or prevented age-related behavioral deficits, but never improved performance above control levels. However, Wu et al. (2002) did describe increased learning in Y maze test, albeit with no differences in memory retention, of rats being submitted to DR, when compared to ad-libitum-fed controls. As mentioned before, our results indicate that dietary restriction seems to protect against the effects of chronic ethanol exposure on rat's exploratory behavior. However, one should consider the possibility that the rats from the restricted groups were simply stressed and hyperactive, not protected. But it is little likely because, were this the case, restricted groups would be above control levels. Clearly, the only significant changes observed in rearing and locomotion activation occurred in the group receiving ad libitum diet and ethanol, thus we concluded that these alterations are indeed due to ethanol alone, which is in accordance with the literature (Harkany et al., 1997). Chronic ethanol is thought to damage the CNS through excitotoxic mechanisms. Since ethanol inhibits the glutamatergic NMDA receptors, they are up-regulated in its chronic consumption; withdrawal of ethanol frees the overnumbered receptors from the inhibition, resulting in glutamatergic hyperactivity and thereby leading to an excessive calcium influx through the receptor channels, causing cytotoxicity (Dodd et al., 2000; Fadda and Rossetti, 1998; Tsai, 1998). According to the oxidative stress hypothesis, this is so because excessive intracellular Ca+2 triggers the activation of cellular proteases and lipases, causing the impairment of mitochondrial oxidative phosphorylation and the generation of free radicals and reactive oxygen species (Choi, 1988; Fadda and Rossetti, 1998), more specifically NO, by means of nNOS activation by Ca+2-Calmodulin kinase. Muscoli et al. (2002) provided further evidence to such oxidative stress hypothesis by antagonizing chronic-ethanol-induced oxidative stress, in

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astroglial cell cultures, with idebenone, a free radical scavenger. Besides, Harkany et al. (1997), using an in vivo model, described increased oxidative stress and lipid peroxidation induced by chronic ethanol consumption. These authors (Muscoli et al., 2002; Harkany et al., 1997) suggest that ethanol-induced injury is mediated by abnormal formation of free radical species. However, they did not measure NOS activity. There is evidence that NO seems to play a role in the mechanisms of ethanol neurotoxicity in different CNS areas (Adams and Cicero, 1998; Baraona et al., 2002; Lancaster, 1992; Pokk et al., 2001) including cerebellum (Xia et al., 1999). Rats previously submitted to a stress situation reduce exploratory open-field activity, an effect that can be modified by nitric oxide synthase inhibitors (Masood et al., 2003). Some authors showed that rats treated with the NOS inhibitor increase (Prickaerts et al., 1998) or decrease (Sandi et al., 1995) the behavioral activity in the open field. Schweighofer and Ferriol (2000) show that NO has a role in cerebellum learning process. We did not find differences in cerebellar nNOS activity between rats receiving ethanol or not, which is in consonance with works carried out by other authors (Ikeda et al., 1999; Naassila et al., 1997). Besides, this absence of chronic ethanol effect on NO production could be due, perhaps, to a ceiling effect. Another possibility could be the differences in the studied brain regions. On the other hand, NO results are in agreement with the working hypothesis of a dietary-restriction-induced reduction in oxidative stress, since restricted groups exhibited lower cerebellar nNOS activities than the adlibitum-fed ones. One possibility is that this reduction made the ceiling effect disappear. It is worthwhile to mention that cerebellar nNOS activity comparison between animals from groups RE and CE is not adequate since animals from RE group consumed significantly more ethanol compared to animals from CE group (see Fig. 1B). Hence, maybe, the cerebellar nNOS production from ethanol source, in these animals (from RE group), could be greater. One could suppose that, if the ethanol consumption of animals from this group was restricted to the amount consumed by animals from CE group, maybe the DR effect observed would be greater. To approach this issue, more detailed studies should be carried out. As pointed out earlier, dietary restriction protects the CNS from excitotoxic insults and oxidative damage, including age-associated protein (Dubey et al., 1996) and DNA (Ingram et al., 1987; Sohal et al., 1994) oxidation, thiamine-deficiency-induced oxidative damage (Calingasan and Gibson, 2000) and kainate-induced excitotoxic damage (Bruce-Keller et al., 1999; Duan et al., 2001; Zhu et al., 1999). DR has been proposed to reduce oxidative stress in several animal models (Lee et al., 2000a; Mattson et al., 2003; Muscoli et al., 2002). DR seems to do so by modulating the expression of genes linked to metabolic regulation, and these are probably related to a response to a mild metabolic stress (Mattson et al., 2004; Pereira et al., 1998). It is important to point out that, in the present study, this indirect assessment of nNOS cerebellar activity is a first step to try to clarify the mechanism of chronic ethanol effect. Thus, further work is necessary to better characterize the observed effects. For instance, besides NO production, other free radical production markers, in cerebellum and other CNS areas, should be measured. Unlike the exploratory behaviors in the open-field, grooming, which is a complex instinctive behavior that is little

