- Email: [email protected]
The comparison of immobility time in experimental rat swimming models
Life Sciences 79 (2006) 1712 – 1719 www.elsevier.com/locate/lifescie
The comparison of immobility time in experimental rat swimming models Caroline Morini Calil, Fernanda Klein Marcondes ⁎ Department of Physiological Sciences, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil Received 15 February 2006; accepted 2 June 2006
Abstract Rat swimming models have been used in studies about stress and depression. However, there is no consensus about interpreting immobility (helplessness or adaptation) in the literature. In the present study, immobility time, glucose and glycogen mobilization, corticosterone and the effect of desipramine and diazepam were investigated in two different models: swimming stress and the forced swimming test. Immobility time was lower in swimming stress than in the forced swimming test. Both swimming models increased corticosterone levels in comparison with control animal levels. Moreover, swimming stress induced higher corticosterone levels than the forced swimming test did [F(2,14) = 59.52; p < 0.001]. Liver glycogen content values differed from one another (swimming stress < forced swimming test < control), [F(2,17) = 32.08; p < 0.001]. The glycogen content values in the gastrocnemius [F(2,16) = 11.35; p = 0.026] and soleus [F(2,16) = 8.68; p = 0.006] muscles were lower during swimming stress in comparison with the forced swimming test and control. The immobility time was recorded and measured in another group treated with desipramine and diazepam in two protocols: a single session of forced swimming test or swimming stress and two sessions (pre- and retest) of forced swimming model or swimming stress. Desipramine decreased the immobility time in the forced swimming test in both the single [F(2,25) = 20.63; p < 0.0001] and retest [F(2,37) = 7.28; p = 0.002] swimming session, without changes in the swimming stress model. Diazepam increased the immobility time in the swimming stress but not in the forced swimming test during the single [F(2,26) = 11.24; p = 0.0003] and retest sessions [F(2,38) = 4.17; p = 0.02]. It was concluded that swimming stress and the forced swimming test induced different behavior, hormonal and metabolic responses and represented different situations to the animal. © 2006 Elsevier Inc. All rights reserved. Keywords: Immobility; Behavior; Swimming; Rat; Anxiety; Adaptation; Learned helplessness; Glycogen
Introduction Swimming is an experimental protocol used for inducing stress in laboratory rodents (Marcondes et al., 1996; Barros and Ferigolo, 1998; Tanno et al., 2002). In these studies, some of the biochemical, hormonal and behavioral changes are recorded to evaluate the animal's physiological responses to a stressful situation (De Boer et al., 1990; Bruner and Vargas, 1994; Marcondes et al., 1996; Tanno et al., 2002). When a swimming session begins, the rats swim actively and afterwards they become less active and immobility is observed. If a rat swims very agitatedly and its behavior is
⁎ Corresponding author. Departamento de Ciências Fisiológicas, Faculdade de Odontologia de Piracicaba-UNICAMP, Av. Limeira, 901-Vila Areião, Piracicaba, São Paulo, Brazil, 13414-903. Tel./fax: +55 19 3412 5212. E-mail address: f [email protected] (F.K. Marcondes). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.06.003
uncoordinated, it dies more easily than a rat that swims rhythmically and sometimes floats (Binik and Sullivan, 1983). Therefore, immobility was interpreted as an adaptive response that increases the rat's chance of survival (Binik and Sullivan, 1983; Bruner and Vargas, 1994). However, the meaning of immobility may vary in accordance with the swimming protocol. In a forced swimming test model (FST), Porsolt et al. (1978a) reported that animals forced to swim in a cylinder (20 diameter, 15 cm of water at 25 °C) stopped trying to escape and adopted a characteristic immobile posture. In this FST, antidepressants shown to be effective in clinical depression, significantly reduced the duration of immobility. These results led to the conclusion that the immobility observed reflected a state of lowered mood or hopelessness in animals (Porsolt et al., 1977, 1978a,b), and this protocol is frequently used to evaluate the efficacy of potential new antidepressive compounds (Barros and Ferigolo, 1998). It is important to note the differences between the
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
two models: the swim–stress model differs with respect to water temperature, water container dimensions, and water depth; in the forced swimming protocol the vessel is smaller and the water is shallower and colder. Stress, adaptive or maladaptive responses to coping with escaping, learning and reluctance to sustain efforts are some of the issues being investigated to explain immobile behavior in rats. How to interpret immobility during swimming is still a matter of debate (Beijamini et al., 1998) and it is extremely important to clarify this. The aim of the present study was to compare the swimming stress and forced swimming test models, to assess the hypothesis that they are two different experimental situations and therefore, the meaning of immobility in fact varies in accordance with the chosen swimming protocol. Behavioral and metabolic responses were recorded, as well as the effect of antidepressive and anxiolytic drugs. Materials and methods Animals Male Wistar rats weighing 300–350 g were used. They were kept under standard conditions in groups of four per cage with lights on from 6 a.m. to 6 p.m and handled at least 10 days before experiments began. Food and water were provided ad lib. All experiments were carried out between 8:00 and 11:00 a.m. to avoid circadian rhythm influence on behavioral and physiological parameters. All animal procedures were approved by the Institute of Biology/UNICAMP Ethical Committee for Animal Research (CEEA- IB- UNICAMP, certificate 13-1). Experiment 1 Since a swimming session has been used as an acute stress model in small laboratory animals (Spadari and De Moraes, 1988; Shors et al., 1999), and the authors have previous experience with a 50-min swimming stress protocol (Bianchi et al., 2001; Tanno et al., 2002), one 50 min session was chosen for comparing this swimming protocol with the Forced Swimming Test (FST). Thus, behavioral, metabolic and hormonal responses were evaluated in the following swimming models: Forced Swimming Test (FST: a 20 × 20 × 50 cm tank, containing 20 cm of water at 25 °C) and Swimming Stress (SS: a 50 × 50 × 50 cm tank, containing 38 cm of water at 30 °C). Rats were assigned to FST, SS or control groups (Table 1). Room temperature (25 °C) was controlled by a split air conditioning system. Both room and water temperature was monitored at the end of each experiment.
1713
Each rat was individually submitted to only one swimming session, and its behavior was recorded on videotape for further analysis. The water was changed after each animal's session, to avoid the influence of alarm pheromones left behind by the previous animal. The immobility time was measured with a stopwatch by a trained observer. The rat was judged to be immobile whenever it stopped swimming and remained afloat in the water, with its head just above water level. Blood sampling, tissue collection and analytic methods Immediately after the swimming session, the rats were anaesthetized under halothane, and blood was collected from the left renal vein (Cunha et al., 2005) into sterilized glass tubes (B and D 4.5 mL) with EDTA and into sterilized glass-gel tubes (B and D 4 mL). Thirty minutes after collection, the blood was centrifuged (15 min, 2500 rpm) and plasma was isolated. The plasma was used for colorimetric glucose dosage with commercially available kits (Laborlab, No. 02200). The corticosterone content of each sample was determined by radioimmunoassay. The intra- and interassay coefficients of variation were 10%. Blood samples were centrifuged at 2000 g at 4 °C and plasma was separated and frozen at −20 °C until it was time to measure the hormones. Plasma corticosterone was determined by radioimmunoassay (RIA) (Niswender et al., 1968, 1969; Genaro et al., 2004). Plasma CT was measured using ethanol extraction (Vecsei, 1979). All samples were measured in the same assay using 3H-corticosterone from NEN Life Science Products. The lowest detection limit was 50 pg/ ml. The intra-assay coefficient of variation was 5.8%. After blood collection, the animals were killed by pneumothorax, and the soleus and gastrocnemious (red portion) were then freed by dissection from the left hindlimb (Cunha et al., 2005). The liver was quickly excised and the tissues were cleared of connective tissue in physiological saline. Next, tissue glycogen content was colorimetrically determined in accordance with Lo et al. (1970) and Cunha et al. (2005). Experiment 2 Since immobility interpreted as meaning helplessness is based on the ability of antidepressant drugs to inhibit this behavior, and since anxiolytics can control the stress level by reducing anxiety, the effect of this class of compounds in this experiment was evaluated in both FSTand SS, in accordance with the original FST protocol, described by Porsolt et al. (1977). For this purpose, another group of male Wistar rats were submitted to the FST or SS, for 15 min on the first day (pre-test) and for 5 min on the
Table 1 Experimental groups for comparative analysis of the responses of rats submitted to a 50-min swimming session, according to the Forced Swimming Test or Swimming Stress Protocols ( Experiment 1). Group
Forced swimming test Swimming stress Control
Tank characteristics
Analysis
Dimensions (cm)
Water depth (cm)
Temperature (°C)
Behavior
Glucose/glycogen
Corticosterone
20 × 20 50 × 50 –
20 38 –
25 °C 30 °C –
X (10) X (10)
X (5–6) X (5–6) X (8)
X (6) X (6) X (5)
The number of observations/group is indicated in parentheses.
