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Influence of the estrous cycle on the behavior of rats in the elevated T-maze
Behavioural Processes 67 (2004) 167–171
Influence of the estrous cycle on the behavior of rats in the elevated T-maze Amauri Gouveia Jr. a,∗ , Ubirajara D. dos Santos b , Fabr´ıcio E. Felisbino b , Taciana L. de Afonseca a , Gabriela Antunes a , Silvio Morato c a
Laboratório de Psicobiologia e Psicopatologia Experimental, Departamento de Psicologia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Av. Edmundo Carrijo Coube, s/n, 17064-032 Bauru, SP, Brazil b Universidade do Sul de Santa Catarina, Av. José Acácio Moreira, 787, 88704-900 Tubarão, SC, Brazil c Faculdade de Filosofia, Ciˆ encias e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14040-901 Ribeirão Preto, SP, Brazil Received 17 October 2003; received in revised form 18 March 2004; accepted 19 March 2004
Abstract The elevated T-maze is an animal anxiety model which can discriminate between anxiety-like and fear-like behaviors. The estrous cycle is an important variable of the response in animal anxiety tests and is known to affect other models. The aim of the present study was to investigate the influence of the estrous cycle on behavior displayed in the elevated T-maze test. Seventeen male and 60 female rats were submitted to one session in this test, with the females being screened for the estrous cycle and divided into groups according to the various phases. The elevated T-maze had three arms of equal dimensions (50 cm × 10 cm), one enclosed by 40-cm high walls and perpendicular to the others, the apparatus being elevated 50 cm above the floor. Each rat was placed in the end of the enclosed arm and the latency for it to leave this arm was recorded. These measurements were repeated three times separated by 30-s intervals (passive avoidance). After trial 3, each rat was placed at the distal end of the right open arm and the latency to exit this arm was recorded. Whenever latencies were greater than 300 s the trial was finished. The results demonstrated females in diestrus exhibited anxiety-like behaviors while females in metaestrus behaved in a similar way as the males. There were no differences between groups in fear-like behaviors. The results also indicate the elevated T-maze to be a sensitive test to measure anxiety. © 2004 Elsevier B.V. All rights reserved. Keywords: Estrous cycle; Sexual differences; Elevated T-maze; Anxiety; Hormones; Rats
1. Introduction The defense reaction is a collection of behavioral strategies selected throughout evolution to increase the possibility of survival in dangerous situations. In mammals, the patterns of response tend to be closely similar ∗ Corresponding author. Tel.: +55-1432037868; fax: +55-1431036097. E-mail address: [email protected] (A. Gouveia Jr.).
in terms of presentation and physiological mechanisms (Blanchard et al., 1993). The defense reaction presents itself as a sequence of steps where the proximity of the aversive stimulus establishes the topographic and physiological variations which can be classified into three levels related to different neural components (Blanchard and Blanchard, 1988; Graeff, 1994). These levels are related to four strategies: freezing, escape, defensive aggression, and submission (Blanchard and Blanchard, 1988; Zangrossi, 1996). Anxiety and
0376-6357/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2004.03.018
168
A. Gouveia Jr. et al. / Behavioural Processes 67 (2004) 167–171
depression are considered to represent non-adaptive exacerbation of the defense reaction (Graeff, 1994). The structure of the elevated T-maze is similar to that of an elevated plus-maze, from which it was derived. The main differences are the absence of one of the enclosed arms in the elevated T-maze and the experimental procedures, which consist of two tests, escape and avoidance, respectively, corresponding to fear and anxiety (Zangrossi and Graeff, 1997). The first type of manipulation is inhibitory avoidance. This consists of latency for the animal to leave the enclosed arm after previous exposures to this arm. The latency tends to increase during the test. Normally, three trials are carried out at 30-s intervals, and a fourth one is carried out 48 or 72 h later to assess memory (Tomaz et al., 1993). The second type of manipulation is escape, which corresponds to the latency to go from the extremity of the open arm to the enclosed arm. Normally, experiments involving animal models utilize males because when females are used, the results are not always similar, or seem to vary abnormally with decreased consistency and increased deviations. This phenomenon is referred to as gender difference or sexual dimorphism. Sex differences have also been reported to occur in other behavioral models. Females and males