Neurobiology of Learning and Memory 96 (2011) 272–279

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Neurobiology of Learning and Memory

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Contribution of the parafascicular nucleus in the spontaneous objectrecognition task

Edwin Castiblanco-Piñeros, Maria Fernanda Quiroz-Padilla ⇑,Carlos Andres Cardenas-Palacio, Fernando P. CardenasLaboratorio de Bases Biológicas del Comportamiento, Facultad de Psicología, Universidad de la Sabana, Bogotá, ColombiaLaboratorio de Neurociencias y Comportamiento, Departamento Psicología, Universidad de los Andes, Bogotá, Colombia

a r t i c l e i n f o

Article history:Received 24 February 2011Revised 11 May 2011Accepted 13 May 2011Available online 23 May 2011

Keywords:Intralaminar thalamic nucleiNMDA lesionsSpontaneous object recognition taskPrefrontal cortexRelational memory

1074-7427/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.nlm.2011.05.004

⇑ Corresponding author. Address: Facultad de PSabana. Campus Universitario del Puente del ComúnDC, Colombia. Fax: +57 861 2721.

E-mail address: mariaqp@unisabana.edu.co (M.F. Q

a b s t r a c t

The parafascicular (PF) nucleus, a posterior component of the intralaminar nuclei of the thalamus, is con-sidered to be an essential structure in the feedback systems of basal ganglia–thalamo-cortical circuitscritically involved in cognitive processes. The specific role played by multimodal information encodedin PF neurons in learning and memory processes is still unclear. We conducted two experiments to inves-tigate the role of the PF in the spontaneous object recognition (SOR) task. The behavioral effects of pre-training rats with bilateral lesions of PF with N-methyl-D-aspartate (NMDA) were compared to vehiclecontrols. In the first experiment, rats were tested on their ability to remember the association immedi-ately after training trials and in the second experiment after a 24 h delay. Our findings provide evidencethat PF lesions critically affect both SOR tests and support its role in that non-spatial form of relationalmemory.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

The parafascicular nucleus (PF) in rats and the centromedianparafascicular complex (CM–PF) in primates and other mammalsare the so-called posterior intralaminar nuclei (pIL) of the thalamus.These nuclei are a critical component of the ascending reticular acti-vating system (ARAS) and the basal ganglia–thalamocortical circuit(Groenewegen & Berendse, 1994; Parent & Parent, 2005; Van derWerf, Witter, & Groenewegen, 2002). In general, damage to intra-laminar nuclei has been associated with signs of amnesia (Bailey &Mair, 2005; Mitchell & Dalrymple-Alford, 2005; Newman & Burk,2005; Quiroz-Padilla, Marti-Nicolovius, & Guillazo-Blanch, 2010;Savage, Castillo, & Langlais, 1998; Van der Werf, Jolles, Witter, & Uy-lings, 2003), and functions as arousal (Mancia & Marini, 1995;Marini, Tredici, & Mancia, 1998; Shirvalkar, Seth, Schiff, & Herrera,2006; Steriade & Deschenes, 1984; Van der Werf et al. 2002), atten-tion (Bailey & Mair, 2005; Burk & Mair, 2001; Kinomura, Larsson,Gulyas, & Roland, 1996; Minamimoto & Kimura, 2002; Newman &Burk, 2005; Newman & Mair, 2007) and pain (Cheng et al., 2009;Dupouy & Zajac, 1997; Gao et al., 2010; Liu, Qiao, & Dafny, 1993;Vogt, Hof, Friedman, Sikes, & Vogt, 2008), in humans and otheranimals.

ll rights reserved.

sicología, Universidad de la, Autopista Norte de Bogotá,

uiroz-Padilla).

