Chronic caffeine consumption prevents cognitive decline...

CHRONIC CAFFEINE CONSUMPTION PREVENTS COGNITIVE DECLINEFROM YOUNG TO MIDDLE AGE IN RATS, AND IS ASSOCIATEDWITH INCREASED LENGTH, BRANCHING, AND SPINE DENSITY OFBASAL DENDRITES IN CA1 HIPPOCAMPAL NEURONS

S. VILA-LUNA,a S. CABRERA-ISIDORO,b L. VILA-LUNA,a

I. JUÁREZ-DÍAZ,b,c J. L. BATA-GARCÍA,a

F. J. ALVAREZ-CERVERA,a R. E. ZAPATA-VÁZQUEZ,d,e

G. ARANKOWSKY-SANDOVAL,a F. HEREDIA-LÓPEZ,a

G. FLORESb AND J. L. GÓNGORA-ALFAROa,d*

aDepartamento de Neurociencias, Centro de Investigaciones Region-ales (CIR) “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán(UADY), Avenida Itzáes 490 � 59, Mérida, Yucatán 97000, MexicobLaboratorio de Neuropsiquiatría, Instituto de Fisiología, UniversidadAutónoma de Puebla, 14 Sur 6301, Puebla 72570, MéxicocLaboratorio de Fisiología, Facultad de Estomatología, UniversidadAutónoma de Puebla, 31 Poniente 1304, Puebla 72410, MéxicodFacultad de Medicina, Universidad Autónoma de Yucatán, AvenidaItzáes 498 � 59-A, Mérida, Yucatán 97000, MexicoeUnidad Médica de Alta Especialidad, Instituto Mexicano del SeguroSocial, Ex-terrenos El Fénix, Mérida, Yucatán 97150, Mexico

Abstract—Chronic caffeine consumption has been inversely

associated with the risk of developing dementia and Alzhei-

mer’s disease. Here we assessed whether chronic caffeine

treatment prevents the behavioral and cognitive decline that

male Wistar rats experience from young (�3 months) to mid-

dle age (�10 months). When animals were young they were

evaluated at weekly intervals in three tests: motor activity

habituation in the open field (30-min sessions at the same

time on consecutive days), continuous spontaneous alterna-

tion in the Y-maze (8 min), and elevated plus-maze (5 min).

Afterward, rats from the same litter were randomly assigned

either to a caffeine-treated group (n�13) or a control group

(n�11), which received only tap water. Caffeine treatment (5

mg/kg/day) began when animals were �4 months old, and

lasted for 6 months. Behavioral tests were repeated from day

14 to day 28 after caffeine withdrawal, a time period that is far

in excess for the full excretion of a caffeine dose in this

species. Thirty days after caffeine discontinuation brains

were processed for Golgi-Cox staining. Compared with con-

trols, we found that middle-aged rats that had chronically

consumed low doses of caffeine (1) maintained their locomo-

tor habituation during the second consecutive day exposure

to the open field (an index of non-associative learning), (2)

maintained their exploratory drive to complete the conven-

tional minimum of nine arm visits required to calculate the

alternation performance in the Y-maze in a greater propor-

tion, (3) maintained their alternation percentage above

chance level (an index of working memory), and (4) did not

increase the anxiety indexes assessed by measuring the time

spent in the open arms of the elevated plus maze. In addition,

morphometric analysis of hippocampal neurons revealed

that dendritic branching (90–140 �m from the soma), length

of 4th and 5th order branches, total dendritic length, and

spine density in distal dendritic branches were greater in the

basal but not the apical dendrites of CA1 pyramidal neurons

from rats chronically treated with caffeine, in comparison

with their age- and littermate-matched controls. Altogether,

the present findings strengthen the epidemiological observa-

tions suggesting that prolonged caffeine intake prevents the

cognitive decline associated with aging, and open the possi-

bility that this process could be mediated by promoting the

growth of dendrites and spines in neurons of the adult mam-

malian brain. © 2011 IBRO. Published by Elsevier Ltd. All

rights reserved.

Key words: caffeine, methylxanthines, aging, memory, neu-

roprotection, dendritic growth.

During the past decade evidence that moderate consump-tion of coffee and other caffeinated beverages delays theonset of dementia (Eskelinen and Kivipelto, 2010) andreduces the risk of developing Alzheimer’s disease (AD) inelderly people (Maia and de Mendonça, 2002; Lindsay etal., 2002; Eskelinen and Kivipelto, 2010) has accumulated.In addition, epidemiological surveys performed in elderlypopulations have found that habitual caffeine consumptionshows a significant association with a lower cognitive de-cline in men (van Gelder et al., 2007) and women (Santoset al., 2010b), a better capacity for word recall and verbalretrieval in women (Johnson-Kozlow et al., 2002; Ritchie etal., 2007), and a better long-term memory for word recall,and faster motor speed in both sexes (Hameleers et al.,2000). However, not all published reports agree with theabove findings, an incongruity that has been attributed tothe lack of a standard methodology among studies (Rossoet al., 2008; Santos et al., 2010a). These discrepancieshave given impetus to animal studies designed to assessthe neuroprotective actions of chronic caffeine administra-tion under controlled experimental conditions.

Thus, the possibility that long-term caffeine consump-tion could prevent AD has been supported by experimentswith transgenic mice (APPsw) bearing a mutated form ofhuman amyloid-� (A�) linked to familial AD, in which long-term treatment with caffeine at a dose equivalent to thatcontained in five cups of coffee per day, afforded protec-tion against cognitive impairment during aging and pre-vented A� deposition in the hippocampus (Arendash et al.,

*Corresponding author. Tel: �52 999-924-6412; fax: �52 999-923-6120.E-mail address: [email protected] (J. L. Góngora-Alfaro).Abbreviations: AD, Alzheimer’s disease; A1R, adenosine A1 receptor;A2AR, adenosine A2A receptor; A�, amyloid-�; DG, dentate gyrus;EPM, elevated plus maze; LTP, long-term potentiation; OF, open field;RM-ANOVA, repeated measures analysis of variance; SAB, sponta-neous alternation behavior.