perform differently in the open-field test (Kennett et al., 1986; Curzon et al., 1990; Alonso et al., 1991), the forced swimming test (Kennett et al., 1986; Alonso et al., 1991), the elevated plus-maze, the social interaction and the Vogel punished drinking tests (Johnston and File, 1991). Some studies (Blanchard et al., 1980; Blanchard and Blanchard, 1990) have found differences in defense reaction between males and females, including ultrasonic vocalizations (Blanchard et al., 1992). The difference between sexes is partially related to the estrous cycle, i.e. the fluctuation of sex hormones present in females, but not in males, which involves physiological and behavioral aspects. Estrous cycle fluctuations have been described in several animal models; during the diestrous phase there is a facilitation of acquisition of two-way conditioned avoidance, and exploratory behavior of the open arms in the elevated plus-maze (Diaz-Veliz et al., 1989, 1997). In memory experiments, the estrous cycle also seems to be important. In the Morris water-maze, females showed better performance in the spatial cues test
during the proestrus phase, and in the spatial learning test during the estrous phase (Warren and Juraska, 1997); Further, in the estrous phase there is an attenuation of the analgesia induced by electric shocks (Ryan and Maier, 1988). In other animal models, such as those involving schizophrenia, there are differences between the phases of the estrous cycle, in latent inhibition, for example (Koch, 1998). The female hormonal variation does not seem to influence feeding behavior after chromic stress (Anderson et al., 1996) although it can alter feeding behavior in the long term (Laviano et al., 1996), and it increases taste reactivity during proestrus and diestrus (Clarke and Ossenkopp, 1998) and intensifies the drinking response induced by insulin, depending on the phase (Fernandez-Trisac et al., 1998). There are no studies about the effect of female estrous cycles on behavior in the elevated T-maze. Thus, the aim of the present investigation was to study the influence of hormonal conditions on the performance of rats in the elevated T-maze.
2. Method 2.1. Animals Eighty-seven Wistar rats (17 males weighing 300–320 g and 60 females weighing 200–220 g) were obtained from the animal houses of the Universidade Estadual Paulista “Júlio de Mesquita Filho”, SP, and Universidade do Sul de Santa Catarina, SC. As they were all of the same age, males and females had different body weights. The animals were housed according to sex in groups of five per cage (41 cm × 35 cm × 33 cm) on a 12-h light/12-h dark cycle (lights from 07:00 to 19:00 h). Cages with females and cages with males were kept in the same room throughout the whole experiment. The rats were divided into groups according to sex or estrous phase (males, N = 17; females in proestrus, N = 19; females in estrus, N = 18; females in diestrus, N = 16; females in metaestrus, N = 17). 2.2. Apparatus The elevated T-maze consisted of two open arms (50 cm × 10 cm) and one enclosed arm (50 cm ×
A. Gouveia Jr. et al. / Behavioural Processes 67 (2004) 167–171
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10 cm × 40 cm) perpendicular to the opened arms; the arms were connected by a square (10 cm × 10 cm) and the apparatus was elevated 50 cm above the floor. The apparatus was made of wood. All the experiments were recorded with a video camera. 2.3. Procedure The females were investigated to establish the estrous cycle by daily collection of vaginal material for at least 5 days before the test. The stage of the estrous cycle was determined by vaginal smears, obtained between 09:00 and 11:00 h. The tails were gently raised and a cotton swab humidified in drops of saline was cautiously inserted into the vagina to obtain vaginal cytology by semicircular movements. The vaginal smears on glass slides were examined under microscope. The animals were divided into groups according to phase of the estrous cycle and sex, as follows: females in estrus, metaestrus, diestrus, or proestrus, and males. The test consisted of three inhibitory avoidance trials and one escape trial held at 30-s intervals. Between the trials the animals were placed in individual cages. The avoidance trials started when the animal was placed at the end of the enclosed arm of the T-maze. The latency for the animal to enter the open arms (a maximum of 300 s) was recorded. The escape trial was run after the inhibitory avoidance test. The animal was placed at the end of one of the open arms and the time the animal took to exit this arm was recorded. An entry was recorded when the animal placed all four paws inside one arm; likewise, an exit was recorded every time the rat removed all four paws from one arm. The experiments were conducted between 9:00 and 14:00 h.