Single-axon tracing studies have indicated that virtually all PFneurons project to both cerebral cortex and striatum (Deschenes,Bourassa, Doan, & Parent, 1996). Anatomically, the PF is a majorsource of excitatory projections to striatum and prefrontal cortices(PFC), mainly prelimbic, and less abundantly to cingulate regions(Berendse & Groenewegen, 1991; Deschenes et al., 1996; Marini,Pianca, & Tredici, 1996; Parent & Parent, 2005; Smith, Raju, Pare,& Sidibe, 2004; Vercelli, Marini, & Tredici, 2003). In relation to stri-atum projections, the lateral PF projects to the putamen, the lateralglobus pallidus and more diffusely to dorsolateral caudate, areasthat are related to sensory-motor function. The medial PF projectsto the ventral pallidus and in lesser amounts to the accumbens andolfactory tubercle, regions both related to associative-limbic func-tions (Otake & Nakamura, 1998; Smith et al., 2004; Van der Werfet al., 2002). The cognitive deficits observed after PF lesions arethought to arise from the combined deafferentation of PFC and stri-atum targets (Van der Werf et al., 2003). The effect of PF manipu-lations on memory functions should be specifically related to theneural systems innerved. The PF has a strategic location to be con-sidered an excellent candidate for investigating memory processes.However, before concluding PF participation in non-spatial rela-tional memory it is necessary to rule out any possible deficits inlocomotion induced by the excitotoxic lesion. So an open field loco-motion assessment was carried out before the spontaneous objectrecognition (SOR).

The link between PF and cognitive processes has traditionallybeen investigated through rat studies of radiofrequency (Nyakas,

E. Castiblanco-Piñeros et al. / Neurobiology of Learning and Memory 96 (2011) 272–279 273

Veldhuis, & De Wied, 1985; v Wimerdma Greidanus, Bohus, & deWied, 1974) or electrolytic (Cardo & Valade, 1965; Guillazo-Blanch et al., 1995; Massanes-Rotger, Aldavert-Vera, Segura-Torres, Marti-Nicolovius, & Morgado-Bernal, 1998; Redolar-Ripollet al., 2003; Shapovalova, Pominova, & Dyubkacheva, 1997;Thompson, Kao, & Yang, 1981; Tikhonravov, 2000) lesions. Thereare only three studies (M’Harzi, Jarrard, Willig, Palacios &Delacour, 1991; Quiroz-Padilla, Guillazo-Blanch, Vale-Martinez, &Marti-Nicolovius, 2006; Quiroz-Padilla, Guillazo-Blanch, Vale-Martinez, Torras-Garcia, & Marti-Nicolovius, 2007) evaluating theeffects of excitotoxic lesions restricted to PF area in four differentmemory tasks: two of procedural memory (the appetitively moti-vated odor discrimination and the aversively motivated two-way ac-tive avoidance tasks) (Quiroz-Padilla et al., 2007), and two ofrelational memory (the object recognition and the social transmis-sion of food preference (M’Harzi et al., 1991; Quiroz-Padilla et al.,2006). The lack of knowledge of the specific participation of PF inmemory processes urges the need to continue researching on its rolein cognitive function.

The main purpose of the present study is to identify the contri-bution of the PF nuclei to non-spatial relational memory by usingthe SOR task. Ennaceur and Delacour (1988) proposed differentialpatterns of exploration for familiar and novel objects maybe as aresult of the natural tendency to explore novel objects. An impor-tant advantage of SOR is that no aversive/stressful stimuli areneeded. Other feature of the task is the requirement of perirhinal(PRh) cortex integrity to discriminate novel objects (Aggleton,Albasser, Aggleton, Poirier, & Pearce, 2010; Brown & Aggleton,2001; Clark & Squire 2010; Murray & Bussey, 1999; Winters,Saksida, & Bussey, 2008). Previous studies (Bussey, Duck, Muir,and Aggleton, 2000; Ennaceur, Neave, & Aggleton, 1996) reportedimpaired object recognition memory in the SOR task followingneurotoxic PRh damage in rat. The PRh cortex is responsible forfamiliarity discrimination however, this kind of memory involvesinteraction with other structures outside the medial temporal lobeas PFC. According to this idea we can speculate that PF lesionsdecreased the cortical activation required to discriminate signifi-cant events in the SOR task, therefore memory can be affected. Thiscould presumably be a result of PF deafferentation of importantsystem components of basal ganglia–thalamic-cortex circuit, par-ticularly PFC (Berendse & Groenewegen, 1991; Deschenes et al.,1996; Heidbreder & Groenewegen, 2003; Hsu & Price, 2007;Macchi & Bentivoglio, 1986; Marini et al., 1996; Otake &Nakamura, 1998; Parent & Parent, 2005; Sadikot & Rymar, 2009;Smith et al., 2004; Van der Werf et al., 2002; Vercelli et al., 2003;Vertes, 2004).