Please cite this article in press as: Vila-Luna S, et al., Chronic caffeine consumption prevents cognitive decline from young to middleage in rats, and is associated with increased length, branching, and spine density of basal dendrites in CA1 hippocampal neurons,Neuroscience (2011), doi: 10.1016/j.neuroscience.2011.11.053

Neuroscience xx (2011) xxx

0306-4522/11 $36.00 - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2011.11.053

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2006). Moreover, when aged APPsw mice already dem-onstrating cognitive impairment were treated with a chroniccaffeine regime, they showed a significant improvement inworking memory that was associated with a significantreduction of A� deposits in the hippocampus and entorhi-nal cortex (Arendash et al., 2009). In addition, other stud-ies have demonstrated that wild-type mice pretreated withcaffeine (30 mg/kg/day) during four days become resistantto the memory deficits caused by the i.c.v. injection of A�peptide fragment 25–35 (Dall’Igna et al., 2007).

However, studies aimed at evaluating whether chroniccaffeine treatment prevents the cognitive deterioration thatoccurs during aging in healthy animals have given incon-sistent results. Thus, in one report 18-month old male micewere evaluated after 12 months of caffeine ingestionthrough their drinking water (�125 mg/kg/day), and it wasfound that their working memory in an object recognitiontask was better than that of their age-matched controls,and similar to the performance of 6-month old animals(Costa et al., 2008). In contrast, another study that evalu-ated 16-month old male mice following 10 months of caf-feine treatment (�60 mg/kg/day) failed to find improvedperformance in a battery of cognitive and motor tests, incomparison with animals that consumed only water (Aren-dash et al., 2009). These last findings agree with those ofan earlier study performed in 10-month old male rats thatconsumed a caffeine solution (0.1 mg/ml) during 12months, and showed no difference in the maintenance oftheir acquired spatial memory in the Lashley III maze whencompared with control animals (Espinola et al., 1997). Itshould be noted that in all these studies behavioral testingwas performed while the animals were still under caffeinetreatment. We are not aware of any study aimed at as-sessing whether the putative cognitive benefits afforded bychronic caffeine treatment extend far beyond its consump-tion period. Such experimental design would be necessaryto disclose whether any memory improvement seen afterprolonged periods of caffeine ingestion are caused by atrue neuroprotective action, and not by its acute cognitivenormalizing effects, which have been consistently ob-served in animal models of memory dysfunction (Taka-hashi et al., 2008).

Another point that deserves consideration is the factthat high doses have been used in the studies that havetested the impact of chronic caffeine intake in healthyanimals (Costa et al., 2008; Arendash et al., 2009). Thishas likely produced brain concentrations affecting multiplemolecular targets other than adenosine receptors (Yu etal., 2009), whose blockade by caffeine has been linked toits protective effects against memory dysfunction (Taka-hashi et al., 2008; Cunha and Agostinho, 2010). However,some reports have supported the possibility that lowerdoses of caffeine can produce beneficial effects on brainfunctioning. Thus, APPsw transgenic mice that received asingle dose of caffeine, as low as 5 mg/kg, had a significantdecrease of A� outflow measured by microdialysis in theinterstitial space of the hippocampus (Cao et al., 2009).Another study found that healthy male rats that consumeda daily caffeine dose of 5 mg/kg during 6 months, followed

by a withdrawal period of at least 2 weeks, developed agreater resistance to the catalepsy induced with the dopa-minergic antagonist haloperidol, suggesting that thischronic caffeine schedule produced perdurable changes inbrain functioning, not attributable to the presence of caf-feine or its metabolites in the cerebral tissue (Góngora-Alfaro et al., 2009).

Consequently, the present study was designed to as-sess whether chronic intake of a low caffeine dose byhealthy adult rats, followed by a 2-week withdrawal period(a) prevents the progressive motor impairment that occursduring normal aging (Willig et al., 1987; Altun et al., 2007);(b) preserves the habituation of motor activity during re-peated exposure to the open field (OF), which is a form ofnon-associative learning (Leussis and Bolivar, 2006) thatdeteriorates during the aging process (Fraley andSpringer, 1981); (c) prevents the aging-associated workingmemory decline in the Y-maze (Willig et al., 1987; Stone etal., 1992); and (d) favors the development of anxiety-likestates as those produced by either acute (Pellow et al.,1985) or chronic (El Yacoubi et al., 2000) high doses ofcaffeine. In addition, the morphometric analysis of Golgi-Cox stained hippocampal neurons from control and caf-feine-treated rats was made because caffeine inducesspontaneous firing oscillations in the hippocampus (Piet-ersen et al., 2009), a nucleus involved in the control of avariety of motor and exploratory behaviors, and previousstudies have shown that chronic caffeine treatment causesmorphological changes in vertebrate pyramidal neurons(Burgess and Monachello, 1983; Juárez-Méndez et al.,2006).

EXPERIMENTAL PROCEDURES

Animals

The present study was performed on 24 male Wistar rats from fivelitters bred in our facilities. Only males were used because thereis evidence that estrogens may counteract some neuroprotectiveactions of caffeine in rodents (Xu et al., 2006). They were accli-matized to the laboratory environment for at least 1 week beforeany experimental manipulation took place, with 12:12 h light/darkcycles (lights on at 07:00), room temperature of 23�2 °C, andfood and water ad libitum. Groups of two to four animals werehoused in acrylic cages (length, 42 cm; width, 32 cm; height, 17.5cm) with the same partners throughout the experimental period.All behavioral tests were carried out during light hours (09:00–15:00 h), in a room with controlled temperature (23�2 °C), and afixed light intensity of approximately 100 lx. When testing beganrats had an average weight of 312�8 g, and at the moment of thelast experiment their weights averaged 489�14 g. This study wasapproved by the Institutional Bioethics Committee of the CIR-UADY, and all efforts were made to minimize animal discomfortaccording to the recommendations of the Guide for the Care andUse of Laboratory Animals of the USA, 1996 revised version.

Treatments

A similar number of animals from the same litter was assigned(see Table 1) to either a caffeine-treated group (5 mg/kg/day,n�13) or a control group (n�11) that received only tap water. Thecaffeine solution was freshly prepared every day and administeredad libitum through the drinking water. In order to ensure that theanimals ingested the expected 5 mg/kg daily dose of caffeine its


S. Vila-Luna et al. / Neuroscience xx (2011) xxx2

concentration was periodically adjusted according to the bodyweight gain of all the rats housed in a given cage and to theiraverage daily liquid ingestion in the preceding 7 days. Followingthis procedure the concentration of caffeine in the solution variedbetween 0.03 and 0.08 mg/ml. A human would receive an equiv-alent dose of caffeine by drinking two to three cups of regularcoffee, which is in the range of normal daily consumption (Fred-holm et al., 1999). Caffeine administration started when the ratswere young adults (�3.9 months old) and lasted for 6 months.This period was the same that was previously used for deliveringan equivalent amount of caffeine that induced perdurable resis-tance to haloperidol-induced catalepsy in this rat strain (Góngora-Alfaro et al., 2009).