Fig. 1. Latencies (mean ± S.E.M.) of the avoidance responses of male rats (Ma) and female rats in the hormonal phases of metaestrus (Me), diestrus (Di), proestrus (Pr), and estrus (Es) in the inhibitory avoidance test in the elevated T-maze. Trial 1 is actually the baseline: (∗) different from the respective trial 1, (◦ ) different from the males in the same trial (Duncan, P < 0.05).
3. Results The results of inhibitory avoidance learning are shown in Fig. 1. ANOVA indicated a significant effect of the hormonal state [F(4, 82) = 2.677, P = 0.037], as well as of the trials [F(2, 164) = 41.593, P < 0.001]. There was no significant interaction between the two factors [F(8, 164) = 1.840, P = 0.073]. Duncan’s test showed that the avoidance latencies in trials 2 and 3 were significantly larger than trial 1 (baseline) in the groups of males and females in metaestrus. Latencies in trial 3 were larger than trial
2.4. Statistics Data from the avoidance trials were subjected to two-way analysis of variance (ANOVA) with one independent factor (hormonal states: estrus, metaestrus, diestrus, proestrus, and males) and repetitions in the other factor (trials: 1, 2, and 3). Data from the escape trial were subjected to one-way ANOVA. These were followed by Duncan’s multiple range post hoc test whenever appropriate. In all cases, the level of statistical significance was set at 0.05.
Fig. 2. Latencies (mean ± S.E.M.) of the escape responses of male rats (Ma) and female rats in the hormonal phases of metaestrus (Me), diestrus (Di), proestrus (Pr), and estrus (Es) in the escape test in the elevated T-maze.
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1 in the groups of females in proestrus and estrus. Finally, the test showed that the latencies of females in diestrus were larger than that of males in trial 1. Fig. 2 shows the results of escape behavior. ANOVA showed no effects due to hormonal state [F(4, 82) = 0.459, P = 0.765). Females in diestrus presented smaller latencies but the differences between groups were not statistically significant.
4. Discussion The present results allow us to conclude that hormonal states constitute an important variable for the behavior studied. First, females in diestrus have presented high latencies in trial 1, indicating higher anxiety levels. These may be caused by the hormones present in this phase. In fact, there are reports of similar high anxiety levels exhibited by females in diestrus in the elevated plus-maze (Mora et al., 1996) and in an inhibitory avoidance test (Diaz-Veliz et al., 1989). Since the elevated T-maze is a good index of anxiety (Zangrossi and Graeff, 1997), the high latencies observed in our avoidance results are caused by the hormones present in the diestrus phase. In the same vein, males and females in metaestrus present the same behavioral profile in all three trials. This indicates that the effects described for diestrus were due to the hormones present in this phase since in metaestrus, characterized by low or no hormonal action, the females presented similar avoidance response latencies as the males. The elevated T-maze is also considered an index of fear (Zangrossi and Graeff, 1997) indicated by the latency to exit the open arm in the escape trial; the lower the latency the higher the fear. Our data show that the lower latencies occurred in the group of females in diestrus but the difference did not reach statistical significance. This indicates that whatever hormone action, which was able to increase anxiety, was not able to cause increases in fear. The present results stress the importance of steroid hormones in mediating the emotional response of anxiety. They also support the claim that the elevated T-maze is sensitive to the same variables that alter behavior in other anxiety models.