The finding of impairments in two different retention delays:immediately and/or 24 h after training, in rats submitted to bilat-eral lesion with N-methyl-D-aspartate (NMDA) should supportthe contribution of PF in the memory process of the SOR task.

2. Materials and methods

2.1. General methods: Experiments 1 and 2

Both experiments were performed taking into account theethical and legal standards required for laboratory animal researchin Colombia (Estatuto Nacional de Protecciòn Animal – Law 84 of1989 and Resolution Number 008430 of 1993 of the Ministerio dela Salud).

Fig. 1. Photograph of the objects used in the spontaneous recognition task.

2.1.1. ApparatusA cylindrical open field (55 � 60 cm; diameter and height) was

used to assess locomotion before and 6 days after surgery. The SORtask was carried out in an open box made of wood

(60 � 60 � 40 cm high) with the floor covered with an acrylicsurface. The objects were made of plastic, aluminum or glass withdifferences in color, height (between 8 and 15 cm), weight (between55 and 226 g), shape and texture (Fig. 1). After each session, theobjects and surface of the open box were cleaned with acetic acid(10%) to avoid olfactory cues.

2.1.2. Analysis of behaviorThe animals were videotaped while performing both the

locomotion tests and the SOR task. Object exploration was opera-tionally defined as physical contact with the object throughforepaws or snout. Distances >2 cm, as well as sitting or standingon the object with the nose facing the ceiling were not consideredexploratory behavior. For the SOR task training the dependentvariables were: the mean time spent exploring the objects andthe mean average frequency of contact with the objects. For theSOR task test, a discrimination ratio was used (discriminationratio = novel object exploration/total interaction with all theobjects) (see Bevins & Besheer, 2006).

The frequency of following behaviors were analyzed in the loco-motion tests: crossing the lines that divided the cylindrical openfield bearing in mind that the animal’s hind paws crossed the linebetween each quadrant; standing on their hind paws in the field(rearing); licking or scratching themselves (grooming); elongationof the head and shoulders followed by retraction to its originalposition (stretching); and remaining stationary showing piloerec-tion (freezing). Each of these behaviors was measured before andafter of the surgery, and a delta score was obtained; this delta scoreis defined as: frequency of the behavior before surgery - frequencyof the behavior after surgery. These dates were collected with thesoftware X-Plo-Rat 2005 1.1. (Taverna-Chaim & Morato, 2008).

2.1.3. Measurements and statistical analysisAll statistical analyses were carried out by using SPSS for

Windows, version 17.0. The comparison of the averages of thegroups for all SOR task measures was done using Student t testfor independent samples. In order to compare the results in loco-motion tests, a Student t test for independent samples was doneusing delta scores for each behavior (crossing, rearing, groomingand stretching). Other analyses were carried out to assess the effectof bilateral PF lesions on object exploratory behaviors and there-fore discard any effect of lesion on the SOR task. In this regard,General Lineal Model for repeated measures was used with corre-sponding contrasts (simple for between-groups effects and polyno-mial and repeated for within-group effects), in which theindependent variables were Group (Lesion and Vehicle) and the

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dependent variables were: approaches 62 cm, contact with fore-paws, and snout. In the statistical procedures used in this studyan alpha of p 6 0.05 were assumed, with a reliability of 95%.

