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Cognitive performance of healthy young rats following chronic donepezil administration

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Cognitive performance of healthy young rats following chronic donepezil administration
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  ORIGINAL INVESTIGATION Cognitive performance of healthy young ratsfollowing chronic donepezil administration Debora Cutuli  &  Francesca Foti  &  Laura Mandolesi  & Paola De Bartolo  &  Francesca Gelfo  & Francesca Federico  &  Laura Petrosini Received: 3 August 2007 /Accepted: 18 January 2008 # Springer-Verlag 2008 Abstract  Rationale  Experimental studies have investigated the effectsof chronic donepezil treatment on the behavioral deficitselicited by reduced activity or the loss of cholinergic neuronsthat occurs in aging or in models of dementia. However, fewstudies have analyzed the effects of chronic donepeziltreatment on the cognitive functions of intact animals. Objectives  The cognitive functions of healthy young ratstreated chronically with the acetylcholinesterase inhibitor donepezil were evaluated using a wide behavioral test battery.  Results  Chronic treatment with donepezil amelioratedmemory functions and explorative strategies, speeded upthe acquisition of localizing knowledge, augmented respon-siveness to the context, and reduced anxiety levels.However, it did not affect spatial span, modify motivationallevels, or influence associative learning. Conclusions  The present findings show the specific profileof donepezil action on cognitive functions in the presenceof unaltered cholinergic neurotransmission systems. Keywords  Acetylcholinesteraseinhibitor .Donepezil.Behavioraltestbattery.Mnesicfunctions.Cognitiveenhancer .Cholinergicneurotransmission Introduction Acetylcholine (ACh) is a pivotal neurotransmitter in learn-ing, memory, and attentive functions (Giovannini et al. 1997;Parent and Baxter  2004; Pepeu and Giovannini 2004). Alteration of cholinergic neurons is one of the primary pathological changes found in the brains of patients affected by Alzheimer  ’ s disease (AD; Birks and Flicker  2006). The “ cholinergic hypothesis ”  (Bartus et al. 1982; Bartus 2000; Freo et al. 2002; Sarter et al. 2003; Phillis 2005), which  postulates that the age-related decline in cognition may becorrelated with reduced cerebral cholinergic function, has ledto the development of pro-cholinergic compounds, such asacetylcholinesterase inhibitors (AChE-Is), which are able toenhance cognitive functioning (Dawson and Iversen 1993;Carey et al. 2001; Takada et al. 2003). Donepezil is a second-generation AChE-I with fewer side effects than earlier drugs of its class and it has a verylong half-life (about 72 h). Currently, it is the first-line palliative treatment for improving cognitive functioning inAD patients afflicted by mild to moderate degrees of dementia (Rogers et al. 1998; Shigeta and Homma 2001; Sugimoto 2001; Bontempi et al. 2003; Seltzer  2007; Winstein et al. 2007). Furthermore, donepezil was reportedto increase frontal activity during a working memory task in patients affected by mild cognitive impairment, and thisincrease wa  s  related to improved cognition (Saykin et al.2004). There are also reports that donepezil reversedmemory deficits in experimental models of learning andmemory (Cheng et al. 1996; Rupniak et al. 1997; Higgins et  PsychopharmacologyDOI 10.1007/s00213-008-1084-0D. Cutuli :  F. Foti :  L. Mandolesi : P. D. Bartolo :  F. Gelfo : F. Federico :  L. PetrosiniIRCCS Santa Lucia Foundation,Rome, ItalyL. Mandolesi : F. GelfoUniversity of Naples  “ Parthenope ” , Naples, ItalyD. Cutuli :  F. Foti :  P. D. Bartolo :  L. Petrosini ( * )Department of Psychology, University of Rome  “ La Sapienza ” ,Via dei Marsi 78,00185 Rome, Italye-mail: laura.petrosini@uniroma1.it F. FedericoUniversity of Siena,Siena, Italy  al. 2002; Tokita et al. 2002; van der Staay and Bourger  2005; Wise et al. 2007). Acute administration of donepezil in experimental models of cholinergic deficits improved performances in the radial arm maze (Ogura et al. 2000;Wise et al. 2007), the Morris water maze (Spowart-Manning and van der Staay 2005), in spatial and visualrecognition tasks (Kirkby et al. 1996; Poorheidari et al.1998; Prickaerts et al. 2005), and in serial reaction time tasks (Kirkby et al. 1996).Some experimental studies have investigated the effectsof chronic donepezil treatment on the behavioral deficitselicited by reduced activity or the loss of cholinergicneurons that occurs in aging (Barnes et al. 2000; Hernandezet al. 2006) or in models of dementia (Dong et al. 