Acknowledgements SM was the recipient of a research fellowship from CNPq (proc. 351317/96-1). References Alonso, S.J., Castellano, M.A., Afonso, D., Rodriguez, M., 1991. Sex differences in behavioral despair—relationship between behavioral despair and open-field. Physiol. Behav. 49 (1), 69– 72. Anderson, S.M., Saviolakis, G.A., Bauman, R.A., Chu, K.Y., Ghosh, S., Kant, G.J., 1996. Effects of chronic stress on food acquisition, plasma hormones and the estrous cycle of female rats. Physiol. Behav. 60 (1), 325–329. Blanchard, D.C., Blanchard, R.J., 1988. Ethoexperimental approaches to the biology of emotion. Annu. Rev. Psychol. 39, 43–68. Blanchard, D.C., Blanchard, R.J., 1990. An ethoexperimental approach to the study of aggression. In: Morato, S., Carobrez, A.P., Lima, T.C.M. (Eds.), Neurosciences and Behavior - 2. Faculdade de Filosofia Ciˆencias e Letras de Ribeirão Preto, Ribeirão Preto, SP, pp. 107–124. Blanchard, R.J., Kleinschmidt, C.F., Fukunagastinson, C., Blanchard, D.C., 1980. Defensive attack behavior in male and female rats. Anim. Learn. Behav. 8 (1), 177–183. Blanchard, R.J., Agullana, R., McGee, L., Weiss, S., Blanchard, D.C., 1992. Sex differences in incidence and sonographic characteristics of antipredator ultrasonic cries in the laboratory rats (Rattus norvergicus). J. Comp. Psychol. 106 (3), 270–277. Blanchard, R.J., Yudko, E.B., Rodgers, R.J., Blanchard, D.C., 1993. Defense system psychopharmacology—an ethological approach to the pharmacology of fear and anxiety. Behav. Brain Res. 58 (1/2), 155–165. Clarke, S.N.D.A., Ossenkopp, K.P., 1998. Taste reactivity responses in rats: influences of sex and the estrous cycle. Am. J. Physiol. Regul. I. 274 (3), R718–R724. Curzon, G., Haaren, D.J., Kennett, G.A., 1990. Sex differences in animal models of depression. In: Spencer, P., Leonard, B. (Eds.), Antidepressants: A Thirty Years on. CSN Publications, London, pp. 101–108. Diaz-Veliz, G., Alarcón, T., Espinoza, C., Dussaubat, N., Mora, S., 1997. Ketanserin and anxiety levels: influences of gender, estrous cycle, ovariectomy and ovarian hormones in female rats. Pharmacol. Biochem. Behav. 58 (3), 637–642. Diaz-Veliz, G., Soto, V., Dussaubat, N., Mora, S., 1989. Influence of the estrous cycle, ovariectomy and estradiol replacement upon the acquisition of conditioned avoidance responses in rats. Physiol. Behav. 46 (3), 397–401. Fernandez-Trisac, J.L., Brime, J.I., López-Sela, P., SánchezGarrido, L., Costales, M., Argüelles, J., Vijande, M., 1998. Effect of castration and gonadal hormones on insulin-induced drinking. Pharmacol. Biochem. Behav. 59 (2), 521–526. Graeff, F.G., 1994. Neuroanatomy and neurotransmitter regulation of defensive behaviors and related emotions in mammals. Braz. J. Med. Biol. Res. 27 (4), 811–819.
A. Gouveia Jr. et al. / Behavioural Processes 67 (2004) 167–171 Johnston, A.L., File, S.E., 1991. Sex differences in animal-tests of anxiety. Physiol. Behav. 49 (2), 245–250. Kennett, G.A., Chaouloff, F., Marcou, M., Curzon, G., 1986. Female rats are more vulnerable than males in animal model of depression—the possible role of serotonin. J. Pharmacol. (Paris) 17 (3), 398–398. Koch, M., 1998. Sensorimotor gating changes across the estrous cycle in female rats. Physiol. Behav. 64 (5), 625–628. Laviano, A., Meguid, M.M., Gleason, J.R., Yang, Z.J., Renvyle, T., 1996. Comparison of long-term feeding pattern between male and female Fisher 344 rats: influences of the estrous cycle. Am. J. Physiol. Regul. I. 270 (2), R413–R419. Mora, S., Dussaubat, N., DiazVeliz, G., 1996. Effects of the estrous cycle and ovarian hormones on behavioral indices of anxiety in female rats. Psychoneuroendocrinology 21 (7), 609– 620.