2.2. Experiment 1

2.2.1. SubjectsTwenty-four male Wistar rats obtained from Inmunopharmos

laboratories were used in the experiment. At the beginning of thestudy the mean age of the animals was 98 days (SE = 3.87) andmean weight of 291.48 g (SE = 8.09). Throughout the period ofthe experiment the animals were housed individually in plastic-bottomed cages (48 � 38 � 20 cm) with sawdust bedding. Duringall stages of the investigation the animals were kept under con-trolled temperature settings (20–24 �C), humidity (40–70%) and a12 h light–dark cycle (lights on at 8:00 a.m.). All experimental pro-cedures were performed during artificial light cycle. During theexperiment the animals were supplied with food and water ad libi-tum, and their weight and cleanliness were monitored.

2.2.2. Surgical procedureBefore surgical procedure, the animals were randomly assigned

to Lesion or Vehicle groups. Rats were anaesthetized withintraperitoneal injection of ketamine hydrochloride (ketamine50�, Holliday-Scott SA, 75 mg/kg) and xylazine (Seton�, Calier;10 mg/kg), and placed in a stereotaxic instrument (Stoelting Co.,Model 51725) with the incisor bar set 3.3 mm below the interauralline. Before the skin incisions, local anesthesia was administeredby subcutaneous application of lidocaine (Roxicaina 1%�, RopsohnTherapeutics Ltda). The skull was exposed through a midline inci-sion and leveled along the bregma-lambda axis. Two holes weredrilled in the cranium of the animals and a 26-gauge needle Ham-ilton syringe was lowered to the targets according to the followingstereotaxic coordinates (Paxinos & Watson, 1997): anteroposterior(AP) – 4.1 mm from bregma, lateral (L) ± 0.6 mm from midline, anddorsoventral (DV) – 6.1 mm and – 5.6 mm from the skull surface.Before the infusion, the needle was left in place for 30 s. Animalsbelonging to the lesion group were infused bilaterally at the PF nu-cleus the excitotoxin NMDA (Sigma–Aldrich�, 0.15 M in sterilephosphate-buffered, saline, pH 7.4). A digital microinjector (Stoel-ting Quintessential Injector, model 5331) was used for allinfusions. The total volume of infusion for each hemisphere was1.2 ll. At the first depth 0.8 at a rate of 0.133 ll per minute wereinfused. While 0.4 at a rate of 0.1 ll per minute were infused atthe second depth. The first infusion was made in the DV deepercoordinate (DV – 0.61 mm). The needle was allowed to sit for10 min before being raised to avoid the spread of NMDA up theneedle tract. The scalp was then sutured and a topical antisepticwas administered to prevent infections (Isodine�, BoehringerIngelheim S.A.). Throughout the procedure, the animals bodytemperature was kept constant with a thermal blanket. Vehiclerats underwent the same procedure, except that sterile phos-phate-buffered saline was infused.

2.2.3. Behavioral tasksIn the first phase of the experiment, each rat was kept in indi-

vidual plastic cages. In order to animals get familiarized to theinvestigator, all rats were handled for 5-min each day for a periodof 4 days. On the fifth day the locomotion pre surgery test wasdone by placing the animals in the cylindrical open field for5 min. Then the rats had 2 days without treatment before the sur-gery. All animals were allowed to recover for 7 days previous tolocomotion post surgery test, which was performed for 5 min inthe same cylindrical open field used in the pre surgery tests. Nextday, the SOR task was applied to the animals. First, each rat had ahabituation session for five minutes in the open field (above

described) without objects. Then, three 5-min training trials weredone, with inter-trial intervals of 2 min. In each trial, the animalswere placed in the corner of the open field where there was notobject, looking at the vertex of the walls. All other three cornershad a different object. These three objects were the same in allthree trials for each subject, but among trials the objects wererotated taking care of do not to repeat the location, to reduce placepreferences. The test session took place two minutes after comple-tion of training trials and lasted five minutes. In this session, one ofthe three objects previously used (familiar objects) was replacedby a new object with which the animal had never had contact.