2005; Yamada et al. 2005). In particular, in Tg2576 mice, anexperimental model of AD, donepezil had ameliorativeeffects on memory-related deficits (Dong et al. 2005).Furthermore, chronic donepezil administration improvedmemory impairment induced by i.c.v. injections of strepto-zotocin (Sonkusare et al. 2005). Thus, research hasaddressed the influence of donepezil on the symptoms provoked by altered cholinergic transmission. However,very few studies have investigated the putative influence of donepezil treatment on normal subjects. Acute donepezil-treated normal mice exhibited an increased rate of spontane-ous alternations compared with controls (Spowart-Manningand van der Staay 2004). Moreover, acute donepeziladministration before a spatial test elicited memory enhanc-ing effects in rodents (Wise et al. 2007). These experimentaldata integrate a few human studies reporting the effects of cholinergic stimulation in healthy adult subjects. Davis et al.(1978) reported that acute physostigmine administrationenhances storage of information in long-term memory aswell as its retrieval, without alterations of short-termmemory. More recently, it has been demonstrated that modulation of the cholinergic system by physostigmineinfusions improved working memory efficiency and reducedactivation of the cortical regions associated with workingmemory (Furey et al. 2000). Furthermore, Sun et al. (1999) reported improved memory and learning functions inadolescent Chinese students following a 4-week administra-tion of an AChE-I (huperzine-A). Recent studies in healthyaircraft pilots reported beneficial effects on the retention of complex tasks (flight simulator) after 30 days of donepeziltreatment (Yesavage et al. 2002; Mumenthaler et al. 2003). However, the tasks were very complex and tapped acombination of cognitive functions that were difficult todistinguish. Moreover, Grön et al. (2005) reported theselective enhancement of episodic memory performance inhealthy young subjects following 30 days of donepeziltreatment, suggesting that the hippocampal region is themajor target of cholinergic enhancement elicited by long-term inhibition of AChE. Overall, these findings suggest that AChE-Is, and donepezil in particular, may act as  “ cognitionenhancers ”  in normal subjects.Based on these findings, we set out to assess the specificeffects of chronic donepezil treatment on unaltered cholin-ergic neurotransmission systems. To this aim, healthyyoung rats chronically treated with donepezil were evalu-ated using a behavioral test battery that tapped different cognitive functions. Materials and methods AnimalsTwenty male Wistar rats (300  –  350 g) kept in standardlaboratory conditions (08:00  –  20:00 light, food and water adlibitum) were used in the present experiments. The animalswere maintained according to the guidelines for ethicalconduct developed by the European Communities CouncilDirective of 24 November 1986 (86/609/EEC) and ap- proved by the Ethical Committee on animal experiments of the University of Rome  “ La Sapienza ” . Rats were randomlyassigned to two experimental groups: the treated group (D; n =10) received daily i.p. injections of donepezil and thecontrol group (C;  n =10) daily injections of saline.DrugFor 3 weeks prior to the behavioral testing, donepezil (EisaiInc.) was administered daily at a dosage of 0.5 mg/kg; then,for the next 7 weeks it was administered daily at a dosageof 0.2 mg/kg dissolved in 0.5 ml of 0.9% NaCl solution.The same volume of saline, but without the drug  ,  wasadministered daily to the control animals. All injectionswere administered at the end of the testing sessions to avoidany acute drug effects. According to clinical (Rogers et al.1998; Rogers and Friedhoff  1996) and experimental (Wise et al. 2007; Ogura et al., 2000; Dong et al. 2005) studies, a 0.2 mg/kg dose of donepezil is sufficient to influencerepeated behavioral testing but not to elicit heavy sideeffects. The initial dose of 0.5 mg/kg was chosen tohabituate the rats to a high drug dosage that would bereduced during the behavioral testing phase.Behavioral testingAs shown in Fig. 1, a battery of six tests was administeredin the following order: Morris water maze (MWM), toanalyze competence in building a spatial cognitive map andin using navigational strategies; radial arm maze (RAM), toanalyze spatial reference and working memory; seriallearning task (SLT), to analyze cognitive flexibility; openfield (OF), to evaluate the ability to develop spatial and Psychopharmacology  discriminative competence; elevated plus maze (EPM), toassess anxiety levels; shuttle box (SB), to analyze activeavoidance learning.Morris water maze (MWM)The rats were placed in a circular white pool (diameter 140 cm) filled with 24°C water (60 cm deep), made opaque by the addition of 2 l of milk. An escape platform (diameter 10 cm), submerged 2 cm below or elevated 2 cm above thewater level, was placed in the middle of one cardinalquadrant, 30 cm from the pool walls. The rat was releasedinto the water from randomly varied starting points and wasallowed to swim around for 120 s to find the platform. Eachrat underwent two sessions of four trials per day, with a4-h inter-session interval. When the rat reached the platform,it was allowed to remain there for 30 s. In the first four sessions, the platform was hidden in the northwest quadrant (place I); in the next two sessions, the platform was visible inthe northeast quadrant (cue phase); in the final four sessions,the platform was hidden in the northeast quadrant (place II;Morris et al. 1982; Petrosini et al. 1996; Federico et al. 2006; Leggio et al. 2006). This protocol allowed investigatingnumerous components of spatial function. Place I analyzedthe sequence of navigational strategies (spatial procedurallearning) put into action to explore the pool and to find the platform, as well as the ability to build a spatial map (spatialmemory) by using extra-maze cues to locate the hidden platform. The cue phase analyzed the development of stimulus  –  response (platform/reaching) associative learningand employment of the procedural knowledge acquiredduring place I. Finally, place II analyzed the ability toremodel the spatial map by exploiting the intra-mazeinformation acquired in the cue phase in the presence of already acquired procedural strategies and also providedinformation about the plastic properties of spatial learning processes . The rats ’  trajectories in the pool were monitored by avideo camera mounted on the ceiling. Video signals wererelayed to a monitor and to an image analyzer (Ethovision, Noldus, Wageningen, the Netherlands).The following behavioral parameters were considered inanalyzing the MWM performances: latencies to find the platform, total distance swum in the entire pool, distancetraveled in a 20-cm peripheral annulus, heading angles (theangle formed by the direction of the rat  ’ s head whenleaving the pool edge and a straight line from the startinglocation to the platform), and swimming velocity.Radial arm maze (RAM)The apparatus consisted of a central platform (diameter 30 cm) from which eight arms (12.5 cm wide×60 cm long)radiated like the spokes of a wheel. A food well  (  5 cmdeep) was located at the end of each arm (Mandolesi et al.2001). Prior to the habituation phase, the rats were foodrestricted to decrease their weight by 20%.  Full-baited maze procedure  In each session, all maze armswerebaitedwithapieceofPurinachow.Theratwasplacedonthe central platform and allowed to make eight correct visits,16 (correct or incorrect) visits, or to explore the maze for 15 min. The animals were submitted to two sessions a day for 5 consecutive days. The inter-session interval was 4 h.The following parameters were considered: total entries(number of visits, either correct or incorrect), total errors(number of re-visited arms), spatial span (longest sequenceof correctly visited arms), and percentage of 45° angles(45° angles made in each session divided by the totalnumber of angles made × 100).  Forced-choice procedure  Forty-eight hours after the end of the above-described protocol, all animals were submitted tothe forced-choice paradigm. In the first phase, only four arms (for example, arms 1, 3, 4, and 7) were opened and baited and the remaining arms were closed. The baited armswere separated by different angles to prevent the animalfrom solving the problem by adopting a stereotyped pattern.   FLOW DIAGRAM 3 rd w2 nd w1 st wMWMRAM F-b RAM F-c SLTOFEPMSB0.5 mg/kg0.2 mg/kg Donepezil  food deprivation   3 rd w2 nd w1 st wMWMRAM F-b RAM F-c SLTOFEPMSB0.5 mg/kg0.2 mg/kg Donepezil  food deprivation Fig. 1  Flow diagram of experimental procedures. After the first 3 weeks of 0.5 mg/kg donepezil administration, a battery of six tests,namely,  MWM   Morris water maze,  RAM   radial arm maze (  F-b  full- baited and  F-c  forced-choice procedures),  SLT   serial learning task,  OF  open field,  EPM   elevated plus maze,  SB  shuttle box, was performedwith concomitant 0.2 mg/kg donepezil administration. All rats werefood deprived during RAM and SLT tests.  