Experimental design

Before starting treatments each rat was evaluated in three behav-ioral tests, at weekly intervals, in the following order: (1) motoractivity in the OF, (2) spontaneous alternation in the Y-maze, and(3) elevated plus-maze. In order to minimize the impact of stres-sors on behavioral outcomes, the animals of both treatmentgroups were housed under similar conditions, they were moved tothe testing room 24 h before evaluation, and all of them wereconsistently habituated to handling by the experimenter during the3–4 days preceding the experiment. In addition, to guarantee thatthe environmental conditions were comparable for both treatmentgroups, animals scheduled to caffeine and water were alterna-tively evaluated with the test performed on a given day. Caffeineadministration started 1 week after the plus-maze test, when therats were �3.9 months old (113–119 days from birthdate), andcontinued until they were �9.9 months old (294–299 days frombirthdate), when treatment was discontinued. As 6 months repre-sent about 20% of the mean survival age of male Wistar rats(Altun et al., 2007), this was considered a sufficiently long periodto uncover perdurable behavioral and morphological brainchanges induced by chronic caffeine treatment. The control ani-mals received only tap water during this same period. Two weeksafter caffeine withdrawal the animals were evaluated in the batteryof behavioral tests, which were applied in the same order andintervals as before. As previously discussed (Góngora-Alfaro etal., 2009), the 2-week washout period was judged adequate toallow for a complete elimination of caffeine or its metabolites in thebrain tissue of rats.

Open field test

The first OF test was performed when animals were �3.2 monthsold (93–98 days from birthdate), and the second one 2 weeks aftercaffeine withdrawal, when rats were �10.4 months old (307–315days from birthdate). The spontaneous motor activity was auto-matically recorded with a custom-made device consisting of anuncovered transparent acrylic cube (50 cm � 50 cm � 50 cm),equipped with two sets of nine equidistant (5 cm) infrared beamslocated 5 cm above the floor, and traversing the area in perpen-dicular directions, for a total of 81 x-y coordinates. Beam obstruc-

tions served to determine the horizontal movements of the rat. Theoccurrence of vertical exploration (rat standing on two hind paws,or rearing) was measured with an additional set of beams (z-coordinate) situated 15 cm above the floor. The data file gener-ated with the xyz motion sequence of each rat was recorded witha personal computer using a virtual instrument developed withLabVIEW software (National Instruments, Austin, TX, USA). Thedata were analyzed off-line using a program in the same graphicalprogramming environment. We used the paradigm of habituationin the OF, where the decrease of exploratory activity as a functionof repeated exposure to the same environment is taken as anindex of memory (Leussis and Bolivar, 2006). Consequently, theOF test was performed twice in 30-min sessions at the same houron consecutive days (herein referred to as day 1 and day 2). Theparameters that were measured and compared among experi-mental groups were distance traveled (cm), time spent in verticalexploration (s), and time in center (min). This last variable wasdefined as the sum of the time spent in those x-y coordinatessituated at more than 5 cm from the box walls. The removablealuminum tray placed at the box bottom was thoroughly cleanedbetween trials.

Spontaneous alternation behavior

Spatial working memory performance was assessed by recordingcontinuous spontaneous alternation behavior (SAB) during a sin-gle session in a Y-maze (Stone et al., 1992; Hooper et al., 1996).The first test was performed when animals were �3.4 months old(100–104 days from birthdate), and the second one 3 weeks aftercaffeine withdrawal, when rats were �10.6 months old (314–321days from birthdate). The device was constructed of black acrylic,with three identical arms (45 cm long, 14 cm wide, and 16 cm high)converging on a triangular area (the neutral zone). The sequenceof arm entries performed by the rat in the maze was automaticallydetermined by means of three sets of infrared beams positioned at3.5, 10, and 26 cm from the neutral zone, and 5 cm above themaze floor. Consecutive entries to the same arm (visiting theneutral zone between entries, but not the other arms) were con-sidered as a single visit. A record of the order of arm visits waskept in an electronic memory for subsequent transfer to a personalcomputer.

Rats were placed in the starting arm facing the closed endand allowed to freely explore the maze for 8 min. It was consid-ered that a minimum of nine arm visits was needed to calculate areliable alternation performance value. Thus, animals unable tomeet this criterion were classified as non-alternating. Every threeconsecutive arm visits made up a triplet set. An alternation wasdefined as the consecutive entry into three different arms onoverlapping triplet sets. Percentage alternation was calculatedwith the formula: % Alternation�{(Number of alternations)/(Totalarm entries�2)}�100 (Stone et al., 1992). In addition, the totalnumber of arm entries was used as an index of locomotor activity.The maze was thoroughly cleaned between trials.

Elevated plus maze

The elevated plus maze (EPM) (50 cm height) was made of blackacrylic, and consisted of two open arms (50 cm � 10 cm), and twoenclosed arms (50 cm � 10 cm � 40 cm) (Pellow et al., 1985).The four arms converged on a square central area (neutral zone),and were arranged in such a way that the two open arms wereopposite to each other, as were the enclosed arms. The first testwas performed when animals were �3.7 months old (107–111days from birthdate), and the second one 4 weeks after caffeinewithdrawal, when rats were �10.8 months old (321–328 daysfrom birthdate). Initially rats were placed in the neutral zone al-ways facing the open arm opposite to the location of the experi-menter. Animals were allowed to freely explore the maze for 5min, and their behavior was videotaped for posterior analysis on a

Table 1. Assignment of rats from the same litter to treatments

Treatment rats

Litter Water Caffeine

A 3 3B 1 2C 1 2D 2 2E 4 4Total 11 13


S. Vila-Luna et al. / Neuroscience xx (2011) xxx 3

screen by a trained observer. Time spent in the open or closedarms was counted only when the rat introduced its four paws intoan arm, whereas time in the central square was counted everytime the rat placed any paw in this zone. The measures recordedfrom all the subjects were time spent in open arms, closed arms,and neutral zone, and number of entries in open and closed arms.The maze was thoroughly cleaned between trials.