2.2.4. HistologyAfter the behavioral procedures, all animals were deeply anes-

thetized with an overdose of sodium pentobarbital (Euthanex�,Invet s.a., 200 mg/kg) and were perfused transcardially with 0.9%saline followed by 10% formalin-saline. Brains were extractedand post-fixed in formalin for at least 24 h and then submergedin a 30% sucrose solution prior to sectioning. Coronal 40-lmsections were cut out on a cryostat (Leica Microsystems�, modelCM1850), mounted and stained with Cresyl violet. The sectionswere examined under a light microscope (Nikon� Eclipse 80i)and microphotographs were taken with a digital camera (NikonDs-Fi1). The tissue evaluation was done using a blind strategy:two observers who were not aware of the behavioral results inde-pendently examined the brain sections. The lesions were recon-structed on standardized sections of the rat brain (Paxinos &Watson, 1997).

2.3. Experiment 2

2.3.1. SubjectsTwenty-four male Wistar rats obtained from Inmunopharmos

laboratories were used in the experiment. At the beginning of thestudy the mean age of the animals was 100 days (SE = 4.7) andmean weight of 295,98 g (SE = 12.16). During all stages of thestudy, the animals were handled with the same parameters formaintenance and control described in Experiment 1.

2.3.2. Surgical procedureBefore surgical procedure, rats were randomly assigned to

LESION or VEHICLE groups. The surgical protocol was the sameas in Experiment 1,

2.3.3. Behavioral tasksThe experimental conditions were similar to those used in

Experiment 1, except that the test session took place 24 h aftercompletion of training trials.

2.3.4. HistologyThe histological procedures and the criteria applied were the

same as in Experiment 1.

3. Results

3.1. Histology

Fig. 2A and B shows the maximum and minimum extents ofsuccessful PF lesions for each plate (figures modified from Paxinos& Watson, 1997). Final sample included only rats with bilaterallesions P40% in the PF, damage that extended from �3.80 mm to�4.52 mm posterior to bregma (Paxinos & Watson, 1997). Thelesions were characterized by loss of neurons, glyosis and tracesof necrosis adjacent to the tracks made by the needle (Fig. 3A–D). The axons in the fasciculus retroflexus remained visible in all

Fig. 2. Schematic drawing of the smallest (gray area) and the largest (striped area) PF lesions in successive anterior/posterior coronal sections in Experiment 1 (A) andExperiment 2 (B). The extent of the lesions is superimposed on figures modified from Paxinos and Watson (1997).

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subjects included in the final sample. For both experiments, histo-logical analyses were performed by two independent blindobservers.

3.2. Behavior

3.2.1. Experiment 1The final sample was made up of 18 rats distributed into LESION

(n = 8) and VEHICLE (n = 10) groups.

3.2.1.1. Locomotion test. In order to assess any possible effect of sur-gery upon locomotion and motility, Student t tests were conductedon delta scores (differences between data at the pre and post surgerysessions) (see page 8 for description) for crossings, grooming, rear-ing, stretching and freezing. Independent samples t-test analysisshowed that there were not differences between the groups in deltascores for crossings [t(16) = �0563; p = 0581], rearing [t(16) = �0.07;p = 0945], grooming [t(16) = �0989; p = 0337] and stretching[t(16) = 0243; p = 0811]. None of the subjects regardless of the groupshowed immobility or piloerection (freezing). These results indicatethat lesions did not affect the locomotor activity of animals.