1714
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
Table 2 Experimental groups and number of observations/group for comparative analysis in the effect of desipramine and diazepam in the immobility time of rats submitted to two swimming sessions of FST or SS ( Experiment 2) or to a single swimming session ( Experiment 3) Groups
Dimensions (cm)
Depth (cm)
Temperature (°C)
Experiment 2 (n)
Experiment 3 (n)
FST saline/3 doses FST saline/1 dose FSTdesipramine/3 doses FST desipramine/1 dose FST diazepam/ 1 dose SS saline/3 doses SS saline/ 1 dose SS desipramine/3 doses SS desipramine/ 1 dose SS desipramine/1 dose
20 × 20 20 × 20 20 × 20 20 × 20 20 × 20 50 × 50 50 × 50 50 × 50 50 × 50 50 × 50
20 20 20 20 20 38 38 38 38 38
25 °C 25 °C 25 °C 25 °C 25 °C 30 °C 30 °C 30 °C 30 °C 30 °C
7 8 14 − 11 7 8 13 − 13
– 9 9 9 10 – 9 9 9 11
n = number of experiments/group.
second day (test), 24 h later (Table 2). Since antidepressant therapy effectiveness becomes evident after 2–3 weeks, the usefulness of a similar dosing schedule was investigated (Borsini and Meli, 1988) and each rat received desipramine (15 mg/kg, i.p) or vehicle administrations 24 h, 5 h, and 1 h before re-test (Porsolt et al., 1977, 1978a). Another group of rats received diazepam (1 mg/kg) or a vehicle 30 min before the test, as the version of the Swimming Stress (Calil et al., 2002) and experiments with anxiolytics in the present study were conducted with the use of single doses (Martí and Armario, 1993). After the first swimming sessions, the rats were thoroughly dried with towels and warmed under a heat source. The swimming sessions were videotaped for further analysis by a trained observer who remained blind to treatments, as described in Experiment 1.
single 15-minute session. Another group of Wistar rats were randomly assigned to 3 groups: vehicle, desipramine (15 mg/kg) or diazepam (1 mg/kg). The animals were treated 30 min before the swimming session, and their behavior was recorded and analyzed as described in Experiment 1. The protocols are shown in Table 2, however, instead of two swimming sessions, in Experiment 3, rats were submitted to only one 15-min swimming session.
Experiment 3
Results
Since the Swimming Stress protocol is based on only one swimming session, the meaning of immobility is judged according to an acute exposure to the stressor (swimming), and the FST protocol is based on two sessions, the first one lasting for 15 min; a third experiment was conducted to evaluate the effect of desipramine and diazepam on immobility behavior during a
Fig. 1 shows total immobility time of animals submitted to the two different protocols. During the swimming session, the rats submitted to swimming stress (SS) presented lower immobility time than those submitted to forced swimming test (FST, p < 0.05). Furthermore, the total immobility time was lower in SS than in the FST protocol (Fig. 1; p < 0.05). Both SS (43.1 ± 1.8 μg/mL) and FST (27.5 ± 2.7μg/mL) induced increased plasma corticosterone levels in comparison with control animals (16.1 ± 4 μg/mL, Fig. 2; p < 0 .05). Moreover, rats
Fig. 1. Immobility time of rats submitted to one 50 min-swimming session in the following swimming models: Forced Swimming Test (tank of 20 × 20 × 50 cm, containing 20 cm of water at 25 °C) and Swimming Stress (tank of 50 × 50 × 50 cm, containing 38 cm of water at 30 °C). Total immobility time is presented in legend. Means and SEM (n = 10/group) are presented. ⁎Statistically different from FST (Student t test, p < 0.05).
Statistical analysis The statistical significance of differences between groups was determined by Student t test (immobility time — Experiment 1) or Analysis of Variance followed by the Tukey test. Statistical significance was set at p < 0.05.
Fig. 2. Effect of 50-min of FST (tank of 20 × 20 × 50 cm, containing 20 cm of water at 25 °C) or Swimming Stress exposure (tank of 50 × 50 × 50 cm, containing 38 cm of water at 30 °C) on plasma corticosterone concentrations (mg/dL). Means and SEM (n = 5–6) are presented. Groups labeled with different letters are statistically different (p < 0.05).
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
1715
Fig. 3. Glucose plasma concentration (A), and glycogen tissue concentration in the liver (B), soleus (C) and red portion of gastrocnemious muscles (D) of rats submitted to 50 min of swimming in the swimming models: Forced Swimming Test (tank of 20 × 20 × 50 cm, containing 20 cm of water at 25 °C) and Swimming Stress (tank of 50 × 50 × 50 cm, containing 38 cm of water at 30 °C). Control Group was not submitted to swimming. Means and SEM (n = 5–8) are presented. Groups labeled with different letters are statistically different (p < 0.05).
submitted to SS presented higher corticosterone levels compared with animals submitted to FST [F(2,14) = 59.52, p < 0.001]. Immediately after the swimming session, glycemia values were higher in rats submitted to both swimming protocols in comparison with the control group (112± 4 mg/dL). Animals submitted to FST (230 ± 3 mg/dL), presented higher glycemia values than those submitted to SS (175 ± 7 mg/dL; Fig. 3A.), [F(2,15) = 18.94; p < 0.001]. Liver glycogen content (mg/100 mg tissue) is shown in Fig. 3B. Rats submitted to SS and FST presented significantly lower glycogen content values than control animals [F(2,17) = 32. 08; p < 0.001]. Moreover, rats submitted to SS presented significantly lower glycogen content than animals in FST (p < 0.05), (FST = 4.3 ± 0.3, SS = 1.8 ± 0.2, C = 6.2 ± 0.4 mg/100 mg). In Fig. 3C and D the muscular glycogen content after 50 min of swimming is illustrated. Glycogen stores in the soleus were significantly reduced in the SS group (0.17 ± 0.02 mg/100 mg) in comparison with the control group [0.51 ± 0.07 mg/100 mg; F(2,16) = 8.68, p = 0.006], and were unchanged in the FST group (0.39 ± 0.05 mg/100 mg). SS also significantly reduced the glycogen content in the red portion of the gastrocnemious of rats in comparison with animals in the FST and control groups [F(2,16) = 11.35, p = 0.026]. There were no significant differences between the FST and the control groups (FST = 0.47 ± 0.05, SS = 0.13 ± 0.01,C = 0.56 ± 0.07 mg/100 mg). In Experiment 2, three doses of desipramine, and only one dose of diazepam were used. The control groups received three
Fig. 4. Effect of desipramine (15 mg/kg), diazepam (1 mg/kg) or vehicle (saline) on immobility time of animals submitted to the Forced swimming test (A) or Swimming Stress Model (B), tested for 5 min, after a previous 15 min test the day before. Means and SEM (n = 11–15) are presented. Groups labeled with different letters are statistically different (p < 0.05).
1716
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
Discussion
Fig. 5. Effect of desipramine (15 mg/Kg), diazepam (1 mg/kg) or vehicle (saline) on immobility time in a single 15 min exposure to Forced Swimming Test (A) or Swimming Stress model (B). The rats did not have a pre-test swimming session. Means and SEM (n = 9–11) are presented. Groups labeled with different letters are statistically different (p < 0.05).
doses or one administration of vehicle respectively. Since there was no difference in the behavior between the two vehicle groups (data not shown), these data were gathered into one vehicle group, in order to make the comparisons with desipramine- and diazepam-treated rats. Therefore there was only one vehicle group for FST and for SS. Desipramine administered to rats tested for 5 min, after a previous 15-min test on the day before, significantly reduced immobility behavior in FST (Fig. 4A); [F(2,37) = 7.28; p = 0.002], in comparison with vehicle- and diazepam-treated animals. There were no significant differences between diazepam and vehicle-treated animals submitted to FST (desipramine = 208 ± 12, diazepam = 261 ± 8, saline = 246 ± 8 s). In Fig. 4B, diazepam administered to rats tested under SS for 5 min, after a previous 15-min test the day before, significantly increased immobility [F(2,38) = 4.17, p = 0.02], in comparison with vehicle-treated animals. There were no significant differences between vehicle- and desipramine-treated animals submitted to SS (desipramine = 129 ± 21, diazepam = 171 ± 12, saline = 170 ± 13 s). The effect of a single dose of desipramine or diazepam on immobility time of rats submitted to a single 15-minute swimming session, is represented in Fig. 5A. Desipramine significantly decreased immobility in animals submitted to FST [F (2,25)= 20.63; p < 0.0001], in comparison with vehicle- and diazepam-treated animals (desipramine = 777 ± 8, diazepam = 844 ± 3, SAL = 842 ± 6 s). On the contrary, in Fig. 5B, diazepam induced increased immobility time in rats submitted to SS [F (2,26)= 11.24; p = 0.0003] in comparison with desipramine- and vehicle-treated rats (desipramine = 170 ± 22, diazepam = 240 ± 31, saline = 127 ± 7 s). No changes were observed between initial and final room and water temperature, in any of the experiments.