affected by individual experience, was not affected by ethanol consumption. However, animals from groups receiving standard diet showed significantly higher frequencies when compared to the ones receiving restricted diet. One could think that the animals' open-field behavior reflects spatial memory deficits of rats from CE group. This, however, seems unlikely because neither chronic ethanol consumption for 21 days nor DR had any effect on either reference or working spatial memory as measured in the water maze. Data in the literature regarding chronic ethanol effects on reference and spatial working memory are still controversial. While some describe deficits on both reference and working spatial memory (Arendt et al., 1989), others describe impairments in spatial remote memory (Pereira et al., 1998), but not on recent reference (Pereira et al., 1998; Pires et al., 2005) and working memory (Pereira et al., 1998). Others found spatial working memory and behavioral flexibility deficits, but no reference memory impairments (Santín et al., 2000). Differences in these findings are probably due to different treatment protocols and/or strains used in experiments. One could think that the lack of ethanol effects on spatial memory in the present study might be explained by the short treatment duration (21 days), which might have been insufficient to cause detectable spatial memory impairments. However, Santín et al. (2000), using a longer ethanol treatment duration, did not find changes in spatial reference memory performance. Besides, there are studies showing that chronic ethanol consumption has no effects on reference memory when the subject is submitted to a treatment of 10% v/v ethanol solution lasting for 2 weeks (Boulouard et al., 2002) or a treatment of 20% v/v lasting for 4 weeks (Arendt et al., 1989). In our study, the animals consumed an average of 11.9 to 13.1 g/ kg/day. This amount is similar to that found by other authors. For instance, Santucci et al. (2004) treated rats with a liquid diet for 26 days and observed a 16.1 ± 0.86 g/kg daily consumption. They showed that this short treatment caused neuroanatomical changes and impaired performance in Morris water maze. Harkany et al. (1997) used rats fed ethanol in a liquid diet for up to 21 days. This regimen resulted in an average ethanol intake equivalent to 5.88 ± 0.8 g/kg/day. This amount consumed, which was even smaller than that obtained in our study, was enough to cause significant changes in exploratory behavior, accompanied by changes in the free radical metabolism. In the present work, although a 21-day ethanol treatment was not sufficient to induce any spatial memory deficits, it did induce alterations in exploratory behavior. One of the possibilities is that different cognitive functions seem to have different time-related sensibilities to the chronic ethanol effects. Body weights of control rats were significantly higher than that of those submitted to restricted diet. Markowska (1999) found similar differences in the rats' body weights between restricted and ad-libitum-fed animals, and other authors working with mice (Calingasan and Gibson, 2000; Dubey et al., 1996; Lee et al., 2000c) also found similar significant differences. However, it is important to emphasize that a diet consisting of 15 g of food, as used in the present study, probably did not result in malnourishment since other authors have stated that Wistar rats need a minimum daily food intake of 12–15 g of chow (Warner, 1962). Besides, the albumin serum concentrations were equal for all groups, and the motor