3.2.1.2. Spontaneous object recognition task. Fig. 4A shows thediscrimination ratio of the novel object for all the groups in thetest. Student t-test showed significant differences between thegroups in the discrimination ratio [t(16) = �2162; p = 0.0461

< 0.05]. This finding suggests that lesions in PF change the normalfunctioning of object recognition memory when performing thetest 2 min. after training. There were no significant differences be-tween groups in the total time exploring objects during the trialsessions [t(16) = 1983; p = 0065], in the total frequency of contactwith objects in the trial sessions [t(16) = 0587; p = 0566] and inthe total time exploring objects during the test session[t(16) = �1277; p = 0220] (Fig. 4 B). General lineal model for re-peated measures showed no significant differences in the explora-tion time [F(1,16) = 3935, P = 0.65], as well as the frequencies ofapproaches 62 cm [F(1,16) = 0136, P = 0, 717], and contacts withforepaws [F(1,16) = 2947, P = 0, 105], and the snout [F(1,16) = 0330,P = 0, 574].

3.2.2. Experiment 2The final sample was made up of 17 rats distributed into LESION

(n = 7) and VEHICLE (n = 10) groups.

3.2.2.1. Locomotion test. Independent samples t-test analysisshowed that there were not differences between the groups in deltascores for crossings [t(15) = 0245; p = 0810], rearing [t(15) = �0095;p = 0926], grooming [t(15) = �0373; p = 0715] and stretching[t(15) = �0967; p = 0349]. None of the subjects regardless of thegroup showed immobility or piloerection (freezing). The findingsare consistent with those found in Experiment 1, confirming that

Fig. 3. Photomicrographs of Cresyl violet-stained coronal sections (40 lm thick), about 4.30 mm posterior to bregma, showing the appearance of a typical NMDA bilateral PFlesion (A) and (B) compared to a vehicle-infused subject (C) and (D). Scale bar = 1.00 mm in (A), (B), (C) and (D). PF, parafascicular; fr, fasciculus retroflexus.

Fig. 4. Experiment 1, (A) Discrimination ratio of the novel object two minutes after training trials for both groups. (B) Differences between groups in the total time exploringobjects during the trial sessions (⁄P < 0.05).

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the PF nucleus lesions did not affect the locomotor activity of theexperimental subjects.

3.2.2.2. Spontaneous object recognition task. Analysis of the discrim-ination ratio revealed that there are significant differencesbetween the control group and lesion group [t(15) = �4890;p < 0.001] (Fig. 5 A). This result suggests that lesions in PF producea deficit in object recognition memory at 24 h. Moreover, Therewere no significant differences between groups in the total timeexploring objects during the trial sessions [t(15) = �0238;p = 0815], in the total frequency of contact with objects in the trialsessions [t(15) = 0.091; p = 0929] and in the total time exploringobjects during the test session [t(15) = 1707; p = 0108] (Fig. 5 B).

To complement the analysis of exploratory behavior, generallineal model for repeated measures was conducted for the trial ses-sions. There were no significant differences between groups[F(1,15) = 0057, P = 0815]. This result supports the evidence thatthe PF nucleus lesion did not affect exploratory behavior. In theanalysis of object exploration during the trial phases there wereno differences groups for the frequency of approaches 6 2 cm[F(1,15) = 0031, P = 0863], the frequency of contacts with snout[F(1,15) = 0.41, P = 0842], and with forepaws [F(1,15) = 1282,P = 0275]. These results, together with those obtained in Experi-ment 1, suggest that bilateral lesions of the PF nucleus did notaffect object exploration and, therefore, did not influence animalperformance in SOR task.

Fig. 5. Experiment 2, (A) Discrimination ratio of the novel object 24 h after training trials for both groups. (B) Differences between groups in the total time exploring objectsduring the trial sessions (⁄⁄⁄P < 0.001).

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4. Discussion

The present study evaluated the effects of bilateral NMDAlesions of the PF nucleus on a SOR task. In this paradigm, rats wererequired to remember familiar objects immediately or 24 h after.The main results showed that PF lesions impair SOR at both reten-tion delays. These findings suggest that PF is involved in the objectrecognition memory.

Regarding to exploration or motor behavior in both experi-ments, there were no significant differences between groups incrossing, rearing and stretching frequency in the open field. Therewere no significant differences between groups in both frequencyor time spent exploring the sample objects in the SOR task or inother exploratory behaviors (approaches 6 2 cm, contact with fore-paws and snout). No significant changes were observed in the over-all exploration time in the test session.