The results of this study support the hypothesis that the Forced Swimming Test (FST) and the Swimming Stress Model (SS) induce different physiological responses in rats. Rats put into the water initially exhibited escape-directed behavior, such as climbing the wall or jumping, and gradually became immobile (Bianchi et al., 2001). The higher immobility time observed in FST in comparison with SS, may have occurred because animals exposed to 20 cm deep water (FST) can touch the bottom of the tank with their hind paws and tail. Whereas, deeper water levels, as presented in the SS model, seem to limit their ability to use their rear paws and tails to stay immobile (Barros and Ferigolo, 1998; Calil et al., 2002), and thus, the animals needed a different, probably more difficult strategy to keep their heads above the water. However, in the literature there are modified versions of FST, in which the animals are unable to touch the bottom of the tank, but nevertheless they present significant immobility behaviors [Abel, 1993, Borsini et al., 1986]. Tank characteristics, other than water depth, contribute to the different responses observed in FST and SS. Tank size seems to affect immobility, and other investigators have also noted this (De Pablo et al., 1989; Borsini et al., 1986). In FST, the small tank size allowed the animals to balance themselves comfortably on their paws against the wall (Barros and Ferigolo, 1998). Whereas, in SS, owing to the large water surface area and wall to wall distances, the animals were forced to move their extremities incessantly to keep their noses above the water. In the present study, the hypothesis is that these different apparatus sizes present the animals with different situations and may therefore change the interpretation of behavior patterns (Borsini et al., 1986; Hilakivi and Hilakivi, 1987). It has also been reported that swimming performance in the rat is substantially influenced by water temperature (Baker and Horvath, 1964; Vanderwolf, 1991; Arai et al., 2000; Brown et al., 2001). Accordingly, when the water is much warmer than the rat's core temperature (i.e., 42 °C or greater), the animal becomes hyperthermic, its exercise performance diminishes greatly, and death may ensue (Bargiel et al., 1981). In contrast, when the water is significantly cooler than the rat's core temperature (i.e., 20 °C or lower), the animal becomes hypothermic, its exercise performance is greatly reduced (Dawson et al., 1968), and death may ensue. If the water is maintained slightly lower than the animal's core temperature (i.e., between 33 and 36 °C), the rat can maintain its core temperature throughout the exercise bout. Moreover, rats swimming in water in this temperature range do not experience decrements in various cardiovascular parameters (e.g., cardiac output, heart rate, mean arterial pressure) that could influence exercise performance (Dawson et al., 1968). In the present study, significant differences in the animals' behavior were observed between the swimming sessions with water at 25 °C and 30 °C. Animals that swam in the FST at 25 °C presented higher immobility than animals that swam in the SS at 30 °C. With regard to these water temperature characteristics, the results of the present study are in accordance with recent studies, (Arai et al., 2000; Brown et al., 2001) which investigated the relation between lowering of body temperature and behavioral response in mice and significant differences were observed
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
between water of 25 °C and 35 °C as regards immobility responses in the FST. Peeters et al. (1991) also reported that animals stayed immobile for most of the time at 25 °C, in contrast with the situation at 35 °C, where they showed greater activity. In this context, Smith in 1979 reported that homeothermic organisms must maintain optimal body temperature levels; exposure to a cold environment means that they must decrease their heat losses by increasing their heat production (Smith, 1979). In the present study, animals submitted to FST in water at a temperature of 25 °C, presented with pilo-erection during the swimming time. In addition, although animals in the FST were more immobile than rats submitted to SS, a marked increase in their paw muscle tone was observed during analysis. They probably adopted these behaviors to avoid heat losses. Whereas in SS, instead of presenting with pilo-erection, the animals were seen doing more vigorous physical exercise. Moreover, Bruner and Vargas (1994) and Marcondes in 2002 (data not published) observed that water temperatures around 30 °C prevented exchange between water and body temperatures, which also helps to maintain the body temperature. In spite of the influence of the water temperature on core body temperature, the authors believe that the different behavior patterns between the FST and SS are due to association among the three factors: tank size, water depth and temperature (Martí and Armario, 1994), and that they cannot be evaluated separately, since FST and SS are two different situations. In addition to the water temperature in SS, water depth was another important swimming test factor that led to reduced immobility to keep the animal above the water surface, and as described above, the tank dimensions also affected immobility. These observations lead to the conclusion that the two models presented in this study appear to represent different situations to the laboratory rat and the meaning of immobility could vary, depending on the model chosen (Calil et al., 2002). Some authors (Thierry et al., 1984; Abel, 1994) believe that the development of immobility in FST could represent either failure to persist with escape-directed behavior or to develop passivity in response to the situation. Although the FST model represented a less stressful situation, compared with the swimming stress model, the authors observed higher corticosterone levels in animals submitted to FST, compared with the control group. This increased corticosterone was possibly due to the partial immersion in water and the inescapability that faced the animal in FST. Moreover, the water temperature in this model was established at 25 °C. This lower water temperature could interfere in the animal's body temperature during swimming. In this context, it has been reported that lower water temperature can cause elevations in corticosterone due to hypothermia (Arai et al., 2000; Taltavull et al., 2003; Drugan et al., 2005). On the other hand, the Swimming Stress model seems to be a more stressful situation than FST, because of the menace of drowning. The higher increase in the corticosterone response in rats submitted to SS, in comparison with those submitted to FST supported this hypothesis. Moreover, Drugan et al. (2005) demonstrated that although the rise in corticosterone in rats submitted to water temperature of 30 °C were not as high as at cooler temperatures, this rise was still significantly higher than that in control groups. Therefore, when the water temperature, water
1717
depth and tank dimensions in the SS are taken together, it is a different situation compared with the FST which, according to the results of the present study, causes different behavior and endocrine responses in rats. This result is consistent with previous finding that reported HPA axis activation in response to swimming stress (Abel, 1994). The immobility behavior of rats exposed to Swimming Stress is perhaps the result of an adaptive response to a stressful situation, as there is danger of sinking and drowning and consequently danger of death (Bruner and Vargas, 1994; Bianchi et al., 2001; Calil et al., 2002). The higher intensity of stress response to SS shows that the physiological stress system activation differs between SS and FST models, as was hypothesized in this study. It is also noteworthy that plasma corticosterone is a well-accepted fear/anxiety index under stressful conditions (Martí and Armario, 1994; De Boer et al., 1990) and the data in this study present evidence that the behavior of rats submitted to SS might be related to fear/anxiety rather than being “learned helplessness”. Since the animals were anesthetized with halothane and blood was collected from the left renal vein, corticosterone levels were expected to increase even in the control animals (Abel, 1993). Although the control corticosterone plasma levels observed in the present study are higher than those previously described in the literature, observed after decapitation, the method of blood collection was the same in the control and swimming groups. Therefore the hormonal differences between FST and SS appear to be related to the differences between the swimming models. The glycemia and tissue glycogen content results show that metabolic responses of animals submitted to FST are different from those observed in animals submitted to SS. As part of stress response, there is increased glucose use by the organism, and catecholamines and glucocorticoids stimulate hepatic glycogenolysis, the main source to maintain glycemia (Marliss et al., 2000). The higher glycemia and hepatic glycogen mobilization observed after SS suggest a higher energy demand in this model and also confirm that SS appears to be a more stressful situation, in comparison with FST. Furthermore, because of the drowning menace and consequent increase in brain activity in the SS model, the glycemia- and hepatic glycogenolysis-related responses may be regarded as evidence of the organism's adaptation to the situation (Blawacka et al., 1977), since it cannot be ruled out that coping with moderate fatigue during swimming could have a bearing on emotional aspects (Kramer et al., 1991). Thus, Ollenberger et al. (1998) found increased relative blood flow to the head during swimming; this probably reflects a fall in cerebrovascular resistance and corresponding absolute increase in cerebrovascular flow, due to the stress the animals are faced with. In addition to liver glycogen, muscle glycogen is also an essential fuel source used during exercise (Ivy and Kuo, 1998; Cunha et al., 2005), but only muscle cells can use it (Marliss et al., 2000). Exercise sessions are known to result in muscle glycogen depletion. The significant decrease observed in both soleus and gastrocnemius glycogen content in animals submitted to SS, and no change in animals exposed to FST, is in accordance with the immobility time observed. During immobility time recording, the experimenter observed that the