Gray boxes  indicate test-free daysPsychopharmacology  The rat was allowed to explore the open arms. Then, it was put in its cage for 60 s before being returned to the maze. Inthe second phase, the rat was allowed free access to alleight arms, but only the four previously closed arms were baited. This task was repeated for 5 consecutive days with adifferent configuration of arms closed each day to avoid afixed search pattern.The parameters considered were working memoryerrors, considered as re-entries into already visited arms.In the second phase, this parameter was broken downfurther into two error subtypes: across-phase errors, definedas entries into an arm entered in the first phase; within- phase errors, defined as re-entries into an arm visited earlier in the same session.Serial learning task (SLT)The apparatus consisted of a white rectangular wooden box(150×40×40 cm) subdivided into five compartments(30 cm long) by four gray panels with two unidirectionaldoors (height 10 cm, width 8 cm). Each door could be lockedusingapivot;therefore,iftheanimalpushedthedooritopenedabout 2 cm. The small split allowed the rat to introduce itsmuzzlebutpreventeditfromgoingthroughthedoor.Thistrick allowed us to obtain proof that the animals were attempting toopen the  “ incorrect  ”  door. The entire apparatus was closedwith a transparent Plexiglas cover. The final, rewardedcompartment was darkened using a black cover.The rats continued to be food restricted. Following a3-day pre-training session, they were submitted to onetesting session a day for 10 consecutive days. All dailysessions included 12 trials. In each trial, the goal was toreach the fifth compartment and collect the reward by goingthrough the open doors and making no attempt to forceopen the closed ones. Each animal was given a sequence of open doors that remained unchanged for all 12 trials of asession but that changed every session; thus, each animalwas tested in ten different sequences.In each of the 12 trials of a session, the following parameters were analyzed: errors, i.e., the attempt to forceopen the closed door (in each trial this parameter ranged from4 to 0); correct choices, i.e., the longest sequence of correct choices (in each trial this parameter ranged from 0 to 4); perseverations, i.e., the number of errors made at the samedoor in the 12 trials of a session. As the first session gave theanimal time to become accustomed to the actual testing procedure, only the results of S2  –  S9 were considered.Open field (OF)The apparatus consisted of a circular container (diameter 140 cm) delimited by a 30-cm-high wall. Five objects were present simultaneously in the open field: (1) a metal bar with a conical base, (2) a plunger, (3) a long steel rod, (4) ayellow rubber plug, and (5) a black cylinder with a plasticcup turned upside down on top of it.During session 1 (S1), each rat was allowed to movefreely in the empty open field and its baseline level of activity was measured. During S2  –  S4 (habituation phase),four objects were placed in a square arrangement in themiddle annulus of the arena and the fifth one was placed inthe central area. For S5 (spatial change, S5 and S6), thespatial configuration was changed by moving objects 2 and5 so that the initial square arrangement was changed to a polygon-shaped configuration, without any central object.During S7 (novelty), the configuration was modified bysubstituting object 4 with a green plastic object shaped likea half moon. Sessions lasted 6 min; inter-session intervalswere 3 min.All testing was recorded by a video camera whose signalwas relayed to a monitor and to the previously describedimage analyzer.The parameters taken into account were total distance (inmeters) traveled in the arena, distances traveled in the peripheral or central arena sectors, and time spent contact-ing objects (contact was considered to have taken placewhen the rat  ’ s snout actually touched an object or when it sniffed the object for at least 1 s).Elevated plus maze (EPM)The maze, which was raised 90 cm above the ground wasformed by a wooden structure in the shape of a cross withfour 50 cm×10 cm arms. The north and south arms wereopen, but the east and west arms were enclosed by walls36 cm high.The following behavioral parameters were measured:number of defecation boluses, total time spent in the openand closed arms or on the platform, and frequency of entries into the arms.Shuttle box (SB)An active avoidance shuttle box (Ugo Basile type 7532,Comerio-Varese, Italy) with an electrifiable grid floor (steelrods spaced 1.5 cm apart) was used (Molinari et al. 1997).The area inside the box was divided into two compartments by a ceiling-to-floor partition that had a 10-cm centralopening to allow the animals to cross from one side to theother. Each compartment had a 15-W bulb mounted on theceiling. Each daily session consisted of 100 automaticcomputerized