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Ryan, S.M., Maier, S.F., 1988. The estrous cycle and estrogen modulate stress-induced analgesia. Behav. Neurosci. 102 (3), 371–380. Tomaz, C., Dickinson-Anson, H., McGaugh, J.L., Souza-Silva, M.A., Viana, M.B., Graeff, F.G., 1993. Localization in the amygdala of the amnestic action of diazepam on emotional memory. Behav. Brain Res. 58 (1/2), 99–105. Warren, S.G., Juraska, J.M., 1997. Spatial and nonspatial learning across the rat estrous cycle. Behav. Neurosci. 111 (2), 259–266. Zangrossi, H., 1996. Modelos animais de ansiedade. In: Graeff, F.G., Zangrossi, H. (Eds.), Ansiedade e Transtornos de Ansiedade. Editora Cientifica Nacional, Rio de Janeiro, pp. 85–112. Zangrossi, H., Graeff, F.G., 1997. Behavioral validation of the elevated T maze: a new animal model of anxiety. Brain Res. Bull. 44 (1), 1–5.
Influence of the estrous cycle on the behavior of rats in the elevated T-maze Amauri Gouveia Jr. a,∗ , Ubirajara D. dos Santos b , Fabr´ıcio E. Felisbino b , Taciana L. de Afonseca a , Gabriela Antunes a , Silvio Morato c a
Laboratório de Psicobiologia e Psicopatologia Experimental, Departamento de Psicologia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Av. Edmundo Carrijo Coube, s/n, 17064-032 Bauru, SP, Brazil b Universidade do Sul de Santa Catarina, Av. José Acácio Moreira, 787, 88704-900 Tubarão, SC, Brazil c Faculdade de Filosofia, Ciˆ encias e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14040-901 Ribeirão Preto, SP, Brazil Received 17 October 2003; received in revised form 18 March 2004; accepted 19 March 2004
Abstract The elevated T-maze is an animal anxiety model which can discriminate between anxiety-like and fear-like behaviors. The estrous cycle is an important variable of the response in animal anxiety tests and is known to affect other models. The aim of the present study was to investigate the influence of the estrous cycle on behavior displayed in the elevated T-maze test. Seventeen male and 60 female rats were submitted to one session in this test, with the females being screened for the estrous cycle and divided into groups according to the various phases. The elevated T-maze had three arms of equal dimensions (50 cm × 10 cm), one enclosed by 40-cm high walls and perpendicular to the others, the apparatus being elevated 50 cm above the floor. Each rat was placed in the end of the enclosed arm and the latency for it to leave this arm was recorded. These measurements were repeated three times separated by 30-s intervals (passive avoidance). After trial 3, each rat was placed at the distal end of the right open arm and the latency to exit this arm was recorded. Whenever latencies were greater than 300 s the trial was finished. The results demonstrated females in diestrus exhibited anxiety-like behaviors while females in metaestrus behaved in a similar way as the males. There were no differences between groups in fear-like behaviors. The results also indicate the elevated T-maze to be a sensitive test to measure anxiety. © 2004 Elsevier B.V. All rights reserved. Keywords: Estrous cycle; Sexual differences; Elevated T-maze; Anxiety; Hormones; Rats
1. Introduction The defense reaction is a collection of behavioral strategies selected throughout evolution to increase the possibility of survival in dangerous situations. In mammals, the patterns of response tend to be closely similar ∗ Corresponding author. Tel.: +55-1432037868; fax: +55-1431036097. E-mail address: [email protected] (A. Gouveia Jr.).