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activity of animals from restricted group is not different from those control animals (water and ad libitum diet). The lower food ingestion by the animals receiving ethanol when compared with animals receiving water is in accordance with previous results obtained by our group (Fernandes et al., 2002; Pires et al., 2001) and might be explained by the fact that ethanol provides extra calories and therefore could reduce food intake by increasing satiety (Strbak et al., 1998). This mechanism, however, is not well known and cannot be explained by alterations in hormonal levels (Strbak et al., 1998). This difference was not observed in rats receiving restricted diet as they all received the same amount of chow. Therefore, restricted rats receiving ethanol were submitted to a relatively milder caloric restriction than the ones receiving water. Dietary-restricted rats receiving water had lower fluid intake than their controls, which might be explained by the fact that a greater amount of food induces greater thirst because the chow is dry. On the other hand, restricted rats drank more ethanol than their controls, in accordance with DiBattista and Joachim (1998), who described increased ethanol consumption also in golden hamsters submitted to DR. This result suggests that the rats used ethanol to compensate for the caloric restriction. The groups did not differ in cortical and hippocampal AChE activity, indicating that this parameter is not affected by either chronic ethanol consumption for 21 days or caloric restriction. Our findings are in accordance with previous data obtained by our group (Pereira et al., 1998; Pires et al., 2001), in which we did not find ethanol-induced alterations in cortical AChE activity. Pires et al. (2001) found a lower AChE activity in the hippocampus of rats submitted to a 4-month chronic ethanol treatment. In the above-mentioned study (Pires et al., 2001), besides being submitted to a longer treatment, the animals were sacrificed while still consuming ethanol, whereas in the present study they were sacrificed after a 2-week ethanol withdrawal period. Arendt et al. (1989) reported that an 8-week ethanol treatment caused a decrease in all the cholinergic parameters studied (ACh content, synthesis and release, choline acetyl-transferase and AChE activity and choline uptake) and that after 4 weeks of ethanol withdrawal all these parameters were returned to control levels. It is worth mentioning, however, that, in the present study, AChE activity measurement was done soon after behavioral tests, in which some behavioral alterations were observed. Therefore, these alterations cannot be explained by change in this specific cholinergic parameter. The hypothesis of an involvement of the cholinergic system cannot be discarded, however, as immunolesions in the septal and diagonal band nuclei, two cholinergic basal nuclei, have been related to a decline in exploratory behavior measured in the open field (Lamprea et al., 2003; Torres et al., 1994). Thus, to verify a possible role of the cholinergic system in the chronic-ethanolinduced changes in the exploratory behavior of rats, it is necessary to investigate other brain areas and also more specific cholinergic parameters. There are also several other neurotransmitter systems that might be involved as chronic ethanol consumption induces alterations in various systems besides the cholinergic (Fadda and Rossetti, 1998). More specifically, both noradrenergic and serotoninergic systems seem to be involved in exploratory and novelty seeking behavior (see, for instance, Mogensen et al., 2003 and Sara et al., 1995).

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In short, our study demonstrates that a 3-week ethanol treatment does not have any detectable effect on reference and working spatial memory, but it does alter exploratory behavior. Besides, ethanol acts differently on dietary-restricted and ad-libitum-fed rats. Some of the chronic ethanol effects seem to be prevented by DR treatment. Mechanisms by which DR exerts its effects are not clear but apparently involve a reduction in oxidative stress. The observed changes in exploratory behavior were not correlated to both AChE and nNOS activity (data not shown). The present study is a first step towards a more detailed characterization of DR's counteraction against chronic ethanol effects. Longer ethanol treatment effects associated to dietary restriction are not yet known, and, besides, molecular mechanisms underlying the present findings still need further investigation.

4.

Experimental procedures

4.1.

