The “Anxiety State” and Its Relation with Rat Models of Memory and Habituation

The “Anxiety State” and Its Relation with Rat Models of Memory and Habituation

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  The “Anxiety State” and Its Relation with RatModels of Memory and Habituation Rafaela L. Ribeiro,* Roberto Andreatini,* Claudia Wolfman,† Haydee´ Viola,†Jorge H. Medina,† and Claudio Da Cunha* *  Laborato´rio de Fisiologia e Farmacologia do SNC, Dep. Farmacologia, UFPR, 81.531-990Curitiba, PR, Brazil; and  †  Instiuto de Biologia Celular, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Buenos Aires, Argentina Rats selected as “anxious”, “nonanxious,” or normal according to their behavior in anelevated plus maze were submitted to memory tasks and the densities of centralbenzodiazepine receptors in the amygdala and the hippocampus were studied. Anxiousrats exibited better retention scores in the inhibitory avoidance task while nonanxiousrats exibited worse retention scores in inhibitory and two-way active avoidance taskscompared to normal rats. No significant differences were detected in the retentionscores for habituation to an open field. Nonanxious rats presented a lower benzodiaz-epine receptor density in the hippocampus but not in the amygdala compared to theother groups. These data suggest that the benzodiazepine receptors are involved in theeffect of “anxiety” or emotional states on memory storage processes.  © 1999 Academic Press INTRODUCTION It is well established that benzodiazepines (BZ), the most prescribed anxio-lytics, cause anterograde amnesia (Bates, 1996; Cahill, Babinsky, Markow-itsch, & McGaugh, 1995; Izquierdo & Medina, 1991; Izquierdo & Medina,1997). Other GABA   A  -receptor complex-binding drugs like barbiturates (Com-missaris, 1993; Devinsky, 1995), muscimol (Izquierdo & Medina, 1991; Izqui-erdo & Medina, 1997; Shephard, 1987), and ethanol (Commissaris, 1993;Melia, Ryabinin, Corodimas, Wilson & Ledoux, 1996) also present anxiolyticand amnestic properties. This double effect is also common to competitive(Bennett & Amrick, 1987; Blanchard, Blanchard, Carobrez, Veniegas, Rodg-ers, & Sheferd, 1992; Izquierdo & Medina, 1991; Kehne, McClosky, Baron, Chi,Harrison, Whitten, & Palfreyman, 1991; Przegalinski, Tatrczynka, Derenwe-solek, & Chojnackawojcik, 1996) and noncompetitive (Bennett & Amrick, 1987;Izquierdo & Medina, 1997; Khene et al., 1991) NMDA-receptor antagonists,anxiolytic glycine-site antagonists at the NMDA receptor (Anthony & Nevins,1993; Trullas, Jackson, & Skolnick, 1989), and   -adrenergic blockers (Cahill &McGaugh, 1996; Commissaris, 1993; Ohno, Yoshimatsu, Kobayashi, & Wa- The authors thank CNPq, Andes-Vitae-Antorchas Foundation, and PRONEX for financialsupport, Dr. Roseli Lacerda for discussing the appropriate selection protocol, Miriam Angelucci forsupport in some behavioral experiments, Miguelina Levi de Stain for support in the binding assays, and Dr. Ivan Izquierdo for discussing the results.Correspondence should be addressed to Claudio Da Cunha, Laborato´rio de Fisiologia e Farma-cologia do SNC, Dep. Farmacologia, UFPR, C.P.:19.031, 81.531-990 Curitiba, PR, Brazil.Fax:  55 41 266-2042. E-mail: of Learning and Memory  72,  78–94 (1999) Article ID nlme.1998.3891, available online at on 1074-7427/99 $30.00Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.  tanabe, 1996). Conversely, the BZ-receptor antagonist flumazenil (Da Cunha,Wolfman, Huang, Walz, Koya, Izquierdo, & Medina, 1990; Herzog, Stackman,& Walsh, 1996; Wolfman, Da Cunha, Jerusalinsky, Stein, Viola, Izquierdo, &Medina,1991),BZinversereceptoragonistslikeanxiogenic  -carbolines(Com-missaris, 1993; Wichlinski & Jensen, 1996), and also  D -cycloserine, an anxio-genic glycine-site agonist at the NMDA receptor (Matsuoka & Aigner, 1996;Ohno & Watanabe, 1996; Schimitt, Coelho, Souza, Guimara˜es, & Carobrez,1995) may improve memory. These facts suggest that memory storage andanxiety processes may interact by using some common neural substrates or bymodulating each other in