1718
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
animals did not perform effective exercise in FST, and could touch the bottom of the tank. Whereas in the SS model, the rats continuously alternated paw movements and floating. Even during floating, they made some movements to keep their noses above the water surface. The results of this study can be associated with the findings of Ollenberger et al. (1998). These authors observed an increased blood flow in rat hindlimb muscles during swimming, and it probably reflects increased locomobile activity. Therefore, there are differences in muscle activity between FST and SS. In this study, the results of behavior analysis during the swimming session and metabolic and hormonal analyses assessment after the swimming session, suggest that the Swimming Stress and Forced Swimming Test induced different physiologic responses and therefore, they presented the laboratory rat with two different situations, confirming the initial hypothesis of this study. Since pharmacological validity is appropriate in behavioral procedures (Martí and Armario, 1993) and the meaning of immobility mainly reflects a state of despair, specifically in FST, (Porsolt et al., 1978b), in addition to considering that this behavior was reduced by a variety of treatments that are therapeutically effective in depression, the effect of the antidepressive desipramine and the anxiolytic diazepam on exposure to both FST and SS was evaluated. As expected, the data presented evidence that immobility in FST is related to a state of despair, or “learned helplessness”, since desipramine significantly decreased immobility time of rats that had previously undergone a 15-min FST and also in rats submitted to only a single 15-min swimming session. It would appear that rats submitted to FST tend to adopt passive strategies when facing inescapable situations, and this behavior was reverted by administering an antidepressive. The findings of the inhibitory effect of desipramine on immobility are in accordance with results in the literature (Martí and Armario, 1993). From this point of view, animals may genetically be highly prepared to adopt immobile responses in water when they learn that more active and aggressive responses do not permit escape (Thierry et al., 1984). Otherwise, the increase in the immobility response of diazepam-treated animals submitted to SS could be interpreted as an adaptive response to stress, which conserves energy output in response to water depth (Abel, 1993). These data are consistent with many previous reports concerning the ability of minor tranquilizers, such as diazepam, to enhance prolonged duration of immobility in rats (Nishimura et al., 1987). These authors indicated that by adopting more immobile behavior during swimming the rats might gain a survival advantage. When rats are observed for extended periods in a swimming chamber, animals that adopt more immobile responses are able to remain afloat for a longer time without sinking. Thus, in terms of survival, immobility during SS appears to represent a form of successful coping rather than failure to cope. Furthermore, in the present study, diazepam did not affect the rats' behavior during exposure to FST, in good agreement with previous reports (Porsolt et al., 1977; Martí and Armario, 1993). These data may help one to understand the differences between
the Forced Swimming Test and the Swimming Stress, which probably represent different situations to the laboratory animal. In conjunction with the findings of Porsolt et al. (1977), Barros and Ferigolo (1998), Hinojosa et al. (2005), the data in the present study show that FST is a suitable animal model to detect antidepressants. However, it was not the purpose of this study to discuss the validity of this experimental animal model. Its aim was to test the hypothesis that changing the swimming session characteristics (tank size, water temperature and water depth) presents the animal with different situation which, in turn, induces different behavior patterns, as well as different metabolic and hormonal responses, and the data of this study confirm this hypothesis. Therefore, the choice of the animal swimming model must be given careful attention. Acknowledgements This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (99/ 00793-1) and FAEP/UNICAMP (71/99 and 27/00). C.M.C. received a FAPESP (99/08286-1) scholarship grant. The authors are grateful to Celso Rodrigues Franci, PhD for the corticosterone determination. The authors thank Maria Claudia de Castro Souza for her laboratory help and Margery Galbraith for editing the English in the manuscript. References Abel, E.L., 1993. Physiological correlates of the forced swim test in rats. Physiology and Behavior 54, 309–317. Abel, E.L., 1994. A further analysis of physiological changes in rats in the forced swim test. Physiology and Behavior 56 (4), 795–800. Arai, I., Tsuyuki, H., Shimoto, M., Satoh, M., Otomoto, S., 2000. Decrease body temperature dependent appearance of behavior despair in the forced swimming test in mice. Pharmacology and Research 42, 171–176. Baker, M.A., Horvath, S.M., 1964. Influence of water temperature on oxygen uptake by swimming rats. Journal of Applied Physiology 19, 1215–1218. Bargiel, Z., Nowicka, H., Wojcikowska, J., 1981. Swim–stress changes of rat adrenal catecholamine level depending on metabolic state and different ambient temperature. Folia Histochemica et Cytochemica (Krakow) 19, 31–37. Barros, H.M.T., Ferigolo, M., 1998. Ethopharmacology of imipramine in the forced-swimming test: gender differences. Neuroscience and Biobehavioral Reviews 23, 279–286. Beijamini, V., Skalisz, L.L., Joca, S.R., Andreatini, R., 1998. The effect of oxcarbazepine on behavioral despair and learned helplessness. European Journal of Pharmacology 347 (1), 23–27. Bianchi, F.J., Tanno, A.P., Marcondes, F.K., 2001. Relationship between the level of stress and supersensitivity to norepinephrine in female rats at proestrus. Brazilian Journal of Pharmaceutical Sciences 37 (3), 391–398. Binik, Y.M., Sullivan, M.J.L., 1983. Sudden swimming deaths: a psychomotor reinterpretation. Psychophysiology 20, 670–681. Blawacka, M., Roth, Z., Wojciechowska, F., Karon, H., 1977. Effect of exercise on glycogen level in muscles and liver in rats. Acta Physiologica Polonica 28 (5), 431–440. Borsini, F., Meli, A., 1988. Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology 94, 147–160. Borsini, F., Volterra, G., Meli, A., 1986. Does the behavioural “despair” test measure “despair”? Physiology and Behavior 38 (3), 385–386. Brown, P.L., Hurley, C., Repucci, N., Drugan, R.C., 2001. Behavior analysis of stress controllability effects in a new swim stress paradigm. Pharmacology, Biochemistry and Behavior 68, 263–272.
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719 Bruner, C.A., Vargas, I., 1994. The activity of rats in a swimming situation as a function of water temperature. Physiology and Behavior 5, 21–28. Calil, C.M., Bianchi, F.J., Tanno, A.P., Cunha, T.S., Marcondes, F.K., 2002. Analysis of the meaning of the immobility time in swimming experimental models. Brazilian Journal of Pharmaceutical Sciences 38 (4), 479–485. Cunha, T.S., Tanno, A.P., Costa Sampaio Moura, M.J., Marcondes, F.K., 2005. Influence of high-intensity exercise training and anabolic androgenic steroid treatment on rat tissue glycogen content. Life Sciences 77 (9), 1030–1043. Dawson, C.A., Nadel, E.R., Horvath, S.M., 1968. Cardiac output in the coldstressed swimming rat. American Journal of Physiology 214, 320–325. De Boer, S.F., Koopmans, S.J., Slangen, J.L., Van der Gugten, J., 1990. Plasma catecholamines, corticosterone and glucose responses to repeated stress in rats: effect of interstressor interval length. Physiology and Behavior 47, 117–1124. De Pablo, J.M., Parra, A., Segovia, S., Guillamon, A., 1989. Learned immobility explains the behavior of rats in the forced swimming test. Physiology and Behavior 46 (2), 229–237. Drugan, R.C., Eren, S., Hazi, A., Silva, J., Christianson, J.P., Kent, S., 2005. Impact of water temperature and stressor controllability in swim stressinduced changes in body temperature, serum corticosterone, and immobility in rats. Pharmacology, Biochemistry and Behavior 82, 397–403. Genaro, G., Schmidekand, W.R., Franci, C.R., 2004. Social condition affects hormone secretion and exploratory behavior in rats. Exploration and hormones in grouped or isolated rats. Brazilian Journal of Medical and Biological Research 37, 833–840. Hilakivi, L.A., Hilakivi, I., 1987. Increased adult behavioral “despair” in rats neonatally exposed to desipramine or zimeldine: an animal model of depression? Pharmacology Biochemistry and Behavior 28 (3), 367–369. Hinojosa, F.R., Spricigo Jr., L., Izidio, G.S., Bruske, G.R., Lopes, D.M., Ramos, A., 2005. Evaluation of two genetic animal models in behavioral tests of anxiety and depression. Behavioral and Brain Research 168 (1), 127–136. Ivy, J.L., Kuo, C.H., 1998. Regulation of GLUT4 protein and glycogen synthase during muscle glycogen synthesis after exercise. Acta Physiologica Scandinavica 162 (3), 295–304. Kramer, K., Dijkstra, H., Bast, A., 1991. Control of physical exercise of rats in a swimming basin. Physiology and Behavior 58, 271–276. Lo, S., Russell, J.C., Taylor, A.W., 1970. Determination of glycogen in small tissue samples. Journal of Applied Physiology 28 (2), 234–236. Marcondes, F.K., Vanderlei, L.C.M., Lanza, L.L.B., Spadari-Bratfisch, R.C., 1996. Stress-induced subsensitivity to catecholamines depends on the estrous cycle. Canadian Journal of Physiology and Pharmacology 74, 663–669. Martí, J., Armario, A., 1993. Effects of diazepam and desipramine in the forced swimming test: influence of previous experience with the situation. European Journal of Pharmacology 236, 295–299. Martí, J., Armario, A., 1994. Forced swimming behavior is not related to the corticosterone levels achieved in the test: a study with four inbred rat strains. Physiology and Behavior 59 (2), 369–373. Marliss, E.B., Kreisman, S.H., Manzon, A., Halter, J.B., Vranic, M., Nessim, S., 2000. Gender differences in glucoregulatory responses to intense exercise. Journal of Applied Physiology 88 (2), 457–466.