Animals and treatment

Forty-eight male Wistar rats aged 3 months were used. Initially, they were housed individually in standard cages and were maintained on a 12/12-h light/dark cycle. All animals received ad libitum commercial chow and water. During 1 week before the beginning of the experiment, food intake was recorded in order to know the daily average consumption of each rat. The subjects were then divided into two groups treated for 1 week as follows: (i) group C (control diet, n = 24) in which the animals received ad libitum commercial chow and, (ii) group R (restricted diet, n = 24) in which the animals received 15 g of commercial chow, which corresponded to 50% of the average amount of chow consumed by the animals in the previous week. Animals from both groups received tap water ad libitum. In the second week, the groups were further divided into four subgroups (n = 12 each): control diet-water (CW), control diet-ethanol (CE), restricted diet-water (RW) and restricted diet-ethanol (RE). Groups CW and RW received tap water ad libitum, groups CE and RE received a 20% v/v ethanol solution ad libitum as the only source of fluid available. The initial ethanol solution concentration was 5% v/v and was increased daily by 5%, until reaching the final concentration of 20% v/v. Treatment lasted for 21 days, and ethanol withdrawal was done by progressively decreasing concentration 5% per day. Food restriction for group R lasted the whole experiment, which took 41 days. Food and fluid intake were recorded daily during the whole experiment. The care and use of animals in this study were done according to the National Institute of Health Guide for Care and Use of Laboratory Animals (National Research Council et al., 1985).

4.2.

Behavioral studies

The behavior tests started 2 days after complete withdrawal of ethanol. All animals underwent all the below described tests.

4.2.1.

Spatial memory assessment

Spatial reference memory was assessed from day 3 to 7 postethanol withdrawal, whereas spatial working memory tests took place on days 8 to 12. Both reference and working spatial

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memory were assessed using the Morris water maze test (Morris, 1984).

4.2.1.1. Apparatus. The apparatus consisted of a 1.80 m diameter fiberglass round pool, filled with water at the depth of 27.5 cm and controlled temperature of 26 ± 2 °C. A 15 cm diameter transparent Plexiglas round platform was placed 2 cm below water surface in the center of one of the pool quadrants (southeast, southwest, northeast or northwest). In order to avoid visual location of the platform, 60 g of powder milk was dissolved in the water, making it turbid. The pool was placed in a 3 × 3 m room, with extra-maze cues (e.g. posters, TV and video equipment, a table). All sessions were recorded with a wide-angle video camera fixed to the ceiling. 4.2.2.

Procedure

4.2.2.1. Spatial reference memory. Each animal had one daily session for four consecutive days, and each session consisted of four trials. The intertrial interval was the same for all rats for each session (30 min). At the start of each trial, animals were put in the pool, facing its wall at each of the four quadrant edges alternated in a pseudo-random fashion. The platform was located in the southeast quadrant in all sessions. The subject was allowed to swim around the pool until it found the platform. If the animal did not find the platform in 60 s, it was gently guided to it by the experimenter. Once on the platform, the animals were allowed to stay there for 20 s. The latency for finding the platform was recorded. 4.2.2.2. Probe trial. On the fifth day of the reference memory test, the animal was left in the pool without the platform for 120 s. The time spent on the target quadrant, southeast, was recorded afterwards, when the videotape of the session was viewed by an observer using a stopwatch, which was started every time the rat entered the quadrant as demarked on the TV screen and stopped every time it left it. 4.2.2.3. Spatial working memory. For the assessment of working memory, we used the same procedures as described for reference memory, except for the location of the platform, which was changed daily, alternating the four quadrants, in a pseudo-random fashion. The number of sessions was 5 instead of 4, and no probe trial was carried out. 4.2.3.

Exploratory behavior assessment

This test began on day 13 after ethanol withdrawal and lasted for 3 days, i.e., ended on day 15 after ethanol withdrawal.

4.2.3.1. Apparatus. The apparatus consisted of a wooden box measuring 80 × 100 × 40 cm with a gray floor and white walls. Three wide-angle video cameras, one fixed to the ceiling and two on the top of the smaller sides of the box at an inclination angle of 60°, were used to record the sessions. 4.2.3.2. Procedure.