an adaptative way.The success obtained in the treatment of anxiety with BZ (Bates, 1996) andthe discovery of endogenous ligands to the BZ receptor in mammalian brain(Medina, Pen˜a, Piva, Wolfman, Stein, Waskowski, & Da Cunha, 1992) stronglyindicate that BZ are candidates to mediate an important role in the interactionof memory storage and anxiety processes. In a previous study we showed thatthe “degree of anxiety” induced by memory tasks in rats, evaluated by theirbehavior in an elevated plus maze, is negatively correlated with the amountsof immunoreactivity to a BZ monoclonal antibody in the amygdala and hip-pocampus (Da Cunha, Stein, Wolfman, Koya, Izquierdo, & Medina, 1992a). Wealso showed that more “anxiogenic” tasks induced changes in BZ-receptordensity in the hippocampus (Da Cunha et al., 1992a). In the present investi-gation we decided to further examine the relationship between memory andanxiety by studying how rats that behave as normal, “anxious,” or “nonanx-ious” in an elevated plus maze perform memory tasks with different degrees of stress. The possible mediation of BZ receptors in this interaction was alsoinvestigated by a study of their density in the hippocampus and amygdala of plus maze selected animals. METHODS Subjects and procedures.  Two hundred male Wistar adult rats from ourown breeding stock, weighing 200  2 g at the beginning of the experiments,were used. The animals were maintained in a temperature-controlled room(22    2°C) on a 12/12-h dark/white cycle (lights on 7:00 AM) with food andwater available ad libitum. All the behavioral experiments were conductedbetween 7:00 and 12:00 AM. During all the tests the animals were maintainedin plexiglass home cages (60  25  25 cm), and the same five cagemate ratswere maintained until the end of the behavioral experiments. During the firstweek the animals were selected according to their behavior in an elevated plusmaze. One week later the selected animals were tested in the habituation to anopen-field task for 1 additional week, 3 days later in an inhibitory (passive)avoidance task (3 more days), and 3 days later in a two-way active avoidancetask (1 more week). Thirty days later the animals were sacrificed by decapi-tation immediately after being taken out of their home cages. Brains wererapidly removed on ice and frozen at   70°C until used for membrane prepa-rations of amygdala and hippocampus samples used in the BZ-receptor-bind-ing assays. Selection of the groups in the elevated plus maze.  Two hundred animalswere tested in an elevated plus maze according to a well-known protocol79 MEMORY AND HABITUATION MODELS  (Belzung & Le Pape, 1994; Bennett & Amrick, 1987; Commissaris, 1993; Cruz,Frei, & Graeff, 1994; Da Cunha et al., 1992a; Da Cunha, Wolfman, Stein,Ruschel, Izquierdo, & Medina, 1992b; Dawson & Tricklebank, 1995; Hogg,1996; Lister, 1990; Pellow, Chopin, File, & Brieley, 1985). The most frequentlyreported parameter in the literature to describe the behavior of rats in theelevated plus maze is percentage of time spent in the open arms. In the presentstudy we opted to select the animals according to the percentage of time theyspent in the closed arms (TC) because more than 20 rats did not enter the openarms but the time they spent in the closed arms varied as a function of the timethey spent on the central platform of the maze. Three groups of 20 animalswere selected: (1) nonanxious, TC    44%; (2) anxious, TC    86%, and (3)normal rats, 62%    TC    68%. Additional selection was made excluding theanimals with lower or higher locomotion activity in the maze (number of entries into the open arms    entries into the closed arms), with 11 animalsremaining in each group.  Memory and habituation tasks.  