deliveries of conditioned (light, 15 W, 10 s)and unconditioned (foot shock, 0.2 mA, 10 s) stimuli.Sessions were repeated for 5 consecutive days.The following parameters were considered: frequencyand latency of avoidances (shuttle behavior occurring Psychopharmacology  during light time), frequency and latency of escapes (shuttle behavior occurring when the foot shock was on), frequencyof failures (remaining in the start compartment until theshock was turned off), and inter-trial crossings (repeated passing from one compartment to the other during the trialregardless of the light or the foot shock).Statistical analysisMetric unit results were compared by one-, two-, or three-way analyses of variance (ANOVAs) followed by post hocmultiple comparisons using Duncan ’ s test. Differences wereconsidered significant at   p <0.05. Results Morris water mazeAs the sessions went by, all animals displayed a progressivereduction of latencies in reaching the platform (Fig. 2a). Atwo-way ANOVA (treatment × session) on latency valuesrevealed significant treatment (  F  (1,18)=4.1;  p <0.05) andsession (  F  (9,162)=35.1;  p <0.00001) effects. Interactionwas also significant (  F  (9,162)=2.14;  p <0.02). As indicated by post hoc comparisons, at the start of the MWM task there were no significantly different latency values betweenthe two groups of animals. However, in the next two place Isessions the treated animals displayed lower latencies thanthe controls. Interestingly, a significant difference betweengroups was also found in the first session of the cue phase,where the treated animals reached the visible platform morequickly than the controls. A two-way ANOVA (treatment ×session) on the total distance swum in the pool to reach the platform revealed no significant treatment effect (  F  (1,18)=0.01;  p  n.s.), but the session effect was highly significant (  F  (9,162)=27.33;  p <0.00001; Fig. 2 b). Interaction wasalso significant (  F  (9,162)=3.24;  p <0.001). The meanvelocity of the treated animals was 27.14 cm/s, and that of the controls 22.7 cm/s (one-way ANOVA:  F  (1,18)=15.07;  p <0.001). A two-way ANOVA (treatment × session)on velocity values revealed significant treatment (  F  (1,18)=14.99;  p <0.001) and session (  F  (9,162)=3.38;  p <0.001)effects. Interaction was also significant (  F  (9,162)=2.41;  p <0. 01). Post hoc comparisons revealed significant increases in velocity values during the 2nd and 3rd sessionsof place I. Thus, the latency reduction seemed to be relatedto differences in velocity rather than to total distances.Interesting results were found when the distance traveled inthe peripheral annulus was analyzed. Although all animalstended to reduce their percentage of peripheral swimming progressively (  F  (9,162)=5.28;  p <0.00001) as the sessionswent by, the treated animals swam in the peripheral sectorssignificantly less than the controls (  F  (1,18)=46.5;  p <0.00001) throughout the task. Interaction was not signifi-cant (  F  (9,162)=1.38;  p  n.s.; Fig. 2c).To obtain information about the localizatory knowledgeon platform position gained by the animals as the sessionswent by, the heading angles were calculated. Once again,the two experimental groups exhibited the same headingangles at the beginning of the task; however, from the endof place I the treated animals displayed more pointedlocalizing behavior, which was maintained throughout thetask (Fig. 2d). A two-way ANOVA revealed significant treatment (  F  (1,18)=6.41;  p <0.02) and session (  F  (9,162)=9.35;  p <0.00001) effects. Interaction was not significant (  F  (9,162)=1.75;  p  n.s.). 020406080100   p  e  r  c  e  n   t  a  g  e 0306090        d     e     g     r     e     e     s ***   m  e  a  n   t   i  m  e   (  s  e  c   ) Place ICuePlace IIPlace ICuePlace II 0400800IIIIIVVVIVIIVIIIIXX sessionssessions   m  e  a  n   d   i  s   t  a  n  c  e   (  c  m   ) **** MORRIS WATER MAZE DC 020406080 *** 1200IIIIIIIVVVIVIIVIIIIXXII **** a Latencies c Peripheral distance b  Total distance d Heading angles Fig. 2  Effects of chronic donepezil treatment on the MWM perform-ances of healthy young rats. Mean escape latencies to reach the platform ( a) , total distance swum in the whole arena ( b ), percentage of distance swum in a 20-cm peripheral annulus ( c ), and heading angles( d ) displayed by the two experimental groups are depicted. In this andin the following figures,  D  donepezil-treated animals,  C   controlanimals.  Asterisks  indicate post hoc comparisons (Duncan ’ s test) between groups. *  p <0.05, ***  p <0.001.  Vertical bars  indicate SEMPsychopharmacology
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