1719
Nishimura, H., Tsuda, A., Oguchi, M., Tanaka, I., Tanaka, M., 1987. Is immobility of rats in the forced swim test “Behavioral despair”? Physiology and Behavior 42, 93–95. Niswender, G.D., Midgley Jr., A.R., Monroe, S.E., Reichert Jr., L.E., 1968. Radioimmunoassay for rat luteinizing hormone with antibovine LH serum and ovine LH 131I. Proceedings of the Society for Experimental Biology and Medicine 128, 807–811. Niswender, G.D., Chen, C.L., Midgley Jr., A.R., Meites, J., Ellis, E., 1969. Radioimmunoassay for rat prolactin. Proceedings of the Society for Experimental Biology and Medicine 130, 793–797. Ollenberger, G.P., Matte, G., Wilkinson, A., West, N.H., 1998. Relative distribution of blood flow in rats during surface and submerged swimming. Comparative Biochemistry and Physiology 119 (1), 271–277. Peeters, B.W.M.M., Smets, R.J.M., Broekkamp, C.L.E., 1991. The involvement of glicocorticoids in the acquired immobility response is dependent on the water temperature. Physiology and Behavior 51, 127–129. Porsolt, R.D., Le Pichon, M., Jalfre, M., 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266 (5604), 730–732. Porsolt, R.D., Bertin, A., Jalfre, M., 1978a. “Behavioural despair” in rats and mice: strain differences and the effects of imipramine. European Journal of Pharmacology 51 (3), 291–294. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978b. Behavioural despair in rats: a new model sensitive to antidepressant treatments. European Journal of Pharmacology 47 (4), 379–391. Shors, T.J., Pickett, J., Wood, G., Paczynski, M., 1999. Acute stress persistently enhances estrogen levels in the female rat. Stress 3 (2), 163–171. Smith, I.O., 1979. Adaptative changes in the sympathetic nervous system and some effector organs of the rat following long term exercise or cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hypertrophy. Acta Physiologica Scandinavica 477, 53–54. Spadari, R.C., De Moraes, S., 1988. Repeated swimming stress and responsiveness of the isolated rat pacemaker to the chronotropic effect of noradrenaline and isoprenaline: role of adrenal corticosteroids. General Pharmacology 19 (4), 553–557. Taltavull, J.F., Chefer, V.I., Shippenberg, T.S., Kiyatkin, E.A., 2003. Severe brain hypothermia as a factor underlying behavioral immobility during cold water forced swim. Brain Research 975, 244–247. Tanno, A.P., Bianchi, F.J., Moura, M.J.C.S., Marcondes, F.K., 2002. Atrial supersensitivity to noradrenaline in stressed female rats. Life Science 71 (25), 2973–2981. Thierry, B., Steru, L., Chermat, R., Simon, P., 1984. Searching-waiting strategy: a candidate for an evolutionary model of depression? Behavioral and Neural Biology 41 (2), 180–189. Vanderwolf, C.H., 1991. Effects of water temperature and core temperature on rat's performance in a swim-to-platform test. Behavioral and Brain Research 44, 105–106. Vecsei, P., 1979. Glucocorticoids: cortisol, corticosterone and compounds. In: Jaffe, M. (Ed.), Methods of Hormone Radioimmunoassay. Academic Press, San Diego, CA, USA, pp. 767–792.
The comparison of immobility time in experimental rat swimming models Caroline Morini Calil, Fernanda Klein Marcondes ⁎ Department of Physiological Sciences, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil Received 15 February 2006; accepted 2 June 2006
Abstract Rat swimming models have been used in studies about stress and depression. However, there is no consensus about interpreting immobility (helplessness or adaptation) in the literature. In the present study, immobility time, glucose and glycogen mobilization, corticosterone and the effect of desipramine and diazepam were investigated in two different models: swimming stress and the forced swimming test. Immobility time was lower in swimming stress than in the forced swimming test. Both swimming models increased corticosterone levels in comparison with control animal levels. Moreover, swimming stress induced higher corticosterone levels than the forced swimming test did [F(2,14) = 59.52; p < 0.001]. Liver glycogen content values differed from one another (swimming stress < forced swimming test < control), [F(2,17) = 32.08; p < 0.001]. The glycogen content values in the gastrocnemius [F(2,16) = 11.35; p = 0.026] and soleus [F(2,16) = 8.68; p = 0.006] muscles were lower during swimming stress in comparison with the forced swimming test and control. The immobility time was recorded and measured in another group treated with desipramine and diazepam in two protocols: a single session of forced swimming test or swimming stress and two sessions (pre- and retest) of forced swimming model or swimming stress. Desipramine decreased the immobility time in the forced swimming test in both the single [F(2,25) = 20.63; p < 0.0001] and retest [F(2,37) = 7.28; p = 0.002] swimming session, without changes in the swimming stress model. Diazepam increased the immobility time in the swimming stress but not in the forced swimming test during the single [F(2,26) = 11.24; p = 0.0003] and retest sessions [F(2,38) = 4.17; p = 0.02]. It was concluded that swimming stress and the forced swimming test induced different behavior, hormonal and metabolic responses and represented different situations to the animal. © 2006 Elsevier Inc. All rights reserved. Keywords: Immobility; Behavior; Swimming; Rat; Anxiety; Adaptation; Learned helplessness; Glycogen
Introduction Swimming is an experimental protocol used for inducing stress in laboratory rodents (Marcondes et al., 1996; Barros and Ferigolo, 1998; Tanno et al., 2002). In these studies, some of the biochemical, hormonal and behavioral changes are recorded to evaluate the animal's physiological responses to a stressful situation (De Boer et al., 1990; Bruner and Vargas, 1994; Marcondes et al., 1996; Tanno et al., 2002). When a swimming session begins, the rats swim actively and afterwards they become less active and immobility is observed. If a rat swims very agitatedly and its behavior is
⁎ Corresponding author. Departamento de Ciências Fisiológicas, Faculdade de Odontologia de Piracicaba-UNICAMP, Av. Limeira, 901-Vila Areião, Piracicaba, São Paulo, Brazil, 13414-903. Tel./fax: +55 19 3412 5212. E-mail address: f [email protected] (F.K. Marcondes). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.06.003
uncoordinated, it dies more easily than a rat that swims rhythmically and sometimes floats (Binik and Sullivan, 1983). Therefore, immobility was interpreted as an adaptive response that increases the rat's chance of survival (Binik and Sullivan, 1983; Bruner and Vargas, 1994). However, the meaning of immobility may vary in accordance with the swimming protocol. In a forced swimming test model (FST), Porsolt et al. (1978a) reported that animals forced to swim in a cylinder (20 diameter, 15 cm of water at 25 °C) stopped trying to escape and adopted a characteristic immobile posture. In this FST, antidepressants shown to be effective in clinical depression, significantly reduced the duration of immobility. These results led to the conclusion that the immobility observed reflected a state of lowered mood or hopelessness in animals (Porsolt et al., 1977, 1978a,b), and this protocol is frequently used to evaluate the efficacy of potential new antidepressive compounds (Barros and Ferigolo, 1998). It is important to note the differences between the
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
two models: the swim–stress model differs with respect to water temperature, water container dimensions, and water depth; in the forced swimming protocol the vessel is smaller and the water is shallower and colder. Stress, adaptive or maladaptive responses to coping with escaping, learning and reluctance to sustain efforts are some of the issues being investigated to explain immobile behavior in rats. How to interpret immobility during swimming is still a matter of debate (Beijamini et al., 1998) and it is extremely important to clarify this. The aim of the present study was to compare the swimming stress and forced swimming test models, to assess the hypothesis that they are two different experimental situations and therefore, the meaning of immobility in fact varies in accordance with the chosen swimming protocol. Behavioral and metabolic responses were recorded, as well as the effect of antidepressive and anxiolytic drugs. Materials and methods Animals Male Wistar rats weighing 300–350 g were used. They were kept under standard conditions in groups of four per cage with lights on from 6 a.m. to 6 p.m and handled at least 10 days before experiments began. Food and water were provided ad lib. All experiments were carried out between 8:00 and 11:00 a.m. to avoid circadian rhythm influence on behavioral and physiological parameters. All animal procedures were approved by the Institute of Biology/UNICAMP Ethical Committee for Animal Research (CEEA- IB- UNICAMP, certificate 13-1). Experiment 1 Since a swimming session has been used as an acute stress model in small laboratory animals (Spadari and De Moraes, 1988; Shors et al., 1999), and the authors have previous experience with a 50-min swimming stress protocol (Bianchi et al., 2001; Tanno et al., 2002), one 50 min session was chosen for comparing this swimming protocol with the Forced Swimming Test (FST). Thus, behavioral, metabolic and hormonal responses were evaluated in the following swimming models: Forced Swimming Test (FST: a 20 × 20 × 50 cm tank, containing 20 cm of water at 25 °C) and Swimming Stress (SS: a 50 × 50 × 50 cm tank, containing 38 cm of water at 30 °C). Rats were assigned to FST, SS or control groups (Table 1). Room temperature (25 °C) was controlled by a split air conditioning system. Both room and water temperature was monitored at the end of each experiment.