Golgi-cox staining

Thirty days after caffeine withdrawal, animals were deeply anes-thetized with pentobarbital (60 mg/kg) and perfused intracardiallywith 200 ml of 0.9% saline. The brains were removed and pro-cessed with the modified Golgi-Cox staining procedure describedby Gibb and Kolb (1998). The brains were first stored in the darkfor 14 days in Golgi-Cox solution, then 3 days in 15% sucrose.Coronal sections (200 �m) at the level of the hippocampus werecut with a vibroslicer (Camden Instruments, Leicester, England).Sections were collected onto gelatin-coated microscope slides. Themounted tissue was rinsed in distilled water and then placed in a bathof ammonium hydroxide for 30 min in the dark. After rinsing thesections in water they were immersed in Kodak film fixer for 30 minin the dark and subsequently washed with distilled water and dehy-drated in successive baths of 50% (1 min), 70% (1 min), 95% (1 min),and 100% (2�5 min) alcohol, followed by 15 min in xylene. Theslides were covered with balsam resinous medium.

Dendritic morphology was evaluated in neurons from threeareas of the dorsal hippocampus, CA1, CA3, and dentate gyrus(DG), as previously described (Juárez-Méndez et al., 2006). TheGolgi-impregnated pyramidal neurons from the hippocampus[Plates 27–32 of Paxinos and Watson (1986)] were readily iden-tified by their characteristic triangular soma shape, apical dendriticextension toward the pial surface, and numerous dendritic spines.Granule cells of the DG were recognized by their distinctive ovalor spherical soma shape and numerous dendritic spines. Onlycomplete, fully impregnated pyramidal neurons located in theCA1, CA3, or granule cells of the DG fields of hippocampus, withno apparent truncation of the basal dendritic tree were included inour analyses; the ends of the dendrites were positively identifiedby their characteristic conical shape. The following criteria wereused to select pyramidal neurons for analysis: (1) localization ofthe cell soma within the middle of the thickness of the section; (2)full impregnation of the dendritic longitude and spine neurons; (3)presence of at least three primary basilar dendritic shafts, each ofwhich branched at least once; (4) no morphological artifacts at-tributable to Golgi-Cox staining. For each hippocampal region, fiveneurons in each hemisphere were drawn using a camera lucida ata magnification of 250� by a person blind to treatment conditions.For each neuron, the three-dimensional apical and basal dendritictree, including all branches, was reconstructed in a two-dimen-sional plane and the dendritic tracing was quantified by Shollanalysis. Using the center of the soma as the reference point, atransparent grid with concentric rings spaced 10 �m was placedover the drawing and the total number of intersections betweenrings and each dendritic branch was counted separately for apicaland basal dendrites with the purpose of estimating the branchorder of the dendritic arborization and the total dendritic length.The density of dendritic spines was estimated by drawing seg-ments of 10 �m from the terminal tips at high magnification(1000�) and then counting the number of spines.

Data analysis

As the rats assigned to both treatments were tested four times inthe OF, the obtained data were analyzed with two-way repeated-measures ANOVA (RM-ANOVA), with OF session and treatmentas factors. Paired comparisons were made with Bonferroni’s test.The same statistical tests were applied to analyze the results ofthe times spent in the open and closed arms of the EPM, recorded

before and after caffeine treatment. Fisher’s exact test (one-tailed)was used to analyze the change in the proportion of animals thatreached the criteria of nine arm visits in the Y-maze necessary toassess percent alternation. In order to determine whether ratsalternated above chance level, one-sample t-tests on the percentalternations minus 50% (i.e. chance level) were performed pertreatment group, and the resulting difference score was comparedwith zero (Hooper et al., 1996). A significance level of 0.05 waschosen for all tests. Values are reported as mean�SEM.

RESULTS

Fig. 1A shows that the actual caffeine intake of rats sharingthe same home cage was close to the intended dose of 5mg/kg/day during the 6 months of its free ingestion throughdrinking water. Consistent with the mild diuretic action ofcaffeine (Rieg et al., 2005), the bedding of caffeine-treatedrats usually needed more frequent changes because itbecame damp with urine faster than that of control animals.The enhanced urine output, likely induced by caffeine, prob-ably rendered the rats thirsty, which might explain why theydrank more liquid on average than those receiving only water,although this effect was not significant (Fig. 1B). This lastobservation can be taken as indirect evidence that animalsconsuming caffeine at a mean dose of 5 mg/kg/day experi-enced its pharmacological effects continuously. This caffeineregime did not affect the weight gain of animals (Fig. 1C).

Locomotion in the open field

The values of the distance traveled in the OF are shown inTable 2. RM-ANOVA revealed significant effects for treat-ment (F1,66�5.4, P�0.05) and session (F3,66�17.8, P�0.01), but not for treatment�session interaction (F3,66�1.0). At young age, before treatments, the rats assigned tothe water regime walked longer distances during day 1than those allocated to the caffeine schedule (Table 2).Besides, both groups walked significantly less during day 1at middle age than at young age.

The difference between the distances walked duringday 1 and day 2 was taken as an index of locomotionhabituation (higher differences representing greater habit-uation). The mean habituation distance values at youngage were 801�344 cm for the water group and 493�167cm for the caffeine group; whereas at middle age themeans were 49�178 cm for water and 449�186 cm forcaffeine. RM-ANOVA disclosed a marginally significanteffect for age (F1,22�3.8, P�0.06), but not for treatment(F1,22�0.04, P�0.85), or treatment�age interaction (F1,22�3.0, P�0.10). Bonferroni tests indicated that only the ratsassigned to the water regime did not present locomotionhabituation when they were middle-aged (P�0.05).

Vertical exploration in the open field

The values of the time spent exploring the OF are shown inTable 2. Two-way RM-ANOVA only revealed a significanteffect for session (F3,66�16.0, P�0.01), but not for treat-ment (F1,66�0.3, P�0.61) or treatment�session interac-tion (F3,66�0.2, P�0.92). With respect to the session ef-fect, Bonferroni test showed that both treatment groups



spent significantly less time in vertical exploration duringday 1 or day 2 at middle age than on day 1 at young age.

The difference between the vertical exploration timesduring day 1 and day 2 was taken as an index of explora-tion habituation. At young age the mean exploration habit-uation time values were 64�19 s for water and 48�13 s forcaffeine; whereas at middle age the means were 0�12 sfor water and 5�15 s for caffeine. RM-ANOVA disclosed asignificant effect for age (F1,22�14.5, P�0.01), indicatingthat both treatment groups had exploration habituationonly at young age. However, no significant effects fortreatment or treatment�age interaction were found.