in terms of presentation and physiological mechanisms (Blanchard et al., 1993). The defense reaction presents itself as a sequence of steps where the proximity of the aversive stimulus establishes the topographic and physiological variations which can be classified into three levels related to different neural components (Blanchard and Blanchard, 1988; Graeff, 1994). These levels are related to four strategies: freezing, escape, defensive aggression, and submission (Blanchard and Blanchard, 1988; Zangrossi, 1996). Anxiety and
0376-6357/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2004.03.018
168
A. Gouveia Jr. et al. / Behavioural Processes 67 (2004) 167–171
depression are considered to represent non-adaptive exacerbation of the defense reaction (Graeff, 1994). The structure of the elevated T-maze is similar to that of an elevated plus-maze, from which it was derived. The main differences are the absence of one of the enclosed arms in the elevated T-maze and the experimental procedures, which consist of two tests, escape and avoidance, respectively, corresponding to fear and anxiety (Zangrossi and Graeff, 1997). The first type of manipulation is inhibitory avoidance. This consists of latency for the animal to leave the enclosed arm after previous exposures to this arm. The latency tends to increase during the test. Normally, three trials are carried out at 30-s intervals, and a fourth one is carried out 48 or 72 h later to assess memory (Tomaz et al., 1993). The second type of manipulation is escape, which corresponds to the latency to go from the extremity of the open arm to the enclosed arm. Normally, experiments involving animal models utilize males because when females are used, the results are not always similar, or seem to vary abnormally with decreased consistency and increased deviations. This phenomenon is referred to as gender difference or sexual dimorphism. Sex differences have also been reported to occur in other behavioral models. Females and males perform differently in the open-field test (Kennett et al., 1986; Curzon et al., 1990; Alonso et al., 1991), the forced swimming test (Kennett et al., 1986; Alonso et al., 1991), the elevated plus-maze, the social interaction and the Vogel punished drinking tests (Johnston and File, 1991). Some studies (Blanchard et al., 1980; Blanchard and Blanchard, 1990) have found differences in defense reaction between males and females, including ultrasonic vocalizations (Blanchard et al., 1992). The difference between sexes is partially related to the estrous cycle, i.e. the fluctuation of sex hormones present in females, but not in males, which involves physiological and behavioral aspects. Estrous cycle fluctuations have been described in several animal models; during the diestrous phase there is a facilitation of acquisition of two-way conditioned avoidance, and exploratory behavior of the open arms in the elevated plus-maze (Diaz-Veliz et al., 1989, 1997). In memory experiments, the estrous cycle also seems to be important. In the Morris water-maze, females showed better performance in the spatial cues test
during the proestrus phase, and in the spatial learning test during the estrous phase (Warren and Juraska, 1997); Further, in the estrous phase there is an attenuation of the analgesia induced by electric shocks (Ryan and Maier, 1988). In other animal models, such as those involving schizophrenia, there are differences between the phases of the estrous cycle, in latent inhibition, for example (Koch, 1998). The female hormonal variation does not seem to influence feeding behavior after chromic stress (Anderson et al., 1996) although it can alter feeding behavior in the long term (Laviano et al., 1996), and it increases taste reactivity during proestrus and diestrus (Clarke and Ossenkopp, 1998) and intensifies the drinking response induced by insulin, depending on the phase (Fernandez-Trisac et al., 1998). There are no studies about the effect of female estrous cycles on behavior in the elevated T-maze. Thus, the aim of the present investigation was to study the influence of hormonal conditions on the performance of rats in the elevated T-maze.
2. Method 2.1. Animals Eighty-seven Wistar rats (17 males weighing 300–320 g and 60 females weighing 200–220 g) were obtained from the animal houses of the Universidade Estadual Paulista “Júlio de Mesquita Filho”, SP, and Universidade do Sul de Santa Catarina, SC. As they were all of the same age, males and females had different body weights. The animals were housed according to sex in groups of five per cage (41 cm × 35 cm × 33 cm) on a 12-h light/12-h dark cycle (lights from 07:00 to 19:00 h). Cages with females and cages with males were kept in the same room throughout the whole experiment. The rats were divided into groups according to sex or estrous phase (males, N = 17; females in proestrus, N = 19; females in estrus, N = 18; females in diestrus, N = 16; females in metaestrus, N = 17). 2.2. Apparatus The elevated T-maze consisted of two open arms (50 cm × 10 cm) and one enclosed arm (50 cm ×
A. Gouveia Jr. et al. / Behavioural Processes 67 (2004) 167–171
169
10 cm × 40 cm) perpendicular to the opened arms; the arms were connected by a square (10 cm × 10 cm) and the apparatus was elevated 50 cm above the floor. The apparatus was made of wood. All the experiments were recorded with a video camera. 2.3. Procedure The females were investigated to establish the estrous cycle by daily collection of vaginal material for at least 5 days before the test. The stage of the estrous cycle was determined by vaginal smears, obtained between 09:00 and 11:00 h. The tails were gently raised and a cotton swab humidified in drops of saline was cautiously inserted into the vagina to obtain vaginal cytology by semicircular movements. The vaginal smears on glass slides were examined under microscope. The animals were divided into groups according to phase of the estrous cycle and sex, as follows: females in estrus, metaestrus, diestrus, or proestrus, and males. The test consisted of three inhibitory avoidance trials and one escape trial held at 30-s intervals. Between the trials the animals were placed in individual cages. The avoidance trials started when the animal was placed at the end of the enclosed arm of the T-maze. The latency for the animal to enter the open arms (a maximum of 300 s) was recorded. The escape trial was run after the inhibitory avoidance test. The animal was placed at the end of one of the open arms and the time the animal took to exit this arm was recorded. An entry was recorded when the animal placed all four paws inside one arm; likewise, an exit was recorded every time the rat removed all four paws from one arm. The experiments were conducted between 9:00 and 14:00 h.