1713
Each rat was individually submitted to only one swimming session, and its behavior was recorded on videotape for further analysis. The water was changed after each animal's session, to avoid the influence of alarm pheromones left behind by the previous animal. The immobility time was measured with a stopwatch by a trained observer. The rat was judged to be immobile whenever it stopped swimming and remained afloat in the water, with its head just above water level. Blood sampling, tissue collection and analytic methods Immediately after the swimming session, the rats were anaesthetized under halothane, and blood was collected from the left renal vein (Cunha et al., 2005) into sterilized glass tubes (B and D 4.5 mL) with EDTA and into sterilized glass-gel tubes (B and D 4 mL). Thirty minutes after collection, the blood was centrifuged (15 min, 2500 rpm) and plasma was isolated. The plasma was used for colorimetric glucose dosage with commercially available kits (Laborlab, No. 02200). The corticosterone content of each sample was determined by radioimmunoassay. The intra- and interassay coefficients of variation were 10%. Blood samples were centrifuged at 2000 g at 4 °C and plasma was separated and frozen at −20 °C until it was time to measure the hormones. Plasma corticosterone was determined by radioimmunoassay (RIA) (Niswender et al., 1968, 1969; Genaro et al., 2004). Plasma CT was measured using ethanol extraction (Vecsei, 1979). All samples were measured in the same assay using 3H-corticosterone from NEN Life Science Products. The lowest detection limit was 50 pg/ ml. The intra-assay coefficient of variation was 5.8%. After blood collection, the animals were killed by pneumothorax, and the soleus and gastrocnemious (red portion) were then freed by dissection from the left hindlimb (Cunha et al., 2005). The liver was quickly excised and the tissues were cleared of connective tissue in physiological saline. Next, tissue glycogen content was colorimetrically determined in accordance with Lo et al. (1970) and Cunha et al. (2005). Experiment 2 Since immobility interpreted as meaning helplessness is based on the ability of antidepressant drugs to inhibit this behavior, and since anxiolytics can control the stress level by reducing anxiety, the effect of this class of compounds in this experiment was evaluated in both FSTand SS, in accordance with the original FST protocol, described by Porsolt et al. (1977). For this purpose, another group of male Wistar rats were submitted to the FST or SS, for 15 min on the first day (pre-test) and for 5 min on the
Table 1 Experimental groups for comparative analysis of the responses of rats submitted to a 50-min swimming session, according to the Forced Swimming Test or Swimming Stress Protocols ( Experiment 1). Group
Forced swimming test Swimming stress Control
Tank characteristics
Analysis
Dimensions (cm)
Water depth (cm)
Temperature (°C)
Behavior
Glucose/glycogen
Corticosterone
20 × 20 50 × 50 –
20 38 –
25 °C 30 °C –
X (10) X (10)
X (5–6) X (5–6) X (8)
X (6) X (6) X (5)
The number of observations/group is indicated in parentheses.
1714
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
Table 2 Experimental groups and number of observations/group for comparative analysis in the effect of desipramine and diazepam in the immobility time of rats submitted to two swimming sessions of FST or SS ( Experiment 2) or to a single swimming session ( Experiment 3) Groups
Dimensions (cm)
Depth (cm)
Temperature (°C)
Experiment 2 (n)
Experiment 3 (n)
FST saline/3 doses FST saline/1 dose FSTdesipramine/3 doses FST desipramine/1 dose FST diazepam/ 1 dose SS saline/3 doses SS saline/ 1 dose SS desipramine/3 doses SS desipramine/ 1 dose SS desipramine/1 dose
20 × 20 20 × 20 20 × 20 20 × 20 20 × 20 50 × 50 50 × 50 50 × 50 50 × 50 50 × 50
20 20 20 20 20 38 38 38 38 38
25 °C 25 °C 25 °C 25 °C 25 °C 30 °C 30 °C 30 °C 30 °C 30 °C
7 8 14 − 11 7 8 13 − 13
– 9 9 9 10 – 9 9 9 11
n = number of experiments/group.
second day (test), 24 h later (Table 2). Since antidepressant therapy effectiveness becomes evident after 2–3 weeks, the usefulness of a similar dosing schedule was investigated (Borsini and Meli, 1988) and each rat received desipramine (15 mg/kg, i.p) or vehicle administrations 24 h, 5 h, and 1 h before re-test (Porsolt et al., 1977, 1978a). Another group of rats received diazepam (1 mg/kg) or a vehicle 30 min before the test, as the version of the Swimming Stress (Calil et al., 2002) and experiments with anxiolytics in the present study were conducted with the use of single doses (Martí and Armario, 1993). After the first swimming sessions, the rats were thoroughly dried with towels and warmed under a heat source. The swimming sessions were videotaped for further analysis by a trained observer who remained blind to treatments, as described in Experiment 1.
single 15-minute session. Another group of Wistar rats were randomly assigned to 3 groups: vehicle, desipramine (15 mg/kg) or diazepam (1 mg/kg). The animals were treated 30 min before the swimming session, and their behavior was recorded and analyzed as described in Experiment 1. The protocols are shown in Table 2, however, instead of two swimming sessions, in Experiment 3, rats were submitted to only one 15-min swimming session.
Experiment 3
Results
Since the Swimming Stress protocol is based on only one swimming session, the meaning of immobility is judged according to an acute exposure to the stressor (swimming), and the FST protocol is based on two sessions, the first one lasting for 15 min; a third experiment was conducted to evaluate the effect of desipramine and diazepam on immobility behavior during a
Fig. 1 shows total immobility time of animals submitted to the two different protocols. During the swimming session, the rats submitted to swimming stress (SS) presented lower immobility time than those submitted to forced swimming test (FST, p < 0.05). Furthermore, the total immobility time was lower in SS than in the FST protocol (Fig. 1; p < 0.05). Both SS (43.1 ± 1.8 μg/mL) and FST (27.5 ± 2.7μg/mL) induced increased plasma corticosterone levels in comparison with control animals (16.1 ± 4 μg/mL, Fig. 2; p < 0 .05). Moreover, rats
Fig. 1. Immobility time of rats submitted to one 50 min-swimming session in the following swimming models: Forced Swimming Test (tank of 20 × 20 × 50 cm, containing 20 cm of water at 25 °C) and Swimming Stress (tank of 50 × 50 × 50 cm, containing 38 cm of water at 30 °C). Total immobility time is presented in legend. Means and SEM (n = 10/group) are presented. ⁎Statistically different from FST (Student t test, p < 0.05).
Statistical analysis The statistical significance of differences between groups was determined by Student t test (immobility time — Experiment 1) or Analysis of Variance followed by the Tukey test. Statistical significance was set at p < 0.05.
Fig. 2. Effect of 50-min of FST (tank of 20 × 20 × 50 cm, containing 20 cm of water at 25 °C) or Swimming Stress exposure (tank of 50 × 50 × 50 cm, containing 38 cm of water at 30 °C) on plasma corticosterone concentrations (mg/dL). Means and SEM (n = 5–6) are presented. Groups labeled with different letters are statistically different (p < 0.05).