Time in the center of the open field

The values of the time spent in the center of the OF areshown in Table 2. RM-ANOVA revealed a significantlylower mean for caffeine than for water (F1,66�4.6, P�0.05). However, this result should be interpreted with cau-tion because the mean times spent in the OF center werelower for the caffeine group even before treatments wereapplied. A significant effect for session was also found(F3,66�6.7, P�0.01), given that both groups spent moretime in the center at middle age than at young age. Therewas no significant treatment�session interaction (F3,66�0.4, P�0.73).

Spontaneous alternation in the Y-maze

At young age, all rats assigned either to the water (n�11)or caffeine (n�13) treatments completed the minimum ofnine arm visits required to calculate the alternation perfor-mance in the Y-maze. However, when these animals werereevaluated at middle age, only 6 out of 11 rats in the watergroup met this criterion, representing a significant change inproportion (P�0.018, Fisher’s test), whereas 12 out of 13rats of the caffeine group still reached the mark (P�0.5).

Half of the caffeine-treated rats that met the SAB cri-terion scored higher at middle age than at young age(increments of 4, 6, 9, 20, 22, and 30%). In contrast, onlytwo out of six control animals increased their alternationpercentage at middle age vs. young age (increments of 4,and 16%). One-sample t-test analysis of SAB performanceof those rats that reached the criterion at both ages re-vealed that the water group performed significantly abovechance level at young age (68.1�5.3%; t5�3.44, P�0.02),but not at middle age (59.8�4.8%; t5�2.03, P�0.10). Incontrast, the caffeine group performed significantly abovechance level at both young age (67.9�3.7%; t11�4.88,P�0.001) and middle age (66.4�4.4%; t11�3.69, P�0.01).

Regarding the number of arm entries in the Y-maze, nosignificant differences were found between treatment groupseither at middle age (water, 10.9�1.7; caffeine, 12.3�1.1) orat young age (water, 15.0�1.4; caffeine, 15.9�1.1). How-ever, for both treatment groups, the values were significantlylower at middle age vs. young age (P�0.05).

Elevated plus maze test

The values of the parameters measured in the EPM areshown in Table 3. RM-ANOVA of the time spent in the

Fig. 1. Average caffeine intake, fluid consumption, and body weightgain during 6 mon. (A) Caffeine intake by four groups of rats housed inseparate cages. Each symbol represents the average caffeine dosecollectively consumed at weekly intervals by all the animals (n) sharinga given cage. The actual daily caffeine consumption was calculatedaccording to the formula: (concentration of caffeine solution supplied inthe preceding week [mg/ml])�(average daily fluid ingestion by all ratsin a given cage in the preceding week [ml])/(sum of body weights of allrats in a given cage [kg]). (B) Fluid consumption. The daily liquid intakewas calculated with the formula: (average daily fluid ingestion by allrats in a given cage in the preceding week [ml])/(sum of body weightsof all rats in a given cage [kg]). Each symbol represents themean�SEM values for animals housed in four cages, receiving eithercaffeine or water. Two-way RM-ANOVA indicated a significant effect fortime (F25,150�5.1, P�0.01), but not treatment (F1,150�2.0, P�0.21), ortheir interaction (F25,150�0.1, P�1.0) (C) Time course of body weightincrease of rats treated with caffeine (n�13) or water (n�11). Arrowspoint at the average weight values of animals when behavioral tests wereperformed: OF, open field; Y, alternation in the Y-maze; �, elevated plusmaze.



open arms revealed a significant effect for age (F1,22�18.6, P�0.01), given that both treatment groups spentsignificantly less time exploring the open arms at middleage vs. young age (Table 3). The time spent in the openarms showed no significant effect for treatment (F1,22�0.9,P�0.35) or treatment�age interaction (F1,22�0.2, P�0.68). Further, no significant changes were found in thetime spent exploring the closed arms or the neutral zone ofthe EPM.

Regarding the number of open arms entries in theEPM, no significant differences were found between treat-ment groups at both ages (Table 3). However, both groupsperformed less open arms entries at middle age than atyoung age (P�0.05).

Morphometry of hippocampal neurons

The Golgi-Cox impregnation procedure clearly filled thedendritic shafts and spines of hippocampal neurons (Fig.2A). The morphological analysis presented here is basedon a total of 710 neurons from 24 animals (11 vehicle-treated and 13 caffeine-treated). As one brain of the caf-feine-treated rats had insufficient number of stained neu-rons in the CA1 field for a reliable morphometric analysis,only the data of 12 brains are reported for this group. Thus,estimates of spine number, total dendritic length, andlength for each dendritic branching order were obtainedfrom 230 CA1 neurons, 240 CA3 neurons, and 240 DGneurons. Sholl analysis revealed that the dendritic branch-ing (90–140 �m from the soma), the length of the 4th and5th order branches (Fig. 3B–G), and the total dendriticlength (Table 4) were greater in the basal, but not theapical, dendrites of CA1 pyramidal neurons from ratschronically treated with caffeine, in comparison with theirage and littermate-matched controls (Fig. 2B). The densityof spines in distal dendritic branches showed a marginallysignificant increase only in the basal dendrites of CA1neurons (Table 4). In contrast, no significant differenceswere found between caffeine and water-treated rats for thesame morphometric parameters of CA3 and DG neurons.