Fig. 1. Latencies (mean ± S.E.M.) of the avoidance responses of male rats (Ma) and female rats in the hormonal phases of metaestrus (Me), diestrus (Di), proestrus (Pr), and estrus (Es) in the inhibitory avoidance test in the elevated T-maze. Trial 1 is actually the baseline: (∗) different from the respective trial 1, (◦ ) different from the males in the same trial (Duncan, P < 0.05).
3. Results The results of inhibitory avoidance learning are shown in Fig. 1. ANOVA indicated a significant effect of the hormonal state [F(4, 82) = 2.677, P = 0.037], as well as of the trials [F(2, 164) = 41.593, P < 0.001]. There was no significant interaction between the two factors [F(8, 164) = 1.840, P = 0.073]. Duncan’s test showed that the avoidance latencies in trials 2 and 3 were significantly larger than trial 1 (baseline) in the groups of males and females in metaestrus. Latencies in trial 3 were larger than trial
2.4. Statistics Data from the avoidance trials were subjected to two-way analysis of variance (ANOVA) with one independent factor (hormonal states: estrus, metaestrus, diestrus, proestrus, and males) and repetitions in the other factor (trials: 1, 2, and 3). Data from the escape trial were subjected to one-way ANOVA. These were followed by Duncan’s multiple range post hoc test whenever appropriate. In all cases, the level of statistical significance was set at 0.05.
Fig. 2. Latencies (mean ± S.E.M.) of the escape responses of male rats (Ma) and female rats in the hormonal phases of metaestrus (Me), diestrus (Di), proestrus (Pr), and estrus (Es) in the escape test in the elevated T-maze.
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1 in the groups of females in proestrus and estrus. Finally, the test showed that the latencies of females in diestrus were larger than that of males in trial 1. Fig. 2 shows the results of escape behavior. ANOVA showed no effects due to hormonal state [F(4, 82) = 0.459, P = 0.765). Females in diestrus presented smaller latencies but the differences between groups were not statistically significant.
4. Discussion The present results allow us to conclude that hormonal states constitute an important variable for the behavior studied. First, females in diestrus have presented high latencies in trial 1, indicating higher anxiety levels. These may be caused by the hormones present in this phase. In fact, there are reports of similar high anxiety levels exhibited by females in diestrus in the elevated plus-maze (Mora et al., 1996) and in an inhibitory avoidance test (Diaz-Veliz et al., 1989). Since the elevated T-maze is a good index of anxiety (Zangrossi and Graeff, 1997), the high latencies observed in our avoidance results are caused by the hormones present in the diestrus phase. In the same vein, males and females in metaestrus present the same behavioral profile in all three trials. This indicates that the effects described for diestrus were due to the hormones present in this phase since in metaestrus, characterized by low or no hormonal action, the females presented similar avoidance response latencies as the males. The elevated T-maze is also considered an index of fear (Zangrossi and Graeff, 1997) indicated by the latency to exit the open arm in the escape trial; the lower the latency the higher the fear. Our data show that the lower latencies occurred in the group of females in diestrus but the difference did not reach statistical significance. This indicates that whatever hormone action, which was able to increase anxiety, was not able to cause increases in fear. The present results stress the importance of steroid hormones in mediating the emotional response of anxiety. They also support the claim that the elevated T-maze is sensitive to the same variables that alter behavior in other anxiety models.
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