The animals were submitted to five ses-sions of habituation to an open field, one session per day. The open fieldconsisted of a white round arena (1 m diameter, 40 cm walls) with 19 quad-rants marked by black concentric circles and lines. The apparatus is coveredwith a white curtain and illuminated by four 60-W lamps hanging on a whiteroof 80 cm above the floor. The experiments were videotaped with a cameraplaced on the roof of the apparatus. During each session the animal wasallowed to freely explore the open field for 5 min and the number of times theanimal crossed the lines and the number of rearings were computed. Thedecrease in these exploration scores was taken as a measure of retention(Netto, Dias, & Izquierdo, 1986).The inhibitory avoidance test apparatus was an automated 23  50  23-cmshuttle box (GEMINI Avoidance System, San Diego Instruments, San Diego,CA) with a frontal dark glass and a floor made of parallel 5-mm-caliberstainless-steel bars spaced 15 mm apart. The box is divided into an illuminatedcompartment and a dark compartment of the same size by a wall with aguillotine door. In the training session the animal was placed in the illumi-nated compartment facing the closed door. After the animal turned around180° the door was opened and the latency to enter the dark compartment wascomputed. Animals that took more than 20 s to enter the dark compartment inthe training session were eliminated from the experiment. After entering thedark compartment the animal received a 0.3-mA foot shock, 1 s, and wasreturned to it home cage. The test session, held 24 h later, was similar to thetraining session except that the animal received no foot shock and a limit of 300 s for latency of entries into the dark compartment was imposed. Thelatency of entry into the dark compartment in the test session was taken as ameasure of retention (adapted from Da Cunha et al., 1990).The two-way active avoidance task was conducted in the same apparatusused for inhibitory avoidance, except that the two compartments were poorlyilluminated and the door remained opened. In the training session, after 3 minofhabituation,20soundcues(1.5kHz,60dB,5s)werepairedwithsubsequent0.5-mA foot shocks (maximum duration of 5 s) until the animal crossed to theother compartment. The animal could avoid the shock by crossing to the otherside during the presentation of the sound cue. The time between each sound80  RIBEIRO ET AL.  cue presentation varied randomly ranging from 10 to 50 s. The number of avoidances was computed. The test session, conducted 24 h later, was identicalto the training one, except for a 1-min habituation time. Retention was esti-mated by an increase in the number of avoidances (Netto, Oliveira, Gianlupi,Quilfeldt, & Izquierdo, 1987).  BZ-receptor-binding assays.  Amygdala and hippocampal formation weredissected from the brains on ice and all the subsequent procedures wereperformed at 4°C. The tissues were homogenized in 10 vol of ice-cold 0.32 Msucrose and centrifuged for 10 min at 1000  g.  The supernatant was separated,and the pellet was rehomogenized and again centrifuged as above. The twosupernatants were pooled and centrifuged at 40,000  g  for 30 min to give a pelletcorresponding to a crude synaptosomal membrane fraction. These membraneswere homogenized in 20 vol of 25 mM Tris-HCl buffer, pH 7.3, centrifuged at40,000  g  for 30 min, and stored at  70°C until utilized.Central-type BZ receptors were measured using [ 3 H]FNZ binding ([ 3 H]FNZ,84 Ci/mmol, DuPont, NEN) as previously described (Medina & De Robertis,1985). Briefly, for each assay, triplicate membrane samples containing 0.2 mg protein, determined by the Lowry method (Lowry, Rosebrough, Far, & Ran-dall, 1951), were suspended in 1 ml Tris-HCl buffer, pH 7.3. The incubationwas carried out at 4°C for 60 min with 9 nM [ 3 H]FNZ. Nonspecific binding determined in a parallel incubation in the presence of 3   M FNZ amounted to10–20% of the total. Binding was terminated by rapid filtration through GF/Bfilters with three washes of 6 ml each of the incubation buffer. The filters weredried and transferred to vials containing a scintillation cocktail (2.5% diphe-nyloxazole-xylene) and the radioactivity was measured with 40% efficiency. Statistics.  