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
1715
Fig. 3. Glucose plasma concentration (A), and glycogen tissue concentration in the liver (B), soleus (C) and red portion of gastrocnemious muscles (D) of rats submitted to 50 min of swimming in the swimming models: Forced Swimming Test (tank of 20 × 20 × 50 cm, containing 20 cm of water at 25 °C) and Swimming Stress (tank of 50 × 50 × 50 cm, containing 38 cm of water at 30 °C). Control Group was not submitted to swimming. Means and SEM (n = 5–8) are presented. Groups labeled with different letters are statistically different (p < 0.05).
submitted to SS presented higher corticosterone levels compared with animals submitted to FST [F(2,14) = 59.52, p < 0.001]. Immediately after the swimming session, glycemia values were higher in rats submitted to both swimming protocols in comparison with the control group (112± 4 mg/dL). Animals submitted to FST (230 ± 3 mg/dL), presented higher glycemia values than those submitted to SS (175 ± 7 mg/dL; Fig. 3A.), [F(2,15) = 18.94; p < 0.001]. Liver glycogen content (mg/100 mg tissue) is shown in Fig. 3B. Rats submitted to SS and FST presented significantly lower glycogen content values than control animals [F(2,17) = 32. 08; p < 0.001]. Moreover, rats submitted to SS presented significantly lower glycogen content than animals in FST (p < 0.05), (FST = 4.3 ± 0.3, SS = 1.8 ± 0.2, C = 6.2 ± 0.4 mg/100 mg). In Fig. 3C and D the muscular glycogen content after 50 min of swimming is illustrated. Glycogen stores in the soleus were significantly reduced in the SS group (0.17 ± 0.02 mg/100 mg) in comparison with the control group [0.51 ± 0.07 mg/100 mg; F(2,16) = 8.68, p = 0.006], and were unchanged in the FST group (0.39 ± 0.05 mg/100 mg). SS also significantly reduced the glycogen content in the red portion of the gastrocnemious of rats in comparison with animals in the FST and control groups [F(2,16) = 11.35, p = 0.026]. There were no significant differences between the FST and the control groups (FST = 0.47 ± 0.05, SS = 0.13 ± 0.01,C = 0.56 ± 0.07 mg/100 mg). In Experiment 2, three doses of desipramine, and only one dose of diazepam were used. The control groups received three
Fig. 4. Effect of desipramine (15 mg/kg), diazepam (1 mg/kg) or vehicle (saline) on immobility time of animals submitted to the Forced swimming test (A) or Swimming Stress Model (B), tested for 5 min, after a previous 15 min test the day before. Means and SEM (n = 11–15) are presented. Groups labeled with different letters are statistically different (p < 0.05).
1716
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
Discussion
Fig. 5. Effect of desipramine (15 mg/Kg), diazepam (1 mg/kg) or vehicle (saline) on immobility time in a single 15 min exposure to Forced Swimming Test (A) or Swimming Stress model (B). The rats did not have a pre-test swimming session. Means and SEM (n = 9–11) are presented. Groups labeled with different letters are statistically different (p < 0.05).
doses or one administration of vehicle respectively. Since there was no difference in the behavior between the two vehicle groups (data not shown), these data were gathered into one vehicle group, in order to make the comparisons with desipramine- and diazepam-treated rats. Therefore there was only one vehicle group for FST and for SS. Desipramine administered to rats tested for 5 min, after a previous 15-min test on the day before, significantly reduced immobility behavior in FST (Fig. 4A); [F(2,37) = 7.28; p = 0.002], in comparison with vehicle- and diazepam-treated animals. There were no significant differences between diazepam and vehicle-treated animals submitted to FST (desipramine = 208 ± 12, diazepam = 261 ± 8, saline = 246 ± 8 s). In Fig. 4B, diazepam administered to rats tested under SS for 5 min, after a previous 15-min test the day before, significantly increased immobility [F(2,38) = 4.17, p = 0.02], in comparison with vehicle-treated animals. There were no significant differences between vehicle- and desipramine-treated animals submitted to SS (desipramine = 129 ± 21, diazepam = 171 ± 12, saline = 170 ± 13 s). The effect of a single dose of desipramine or diazepam on immobility time of rats submitted to a single 15-minute swimming session, is represented in Fig. 5A. Desipramine significantly decreased immobility in animals submitted to FST [F (2,25)= 20.63; p < 0.0001], in comparison with vehicle- and diazepam-treated animals (desipramine = 777 ± 8, diazepam = 844 ± 3, SAL = 842 ± 6 s). On the contrary, in Fig. 5B, diazepam induced increased immobility time in rats submitted to SS [F (2,26)= 11.24; p = 0.0003] in comparison with desipramine- and vehicle-treated rats (desipramine = 170 ± 22, diazepam = 240 ± 31, saline = 127 ± 7 s). No changes were observed between initial and final room and water temperature, in any of the experiments.
The results of this study support the hypothesis that the Forced Swimming Test (FST) and the Swimming Stress Model (SS) induce different physiological responses in rats. Rats put into the water initially exhibited escape-directed behavior, such as climbing the wall or jumping, and gradually became immobile (Bianchi et al., 2001). The higher immobility time observed in FST in comparison with SS, may have occurred because animals exposed to 20 cm deep water (FST) can touch the bottom of the tank with their hind paws and tail. Whereas, deeper water levels, as presented in the SS model, seem to limit their ability to use their rear paws and tails to stay immobile (Barros and Ferigolo, 1998; Calil et al., 2002), and thus, the animals needed a different, probably more difficult strategy to keep their heads above the water. However, in the literature there are modified versions of FST, in which the animals are unable to touch the bottom of the tank, but nevertheless they present significant immobility behaviors [Abel, 1993, Borsini et al., 1986]. Tank characteristics, other than water depth, contribute to the different responses observed in FST and SS. Tank size seems to affect immobility, and other investigators have also noted this (De Pablo et al., 1989; Borsini et al., 1986). In FST, the small tank size allowed the animals to balance themselves comfortably on their paws against the wall (Barros and Ferigolo, 1998). Whereas, in SS, owing to the large water surface area and wall to wall distances, the animals were forced to move their extremities incessantly to keep their noses above the water. In the present study, the hypothesis is that these different apparatus sizes present the animals with different situations and may therefore change the interpretation of behavior patterns (Borsini et al., 1986; Hilakivi and Hilakivi, 1987). It has also been reported that swimming performance in the rat is substantially influenced by water temperature (Baker and Horvath, 1964; Vanderwolf, 1991; Arai et al., 2000; Brown et al., 2001). Accordingly, when the water is much warmer than the rat's core temperature (i.e., 42 °C or greater), the animal becomes hyperthermic, its exercise performance diminishes greatly, and death may ensue (Bargiel et al., 1981). In contrast, when the water is significantly cooler than the rat's core temperature (i.e., 20 °C or lower), the animal becomes hypothermic, its exercise performance is greatly reduced (Dawson et al., 1968), and death may ensue. If the water is maintained slightly lower than the animal's core temperature (i.e., between 33 and 36 °C), the rat can maintain its core temperature throughout the exercise bout. Moreover, rats swimming in water in this temperature range do not experience decrements in various cardiovascular parameters (e.g., cardiac output, heart rate, mean arterial pressure) that could influence exercise performance (Dawson et al., 1968). In the present study, significant differences in the animals' behavior were observed between the swimming sessions with water at 25 °C and 30 °C. Animals that swam in the FST at 25 °C presented higher immobility than animals that swam in the SS at 30 °C. With regard to these water temperature characteristics, the results of the present study are in accordance with recent studies, (Arai et al., 2000; Brown et al., 2001) which investigated the relation between lowering of body temperature and behavioral response in mice and significant differences were observed
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
between water of 25 °C and 35 °C as regards immobility responses in the FST. Peeters et al. (1991) also reported that animals stayed immobile for most of the time at 25 °C, in contrast with the situation at 35 °C, where they showed greater activity. In this context, Smith in 1979 reported that homeothermic organisms must maintain optimal body temperature levels; exposure to a cold environment means that they must decrease their heat losses by increasing their heat production (Smith, 1979). In the present study, animals submitted to FST in water at a temperature of 25 °C, presented with pilo-erection during the swimming time. In addition, although animals in the FST were more immobile than rats submitted to SS, a marked increase in their paw muscle tone was observed during analysis. They probably adopted these behaviors to avoid heat losses. Whereas in SS, instead of presenting with pilo-erection, the animals were seen doing more vigorous physical exercise. Moreover, Bruner and Vargas (1994) and Marcondes in 2002 (data not published) observed that water temperatures around 30 °C prevented exchange between water and body temperatures, which also helps to maintain the body temperature. In spite of the influence of the water temperature on core body temperature, the authors believe that the different behavior patterns between the FST and SS are due to association among the three factors: tank size, water depth and temperature (Martí and Armario, 1994), and that they cannot be evaluated separately, since FST and SS are two different situations. In addition to the water temperature in SS, water depth was another important swimming test factor that led to reduced immobility to keep the animal above the water surface, and as described above, the tank dimensions also affected immobility. These observations lead to the conclusion that the two models presented in this study appear to represent different situations to the laboratory rat and the meaning of immobility could vary, depending on the model chosen (Calil et al., 2002). Some authors (Thierry et al., 1984; Abel, 1994) believe that the development of immobility in FST could represent either failure to persist with escape-directed behavior or to develop passivity in response to the situation. Although the FST model represented a less stressful situation, compared with the swimming stress model, the authors observed higher corticosterone levels in animals submitted to FST, compared with the control group. This increased corticosterone was possibly due to the partial immersion in water and the inescapability that faced the animal in FST. Moreover, the water temperature in this model was established at 25 °C. This lower water temperature could interfere in the animal's body temperature during swimming. In this context, it has been reported that lower water temperature can cause elevations in corticosterone due to hypothermia (Arai et al., 2000; Taltavull et al., 2003; Drugan et al., 2005). On the other hand, the Swimming Stress model seems to be a more stressful situation than FST, because of the menace of drowning. The higher increase in the corticosterone response in rats submitted to SS, in comparison with those submitted to FST supported this hypothesis. Moreover, Drugan et al. (2005) demonstrated that although the rise in corticosterone in rats submitted to water temperature of 30 °C were not as high as at cooler temperatures, this rise was still significantly higher than that in control groups. Therefore, when the water temperature, water