DISCUSSION

The main finding of the present study was that prolongedcaffeine treatment (from �4 to �10 months of age) givento adult male rats caused a significant attenuation of someindexes of behavioral decline associated with aging: (1) pre-serving their locomotor habituation in the OF, (2) maintainingtheir exploratory drive for completing the minimum of ninearm visits required to calculate the alternation performance inthe Y-maze in a greater proportion than control animals, and(3) maintaining their alternation percentage above chancelevel. As these behavioral outcomes were observed 2 weeksafter caffeine discontinuation, it is unlikely that they were amanifestation of withdrawal symptoms (e.g. hypolocomotion),which in rats does not last more than 5 days after ceasing thechronic administration of high doses (160 mg/kg/d) (Holtz-man, 1983). However, as the time course of mood or mem-

Table 2. Spontaneous motor activity parameters displayed by rats during 30 min in the open field

Parameter Treatment Age

3.2 mon 10.4 mon

Day 1 Day 2 Day 1 Day 2

Distance traveled (cm) Water 4059�345*,��,��,† 3258�247** 2683�320*** 2634�352†

Caffeine 3112�204��,† 2620�236‡ 2376�159** 1927�189‡,†

Vertical exploration (s) Water 155�18��,†,§ 91�16*** 78�16† 84�10§

Caffeine 157�22��,†,§ 102�18** 79�17† 89�10§

Time in center (min) Water 8.3�0.9** 8.5�1.1‡ 12.5�1.6��,‡ 11.3�1.9Caffeine 7.0�1.0 5.0�0.8*** 10.6�1.6*** 8.0�1.2

For each variable, paired comparisons with Bonferroni test of the means (�SEM) arranged in columns showed that the only significant differenceoccurred at young age, when the rats assigned to the water regime traveled a significantly longer distance on day 1 in the OF (* P�0.05) than thoseallocated to caffeine.

Paired comparisons of the means arranged in rows yielded significant differences among those marked with the same symbols. ** P�0.05;*** P�0.01;† P�0.01;‡ P�0.05;§ P�0.01.

Table 3. Time measurements (in seconds and as percentages) andarm entries for the elevated plus maze test

Zone Treatment Age

3.7 mon 10.8 mon

Open arms WaterTime 36.0�8.3 11.4�5.0*% Time 12.0�2.8 3.8�1.7*Entries 2.0�0.3 0.6�0.2*

CaffeineTime 27.3�6.8 7.0�3.6**% Time 9.1�2.3 2.3�1.2**Entries 1.3�0.3 0.4�0.2*

Closed arms WaterTime 182.8�12.7 204.6�12.7% Time 60.9�4.2 68.2�4.2Entries 10.5�1.4 8.2�1.0

CaffeineTime 197.5�9.7 211.6�16.7% Time 65.8�3.2 70.5�5.6Entries 9.6�1.1 6.6�1.3

Central square WaterTime 81.2�10.7 84.0�11.3% Time 27.1�3.6 28.0�3.8

CaffeineTime 75.2�8.1 81.4�16.3% Time 25.1�2.7 27.1�5.4

* P�0.01,** P�0.05; vs. 3.7 mon.



ory-related performance after caffeine withdrawal in rats hasnever been tested, the possibility that the lower behavioraldecline observed in caffeine-treated rats was a manifestationof drug withdrawal cannot be completely discarded.

Besides the behavioral effects, histological analysis ofbrains processed for Golgi-Cox staining 1 month aftertreatment withdrawal revealed that caffeine-exposed ratsdeveloped a persistent increase in the length, branching,and spine density of the basal dendrites of CA1 hippocam-pal pyramidal neurons. The present experimental designdoes not permit discerning whether these morphologicchanges occurred during chronic caffeine administration orafter its withdrawal. However, there is evidence that thechanges in the number of spines measured in dendrites ofaccumbal neurons 2 days after discontinuing the chronicadministration of morphine (Diana et al., 2006) or cocaine(Dobi et al., 2011) are no longer observed 2–4 weeks later.Altogether, these evidences argue against the possibilitythat the morphologic changes observed in CA1 hippocam-pal neurons had occurred during the month elapsed aftercaffeine withdrawal.

It should be noted that chronic caffeine treatment didnot prevent the aging-associated decline in all recordedbehaviors. Specifically, the caffeine-treated rats and their

littermate controls performed similarly at middle age invarious parameters measured in the OF (traveled dis-tance, exploration time), the Y-maze (% SAB), and theelevated-plus maze (time spent in open and closed arms).These findings agree with those of an earlier study show-ing that healthy mice chronically treated with caffeine for10 months (from 5½ to 15½ months of age) did not im-prove their cognitive performance in a battery of memorytests, neither were sensorimotor or anxiety measures af-fected when compared with control animals consumingonly water (Arendash et al., 2009). Unfortunately, theseauthors did not measure the behavioral performance ofmice before starting the caffeine or vehicle treatments,when animals were middle-aged (5½ months), making itimpossible to assess whether caffeine treatment pre-vented any behavioral decline that might have occurred atadvanced age (15½ months). Other experiments with maleWistar rats have shown that their acquired spatial memoryin a Lashley III maze remained unaffected during 1 year ofchronic caffeine treatment in comparison with control ani-mals (Espinola et al., 1997). In the only study that hasreported preservation of working memory in aged miceafter chronic caffeine treatment, very high doses (�125mg/kg/day) were used, and were still delivered at the mo-

Fig. 2. Chronic caffeine treatment selectively modifies the morphology of basal dendrites of hippocampal CA1 pyramidal neurons. (A) Photomicro-graph of a representative Golgi-Cox-impregnated CA1 pyramidal neuron of the right hippocampus from a vehicle-treated rat. Bar�50 �m. (B)Representative schematic drawings of the dendritic arbor of the CA1 pyramidal neurons of the right and left hippocampus. Observe that both neuronsfrom a caffeine-treated rat have longer basal dendrites than those from a control animal.



Fig. 3. Effect of chronic caffeine treatment on dendritic morphometry of hippocampal neurons. Graphs (A–E): Sholl analysis of dendrite ramifications.Graphs (F–J): length of dendritic branches according to their order. Long-term caffeine consumption caused a significant effect only in the basal



ment of behavioral testing (Costa et al., 2008), thus makingit difficult to distinguish whether the effect was because ofa neuroprotective action or because of the acute cognitivenormalizing effects of caffeine (Takahashi et al., 2008;Cunha and Agostinho, 2010). The present results showthat, even after several weeks of caffeine withdrawal, therats that consumed low doses of caffeine during 6 monthsexhibited improved performance in some memory teststhan their controls. These observations indicate thatchronic caffeine intake prevents the decline of some brainfunctions that occur during the aging process.

Effects of chronic caffeine treatment on open field

behavior

In agreement with previous studies showing that aging isaccompanied by a gradual decrease in locomotion andrearing (Willig et al., 1987; Miyagawa et al., 1998; Altun etal., 2007), here we found that rats traveled shorter dis-tances and spent less time in vertical exploration at middleage than at young age. The activity decline associated withaging was also evident as a reduced number of arm visitsmade by rats from both treatment groups in the Y-mazeand the EPM at middle age. Altogether, these findingssuggest that chronic caffeine treatment during the periodspanning from young to middle age does not prevent thenormal decline of locomotion and exploration that occursduring the aging process.