Altogether, these findings dismiss the incidence of variablesother than memory and maybe learning on the performance of le-sioned animals in the SOR task. The explanations for the results ob-tained in this investigation are based on the fact that the PFnucleus participates in the ARAS, as well as in the basal ganglia–thalamic-cortex circuit; both systems related to generalized activa-tion or arousal (Jones, 2003; Quiroz-Padilla et al., 2010), attention(Heidbreder & Groenewegen, 2003; Hulme, Whiteley, & Shipp,2010; Matsumoto, Minamimoto, Graybiel, & Kimura, 2001; Mina-mimoto, Hori, & Kimura, 2005; Minamimoto & Kimura, 2002; Rae-va, 2006; Smith et al., 2004), learning and memory (Guillazo-Blanch et al., 1995; Massanes-Rotger et al., 1998; Quiroz-Padillaet al., 2006; Quiroz-Padilla et al., 2007).

According to this evidence it is hypothesized that the PF lesionsdecreased the cortical activation required to discriminate signifi-cant events in the SOR task, therefore memory can be affected. Thiscould presumably be a result of PF deafferentation of importantsystem components of basal ganglia–thalamic-cortex circuit, par-ticularly PFC (Berendse & Groenewegen, 1991; Deschenes et al.,1996; Heidbreder & Groenewegen, 2003; Hsu & Price, 2007; Mac-chi & Bentivoglio, 1986; Marini et al., 1996; Otake & Nakamura,1998; Parent & Parent, 2005; Sadikot & Rymar, 2009; Smithet al., 2004; Van der Werf et al., 2002; Vercelli et al., 2003; Vertes,2004).

There are some reports on memory impairment after lesions inthe intralaminar nuclei, including the PF nucleus, very similar tothose caused by hypofunction of PFC (Burk & Mair, 1998; Vander Werf et al., 2003). Indeed, there have been reported deficitsin working memory after thalamic damage, including the PF

nucleus, in different animal models used to evaluate this kind ofmemory (Burk & Mair, 1999; Langlais & Savage, 1995; Porter, Koch,& Mair, 2001; Savage et al., 1998). Furthermore, Smith, Country-man, Sahuque, and Colombo (2007) found that the acquisitionand remembering in the socially transmitted food preference task(model of non-spatial relational memory) induces activation of themedial prefrontal cortices (PFCm), specifically in the prelimbic andinfralimbic cortex.

Concerning to object recognition memory, there are studiesshowing connection between this kind of memory and PFCmcortex, especially with the prelimbic area (Barker, Bird, Alexander,& Warburton, 2007; Christoffersen et al., 2008; DeVito &Eichenbaum, 2010; Levallet, Hotte, Boulouard, & Dauphin, 2009).Barker et al. (2007), suggest that object recognition memory in ratsrequires judgments on the previous occurrence of stimuli made onthe basis of the relative familiarity with each object. It has beenproposed that PFCm may be related to the discrimination requiredfor object recognition test in order to establish an order or timingof the objects presentation (Dere, Huston, & De Souza Silva, 2007).

Our results contrast with those reported by M’Harzi, et al.(1991), who found no impairment on object recognition memoryafter ibotenic acid lesion of PF in rats. This divergence in resultsmay be due to some methodological differences such as the kindof the object recognition task used. In the experiments reportedhere, three trials before the test session were done using three ob-jects per session, whereas in the study of M’Harzi et al. (1991),there was only one trial before test and two objects per session.Our variation increases the difficulty of the SOR task, demandingso a greater degree of arousal and attention in order to properly ac-quire and store it. Moreover, this study adds to emerging evidenceof the involvement of PF non-spatial relational memory, as wassuggested in the study reported by Quiroz-Padilla et al. (2006),and complements the work involving PF with other kinds of rela-tional memory (Bailey & Mair, 2005; Burk & Mair, 1998; Lopezet al., 2009; Porter et al., 2001; Savage, Sweet, Castillo, & Langlais,1997; Savage et al., 1998; Van der Werf et al., 2003).