Frequency distribution histograms of the time spent in theclosed arms were constructed and compared to a normal distribution (see Fig.1). Twenty animals were selected from a sample of 200 according to theirposition in this histogram: closer to the mean, lower than the mean  SD, andhigher than the mean    SD. A second selection was made according to thedistribution of the animals in the “total number of entries” histogram (notshown). This time 9 animals with scores highly different from the mean wereexcluded. All the scores of selected animals in the elevated plus maze test werecompared by analysis of variance (ANOVA) followed by the Duncan test, withthe exception of the inhibitory avoidance data. The total number of entries intothe closed    open arms was taken as a covariate in the analysis of thepercentage of time and entries into the closed and open arms of the maze.Inhibitory avoidance data were analyzed by one-way Kruskal-Wallis analysisof variance followed by the Mann-Whitney  U   test. RESULTS Three groups of 20 rats each were selected from a sample of 200 animalsaccording to their scores in an elevated plus maze. Figure 1 shows the fre-quency distribution of the animals according to TC (percentage of time spent inthe closed arms of the maze). Animals in the three groups matched thefollowing scores: normal, 62  TC  68; nonanxious, TC  44; anxious, TC  86. One of the selected animals died during the subsequent experiments.Further selection was done by eliminating 8 or 9 animals from each group81 MEMORY AND HABITUATION MODELS  according to their ambulation in the maze (total number of entries in theclosed  open arms): the scores for each group were ordered and the animalswith the lower and upper scores were discarded. Table 1 shows the scores of the 11 selected animals per group in the elevated plus maze. The three groupsdiffered in the total number of entries into the closed  open arms:  F  (2, 30)  1060.96  p  .001. One-way ANOVA considering the total number of entries asa covariate showed that, independent of this covariate, the three groupsdiffered in percentage of time spent in the closed arms (  F  (2, 20)  156.14,  p  .001), percentage of time spent in the open arms (  F  (2, 29)  21.67,  p  .001),and percentage of entries into the open arms (  F  (2, 29)  3.63,  p  .05).The scores of habituation to an open field for the three groups of animals arepresented in Fig. 2. One-way ANOVA showed that there was no significantdifference among the scores for crossing (  F  (2, 30)    .17  p    .2) and rearing (  F  (2, 30)    .15  p    .2) on the first day of habituation. Two-way ANOVA considering the day of training as a repeated measure showed a significantdecrease in the scores for crossing (  F  (4, 120)  11.61;  p  .01) and rearing (  F  (4,120)    8.26;  p    .01), denoting habituation. This analysis also showed thatthere was no difference in the scores for crossing (  F  (2, 30)    .33  p    .2) orrearing (  F  (2, 30)   .14  p    .2) among the three groups and that the “anxietystate” of the group did not alter the habituation scores (interaction withinfactors) related to crossing (  F  (8, 120)  1.12  p  .2) or rearings (  F  (8, 120)  .83  p  .2).Inhibitory avoidance results are presented in Fig. 3. Animals of the threegroups presented similar latencies to enter the dark compartment in thetraining session: mean    SEM    21.8    4.5 s (one-way ANOVA,  F  (2, 30)   1.45;  p    .2). The anxious group presented a better retention score than thenormal (  H  (2, 33)    6.84;  p    .05, Kruskal-Wallis ANOVA;  U     31,  p    .05,Mann-Whitney  U   test) or nonanxious groups ( U   24,  p  .01, Mann-Whitney U   test). Similar results were obtained in the two-way active avoidance test, asseen in Fig. 4. There was no significant difference between avoidance scores in FIG. 1.  Selection of animals according to their behavior in an elevated plus maze. Twohundred rats were tested in the maze and classified into “nonanxious”, “normal,” or “anxious”groups according to the time they spent in the closed arms of the maze. 82  RIBEIRO ET AL.
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