Long-term habituation to a novel environment in ro-dents is conceptualized as a form of non-associative learn-ing in which the decline of locomotion after repeated ex-posure to the same environment is taken as an index ofmemory (Leussis and Bolivar, 2006). This behavior dete-

riorates during the aging process (Fraley and Springer,1981), and the hippocampus is critically involved in theacquisition of motor habituation to the OF (Leussis andBolivar, 2006). Using this paradigm we found that, atyoung age, both the water- and caffeine-treated rats had asignificant reduction in the distance walked during thesecond consecutive day of testing in the OF, which isconsistent with previous studies (Miyagawa et al., 1998;Bert et al., 2002). However, when animals were evaluatedagain at middle age, those that had been under chroniccaffeine treatment maintained their locomotion habituationat values that were closer to those measured when theywere young, whereas control animals did not. These ob-servations could be considered as evidence that long-termconsumption of caffeine at low doses prevents the declineof locomotion habituation to a novel environment associ-ated with aging (Fraley and Springer, 1981).

Effects of chronic caffeine treatment on anxiety indexes

As caffeine is a drug that has been associated with thedevelopment of anxious states (El Yacoubi et al., 2000), itwas important to assess whether its chronic consumptioncould enhance the anxiety levels of rats. In the OF test, anyexperimental manipulation that decreases the time spentin the central part of the arena is interpreted as anxiogenic,whereas the opposite is considered anxiolytic (Prut andBelzung, 2003). The observation that control and caffeine-treated rats spent similar amounts of time in the OF centerwhen they reached middle age suggests that chronic con-sumption of low amounts of caffeine does not predisposethe subjects to the development of anxiety to open spaces.

In the EPM, the amount of time spent in the open arms isconsidered the most reliable procedure to evaluate emotion-ality in rodents (Walf and Frye, 2007). Using this paradigm nosignificant differences were detected between control ani-mals and those that had consumed caffeine. Although anearlier study reported that chronic caffeine treatment reducedthe time that mice spent in the open arms of an EPM incomparison with their controls, this effect occurred at veryhigh doses (50 mg/kg) (El Yacoubi et al., 2000). Collectively,the above findings argue against the possibility that chroniccaffeine consumption at the dose of 5 mg/kg/day could favorthe development of anxious states.

Effects of chronic caffeine treatment on spontaneous

alternation behavior

SAB in a Y-maze is considered a test of working spatialmemory because animals need to remember the arm thatwas previously visited in order to sequentially explore thethree arms of the maze (Hooper et al., 1996). The expres-sion of SAB requires an intact hippocampal function (Ger-

dendrites of CA1 pyramidal neurons. (B) Two-way ANOVA of Sholl analysis revealed a significant effect of treatment (F1,483�4.8, P�0.05) and circleradius (F23,483�595.4, P�0.0001) on the number of intersections per shell, as well as a significant interaction between the main factors (F23,483�3.7,P�0.0001). Bonferroni test indicated that the number of intersections per concentric ring was significantly greater at distances of 90–140 �m from thesoma. (G) Two-way ANOVA of dendritic length per branch order revealed a significant effect of treatment (F1,126�4.9, P�0.05) and branch order(F6,126�156.2, P�0.001) on branch length, as well as a significant interaction between the main factors (F6,126�4.1, P�0.0001). Bonferroni testindicated that 4th and 5th order dendritic branches were significantly longer in caffeine-treated rats: * P�0.05, and � P�0.01, vs. control animals.

Table 4. Dendritic length and spine density in hippocampal neurons

Dendrites Water Caffeine

CA1 apicalTotal length (�m) 1752�88 1749�70Spines/10 �m 7.8�0.3 7.8�0.4

CA1 basalTotal length (�m) 1562�74 1782�68*Spines/10 �m 7.2�0.3 8.0�0.3**

CA3 apicalTotal length (�m) 1466�43 1383�73Spines/10 �m 6.4�0.3 6.6�0.3

CA3 basalTotal length (�m) 2003�75 2156�78Spines/10 �m 6.0�0.3 6.6�0.2

DGTotal length (�m) 1425�75 1466�90Spines/10 �m 6.9�0.3 7.4�0.3

* P�0.02,** P�0.05, vs. water; unpaired Student’s t-test (one-tailed).



lai, 1998), and it is a behavior that deteriorates during theaging process (Willig et al., 1987; Stone et al., 1992). Herewe evaluated SAB performance of the same animals attwo different ages, and found that when they were youngall of them completed the minimum of nine arm visitsrequired to reliably calculate the alternation performance,irrespective of the assigned treatment. However, whenthey were retested at middle age, about half of the animalsthat consumed only water failed to meet this criterion,which might reflect the loss of exploratory drive usuallyobserved during the aging process (Willig et al., 1987). Incontrast, all but one of the rats that consumed caffeine for6 months reached the norm, suggesting that this treatmentprevented the deterioration of the brain processes thatdrive exploration of the environment.

In addition, the mean percent alternation at young agewas practically the same for animals assigned to the wateror caffeine regimes, which in both cases occurred abovechance level. However, when they were reevaluated atmiddle age, only the rats that had consumed caffeine for 6months maintained their percent alternation above chancelevel, but not the control animals. It is important to empha-size that the improved alternation execution recorded inmiddle-aged rats that had been under caffeine treatmentwas observed 3 weeks after its withdrawal, which impliesthat the previous prolonged caffeine consumption inducedpersistent brain changes (see next section) that preservedthe normal functioning of the neural circuits involved inshort-term spatial memory, and also in the neural pro-cesses that drive the animals to explore their surroundings,effects that could be considered as neuroprotective.

Effects of chronic caffeine treatment on neuronal

morphology in the hippocampus

The most remarkable finding of the present study was thatin the group of rats chronically treated with caffeine theneurons of the CA1 field of the hippocampus had longerand more branched basal dendrites, and with greater num-bers of spines, compared with the same neurons of thecontrol group. As the size of neurites in CA1 pyramidalneurons has reached the adult levels by postnatal age P15(Pokorný and Yamamoto, 1981), this finding can be ex-plained in two possible ways. First, that chronic caffeineconsumption had prevented the initial steps of atrophy inthe basal dendrites of CA1 pyramidal neurons during thetransition from young to middle age. However, although ithas been reported that the apical dendrites of CA1 neu-rons experience degeneration at advanced ages (Lolova,1989), at present there is no evidence that this degenera-tive process occurs at earlier ages.