Given that an optimal level of alertness is required for learningand behavior to occur, and that the PFCm is critically involved inalertness (Robbins & Everitt, 1995; Valdés & Torrealba, 2006), it ispossible that the deficit observed in object recognition test couldbe the result of alterations in glutamatergic neurons projecting fromPF to PFCm, particularly to prelimbic cortex – region directly relatedto ARAS and relational memory processes. The deafferentation of theprelimbic cortex could have decreased the cortical activation re-quired for encoding information and memory formation. This idea

278 E. Castiblanco-Piñeros et al. / Neurobiology of Learning and Memory 96 (2011) 272–279

is consistent with the findings reported by DeCoteau, McElvaine,Smolentzov, and Kesner (2009), according to which the animals withprelimbic/infralimbic cortex lesions displayed a profound and sus-tained deficit, in a go/no-go visual discrimination task.

According to Minamimoto and Kimura (2002), PF is involved intasks requiring shifts in response patterns. Differential exploratorybehavior on near objects could be understood as a response pattern.It could be the case that PF lesioned animals failed to shift theexploratory pattern between new and familiar objects resemblingso an attentional failure leading to alterations in memory forma-tion. From this stand, it is possible that the SOR task used here couldinclude some components similar to those found in flexibility tasks(i.e. enhancement of exploration of novel object and decrease offamiliar objects exploration) and, as a result of PF lesions, the sub-ject is unable to make a behavioral shift between ongoing behaviorand appropriated responses to each kind of object.

The crucial role of PFC has been well documented in this kind ofbehavioral shifts (Egerton et al., 2008; Floresco, Block & Tse, 2008;Parsegian, Glen, Lavin, & See, 2011). As already stated by Brown,Baker, and Ragozzino (2010), the role of PF on PFC activation couldbe indirectly mediated by striatal cholinergic neurons related tobehavioral flexibility. They found that PF lesions cause a decreasein acetylcholine release which concomitantly impaired tasks requir-ing flexibility as reversal learning. This effect could be mediated bythe decreased cholinergic activity in the striatal-cortical projectionto PFC. In fact, it has been proposed that one of the functions of PF re-lates to the processing of stimuli that were previously irrelevant,requiring the selection of different actions (Brown et al., 2010).

It could be also the case that PF lesions lead to a possible atten-tion deficit interfering with the performance in object recognitiontask and not exactly a failure in cortical arousal. As above men-tioned, PF is also related to attentional processes; in fact, someauthors have proposed that the preference for novelty (tradition-ally assessed by SOR tasks) could reflect deficits in attention ratherthan alterations of relational memory (Desimone & Duncan, 1995;Gaskin et al., 2010). However, as already mentioned, in this studysome changes in the SOR task made it more complex and, there-fore, demanded greater attention for the acquisition and retentionof the task. In addition, the analysis of exploratory behavior duringthe three trials and the test session did not show any differencesbetween control group and lesioned animals, so, it is possible thatPF lesion did not imply attention deficits as the only explanationfor the memory deficits observed.

In conclusion, our results suggest that PF may be involved inmodulation of object recognition memory. The contribution of PFin memory may be linked to its role in the basal ganglia–thalamic-cortex circuit. Hence, PF damage may have reduced theactivation of specific brain regions necessary for helping animalsto cope with changing task demands.

Acknowledgments

This research was supported by funds provided by the Fondofinanciero de investigación de la Universidad de la Sabana and byFAPA-GRANT from Universidad de los Andes. The authors thankDr. Angela Trujillo and Dr. Claudio Da Cunha for their helpful com-ments. These data were presented at the 2009 European Brain andBehaviour Society Meeting (Rhodas-Grecia, 13th and 14thSeptember).

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