Each animal had one daily session for three consecutive days. The rats were placed in the open field and had their behavior observed for 5 min in the first two sessions and for 10 min in the third one. In the last 5 min of the third session, a novel object was introduced in the center of

the open field. Five behavioral categories were recorded: (i) rearing, if it rose on its hind paws; (ii) moving, if the rat was walking through the box; (iii) touching, a category recorded only in the second half of the last session, whenever it touched the novel object; using its paws, nose or vibrissae; (iv) grooming, if it was biting its fur, scratching or cleaning whiskers; or (v) other, if none of these was observed. The first three categories are considered exploratory behaviors. To record behavior, the momentary time-sample technique (Powell et al., 1975) was used. The observer, who monitored the test through a TV screen, glanced at the animal every 10 s and recorded manually the behavior displayed at the exact moment of the glance.

4.3.

Biochemical studies

4.3.1.

Biological samples processing

The animals were killed by decapitation for performance of biochemical analyses 1 day after the end of the behavior studies, i.e., on day 16 after ethanol withdrawal. Blood samples were collected immediately after killing. Neocortex and hippocampus were separated from one of the hemispheres. All samples were kept in a −70 °C freezer until the day of the assays, which occurred a maximum of 7 days after the sacrifice. All procedures were done under the temperature of 0–4 °C.

4.3.2.

Biochemical experiments

4.3.2.1. AChE activity. AChE activity was assessed by means of a spectrophotometric method adapted from Ellman et al. (1961). AChE activity was measured separately in neocortex and hippocampus. Aliquots of approximately 20 mg of tissue from each area (neocortex or hippocampus) were cut into 400 μm slices with a McIlwain tissue chopper, mixed with a spatula, transferred to Eppendorf tubes containing 1 ml of borate buffer and frozen at −20 °C until the day of the assay. The samples were homogenized in a Potter Elvehjem and centrifuged for 10 min at 12,300×g. The supernatant containing AChE was separated and kept on ice. Then, 135 μl of the supernatant, 35 μl of 5 mM dithiobisnitrobenzoate (DTNB), 820 μl of borate buffer (pH 8.2) and 10 μl of 1 mM acetyltiocholine were added in a cuvette, in that order. After the addition of acetyltiocholine, the reaction rate was recorded every 10 s, for a total time of 60 s using a Varian® spectrophotometer, mod. Cary 50. The results are expressed as micromoles of acetyltiocholine hydrolyzed/min/g of tissue.

4.3.2.2. Albumin. Albumin levels were measured in the blood serum by a spectrophotometric method using the Bioclin® Albumin Assay Kit. 4.3.2.3. Neural NOS (nNOS) activity. The activity was assessed in the cerebellum of four rats of each group as an index of free radical production. Activity of nNOS was measured in the soluble fraction of cerebellum according to Bredt and Snyder (1989), with some modifications. Briefly, the method consists of obtaining a supernatant fraction, by centrifugation, from homogenized cerebellum tissue. Aliquots of this supernatant were incubated for 30 min. The NO produced by the NOS during incubation is a very unstable

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molecule and is oxidized to nitrate and nitrite. As in previous study (Nims et al., 1996), nitrite, a stable product formed from NO, was measured by the colorimetric method of Griess using spectrophotometry.

4.4.

Statistical analysis

Body weight, food and fluid intake data were analyzed using analysis of variance with repeated measures (Milliken et al., 1998). Normality was tested by the Kolmogorov–Smirnov test (D'Agostino et al., 1986). AChE and nNOS activity and albumin were compared through a two-way ANOVA, in which the first factor is the ethanol and the second factor the food restriction (Anderson and Finn, 1996), with the Newman–Keuls test used as post hoc test to conduct multiple comparisons (Arango, 2001). All the behavioral data were analyzed using 2 × 2 × r analysis of variance with repeated measures to verify if there was any treatment effect (ethanol and/or food restriction) or time effect. All tests were considered statistically significant at the 5% level.

Acknowledgments This study was supported by PROGRAD/UFMG (Pró-Reitoria de Graduação/Universidade Federal de Minas Gerais), FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

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