An alternative explanation is that the prolonged caf-feine consumption had triggered some kind of trophicmechanism that selectively promoted the growth of thebasal dendritic tree and spines in CA1 pyramidal neurons.There is evidence that the release of neurotrophic factorsin the hippocampus is directly proportional to the firing rateof neurons, with the maximal release occurring at frequen-cies that induce long-term potentiation (LTP) (Balkowiecand Katz, 2002). As low caffeine concentrations (�30 �M)

induce spontaneous firing oscillations in the hippocampus(Pietersen et al., 2009) and facilitate the development ofLTP (Costenla et al., 2010; Simons et al., in press), thepossibility exists that during its chronic consumption caf-feine had promoted the activity-dependent release of neu-rotrophic factors that stimulated the growth of morebranches and spines in the basal dendrites of adult CA1pyramidal neurons.

Caffeine reaches an average concentration of �22 �Min the hippocampus of rats that consumed high dosesduring 4 weeks (Costenla et al., 2010). As the animalsused in the present study consumed smaller amounts ofcaffeine, it is conceivable that its brain levels attainedvalues in the low micromolar range, at which caffeine bindspreferentially to A1 and A2A adenosine receptors (A1Rs,A2ARs) (Fredholm et al., 1999). Then, it is plausible thatthe morphologic changes found in CA1 neurons of caf-feine-treated rats were mediated through blockade of ei-ther of these receptors, or both.

In the hippocampus, the blockade of A1Rs facilitatesthe induction of LTP in young animals, but not in those thathave reached either middle or old age, which is explainedby the greater number of A1Rs in the glutamatergic nerveendings at young age in comparison with the older agegroups (Costenla et al., 2011). Remarkably, the greaterexpression of A1Rs occurs in the pyramidal neurons of theCA2 field (Ochiishi et al., 1999), which are refractory to thedevelopment of LTP (Zhao et al., 2007), possibly through astrong inhibitory tone imposed by endogenous adenosine(Ochiishi et al., 1999). However, following in vivo dosingand in vitro application of caffeine, a robust and long-lasting LTP can be elicited in CA2 neurons (Simons et al.,in press). In the present study, chronic caffeine adminis-tration to rats started at �4 months of age, when hip-pocampal A1Rs are more abundant (Costenla et al., 2011).As the majority of synapses made by CA2 neurons occuron the proximal branches of basal dendrites of CA1 neu-rons (Ropireddy and Ascoli, 2011), we suggest that sus-tained blockade of A1Rs in CA2 pyramidal neurons duringchronic caffeine treatment increased their basal firing rate,thus favoring the occurrence of LTP (Costenla et al., 2010;Simons et al., in press), and the release of neurotrophicfactors (Balkowiec and Katz, 2002) that promoted thegrowth of 4th and 5th order branches and spines in thebasal dendrites of CA1 pyramidal neurons. LTP is consid-ered the functional substrate of memory, and both of theseprocesses deteriorate during aging, starting at middle age(Kumar, 2011). The observation that mice carrying a mu-tation that selectively unmasks a robust LTP in CA2 neu-rons exhibit marked enhancement in spatial learning (Leeet al., 2010) leaves open the possibility of a causal rela-tionship between the enlargement of the basal dendritictree in CA1 neurons and the preservation of motor habit-uation and SAB in caffeine-treated rats.

It is unlikely that the morphologic changes seen in CA1neurons of caffeine-treated rats was mediated throughblockade of A2ARs, as this suppresses the firing oscilla-tions induced by adenosine (Pietersen et al., 2009), andinhibits the induction of LTP in the hippocampus (Costenla



et al., 2011). Although many studies have demonstratedthat acute or chronic caffeine treatment attenuates mem-ory impairment through A2AR blockade, these experimentshave been performed in animal models of pathologicalconditions (e.g. emotional stress, diabetes, seizures, Par-kinson’s or Alzheimer’s diseases) that produce noxiousbrain insults which disrupt the inhibitory/facilitatory balanceof adenosine in the hippocampus through down-regulationof A1Rs and up-regulation of A2ARs (Costenla et al., 2010;Cunha and Agostinho, 2010). However, a different picturewould be expected in healthy young animals (as thoseused here), in which adenosinergic tone would be strongerin the more abundant A1Rs expressed in hippocampus atthis age (Costenla et al., 2011). Hypothetically, by promot-ing the activity-dependent growth of dendrites and spinesof hippocampal CA1 neurons in young subjects, caffeinewould compensate the atrophy and loss of dendrites thatoccur at advanced ages (Lolova, 1989), thus providing analternative mechanism by which chronic caffeine con-sumption could provide protection against aging-depen-dent cognitive decline.

At present it is an enigma whether the morphologicalchanges seen in the basal dendrites of CA1 neurons fromcaffeine-treated rats would have persisted beyond the 30-day period of caffeine withdrawal. However, previous stud-ies suggest that this could be the case, as juvenile fishraised during 50 days in water with a low caffeine concen-tration (�7 �M) show a greater number of dendrites andspines in their neurons when they reach the age of 21months, in comparison with their controls of the same age(Burgess and Monachello, 1983). Similarly, neonatal ratschronically treated with high doses of caffeine developprefrontal cortical neurons with longer and more brancheddendrites at puberal and postpuberal ages (Juárez-Mén-dez et al., 2006).

CONCLUSION

These results of the present study agree with previousobservations indicating that chronic caffeine treatmentdoes not produce cognitive enhancement but preventsthe cognitive decline associated with aging (Cunha andAgostinho, 2010). They also strengthen the findings ofepidemiological surveys suggesting that prolonged caf-feine intake is associated with lower cognitive declineand a reduced risk of developing AD in elderly people. Itis suggested that the longer basal dendrites and highernumber of spines seen in CA1 hippocampal neurons ofanimals that consumed caffeine from young to middleage could be another mechanism by which caffeinehelps to prevent the cognitive decline associated withaging.

Acknowledgments—S.V.-L. and S.C.-I. contributed equally to theexperimental part of this report. A scholarship to S.V.-L. and thebehavioral part of this work were supported by CONACYT-Mexicogrant 47763 to J.L.G.-A. The histological part was supported byCONACYT-Mexico grant 138663 to G.F.

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(Accepted 24 November 2011)



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