1717
depth and tank dimensions in the SS are taken together, it is a different situation compared with the FST which, according to the results of the present study, causes different behavior and endocrine responses in rats. This result is consistent with previous finding that reported HPA axis activation in response to swimming stress (Abel, 1994). The immobility behavior of rats exposed to Swimming Stress is perhaps the result of an adaptive response to a stressful situation, as there is danger of sinking and drowning and consequently danger of death (Bruner and Vargas, 1994; Bianchi et al., 2001; Calil et al., 2002). The higher intensity of stress response to SS shows that the physiological stress system activation differs between SS and FST models, as was hypothesized in this study. It is also noteworthy that plasma corticosterone is a well-accepted fear/anxiety index under stressful conditions (Martí and Armario, 1994; De Boer et al., 1990) and the data in this study present evidence that the behavior of rats submitted to SS might be related to fear/anxiety rather than being “learned helplessness”. Since the animals were anesthetized with halothane and blood was collected from the left renal vein, corticosterone levels were expected to increase even in the control animals (Abel, 1993). Although the control corticosterone plasma levels observed in the present study are higher than those previously described in the literature, observed after decapitation, the method of blood collection was the same in the control and swimming groups. Therefore the hormonal differences between FST and SS appear to be related to the differences between the swimming models. The glycemia and tissue glycogen content results show that metabolic responses of animals submitted to FST are different from those observed in animals submitted to SS. As part of stress response, there is increased glucose use by the organism, and catecholamines and glucocorticoids stimulate hepatic glycogenolysis, the main source to maintain glycemia (Marliss et al., 2000). The higher glycemia and hepatic glycogen mobilization observed after SS suggest a higher energy demand in this model and also confirm that SS appears to be a more stressful situation, in comparison with FST. Furthermore, because of the drowning menace and consequent increase in brain activity in the SS model, the glycemia- and hepatic glycogenolysis-related responses may be regarded as evidence of the organism's adaptation to the situation (Blawacka et al., 1977), since it cannot be ruled out that coping with moderate fatigue during swimming could have a bearing on emotional aspects (Kramer et al., 1991). Thus, Ollenberger et al. (1998) found increased relative blood flow to the head during swimming; this probably reflects a fall in cerebrovascular resistance and corresponding absolute increase in cerebrovascular flow, due to the stress the animals are faced with. In addition to liver glycogen, muscle glycogen is also an essential fuel source used during exercise (Ivy and Kuo, 1998; Cunha et al., 2005), but only muscle cells can use it (Marliss et al., 2000). Exercise sessions are known to result in muscle glycogen depletion. The significant decrease observed in both soleus and gastrocnemius glycogen content in animals submitted to SS, and no change in animals exposed to FST, is in accordance with the immobility time observed. During immobility time recording, the experimenter observed that the
1718
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719
animals did not perform effective exercise in FST, and could touch the bottom of the tank. Whereas in the SS model, the rats continuously alternated paw movements and floating. Even during floating, they made some movements to keep their noses above the water surface. The results of this study can be associated with the findings of Ollenberger et al. (1998). These authors observed an increased blood flow in rat hindlimb muscles during swimming, and it probably reflects increased locomobile activity. Therefore, there are differences in muscle activity between FST and SS. In this study, the results of behavior analysis during the swimming session and metabolic and hormonal analyses assessment after the swimming session, suggest that the Swimming Stress and Forced Swimming Test induced different physiologic responses and therefore, they presented the laboratory rat with two different situations, confirming the initial hypothesis of this study. Since pharmacological validity is appropriate in behavioral procedures (Martí and Armario, 1993) and the meaning of immobility mainly reflects a state of despair, specifically in FST, (Porsolt et al., 1978b), in addition to considering that this behavior was reduced by a variety of treatments that are therapeutically effective in depression, the effect of the antidepressive desipramine and the anxiolytic diazepam on exposure to both FST and SS was evaluated. As expected, the data presented evidence that immobility in FST is related to a state of despair, or “learned helplessness”, since desipramine significantly decreased immobility time of rats that had previously undergone a 15-min FST and also in rats submitted to only a single 15-min swimming session. It would appear that rats submitted to FST tend to adopt passive strategies when facing inescapable situations, and this behavior was reverted by administering an antidepressive. The findings of the inhibitory effect of desipramine on immobility are in accordance with results in the literature (Martí and Armario, 1993). From this point of view, animals may genetically be highly prepared to adopt immobile responses in water when they learn that more active and aggressive responses do not permit escape (Thierry et al., 1984). Otherwise, the increase in the immobility response of diazepam-treated animals submitted to SS could be interpreted as an adaptive response to stress, which conserves energy output in response to water depth (Abel, 1993). These data are consistent with many previous reports concerning the ability of minor tranquilizers, such as diazepam, to enhance prolonged duration of immobility in rats (Nishimura et al., 1987). These authors indicated that by adopting more immobile behavior during swimming the rats might gain a survival advantage. When rats are observed for extended periods in a swimming chamber, animals that adopt more immobile responses are able to remain afloat for a longer time without sinking. Thus, in terms of survival, immobility during SS appears to represent a form of successful coping rather than failure to cope. Furthermore, in the present study, diazepam did not affect the rats' behavior during exposure to FST, in good agreement with previous reports (Porsolt et al., 1977; Martí and Armario, 1993). These data may help one to understand the differences between
the Forced Swimming Test and the Swimming Stress, which probably represent different situations to the laboratory animal. In conjunction with the findings of Porsolt et al. (1977), Barros and Ferigolo (1998), Hinojosa et al. (2005), the data in the present study show that FST is a suitable animal model to detect antidepressants. However, it was not the purpose of this study to discuss the validity of this experimental animal model. Its aim was to test the hypothesis that changing the swimming session characteristics (tank size, water temperature and water depth) presents the animal with different situation which, in turn, induces different behavior patterns, as well as different metabolic and hormonal responses, and the data of this study confirm this hypothesis. Therefore, the choice of the animal swimming model must be given careful attention. Acknowledgements This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (99/ 00793-1) and FAEP/UNICAMP (71/99 and 27/00). C.M.C. received a FAPESP (99/08286-1) scholarship grant. The authors are grateful to Celso Rodrigues Franci, PhD for the corticosterone determination. The authors thank Maria Claudia de Castro Souza for her laboratory help and Margery Galbraith for editing the English in the manuscript. References Abel, E.L., 1993. Physiological correlates of the forced swim test in rats. Physiology and Behavior 54, 309–317. Abel, E.L., 1994. A further analysis of physiological changes in rats in the forced swim test. Physiology and Behavior 56 (4), 795–800. Arai, I., Tsuyuki, H., Shimoto, M., Satoh, M., Otomoto, S., 2000. Decrease body temperature dependent appearance of behavior despair in the forced swimming test in mice. Pharmacology and Research 42, 171–176. Baker, M.A., Horvath, S.M., 1964. Influence of water temperature on oxygen uptake by swimming rats. Journal of Applied Physiology 19, 1215–1218. Bargiel, Z., Nowicka, H., Wojcikowska, J., 1981. Swim–stress changes of rat adrenal catecholamine level depending on metabolic state and different ambient temperature. Folia Histochemica et Cytochemica (Krakow) 19, 31–37. Barros, H.M.T., Ferigolo, M., 1998. Ethopharmacology of imipramine in the forced-swimming test: gender differences. Neuroscience and Biobehavioral Reviews 23, 279–286. Beijamini, V., Skalisz, L.L., Joca, S.R., Andreatini, R., 1998. The effect of oxcarbazepine on behavioral despair and learned helplessness. European Journal of Pharmacology 347 (1), 23–27. Bianchi, F.J., Tanno, A.P., Marcondes, F.K., 2001. Relationship between the level of stress and supersensitivity to norepinephrine in female rats at proestrus. Brazilian Journal of Pharmaceutical Sciences 37 (3), 391–398. Binik, Y.M., Sullivan, M.J.L., 1983. Sudden swimming deaths: a psychomotor reinterpretation. Psychophysiology 20, 670–681. Blawacka, M., Roth, Z., Wojciechowska, F., Karon, H., 1977. Effect of exercise on glycogen level in muscles and liver in rats. Acta Physiologica Polonica 28 (5), 431–440. Borsini, F., Meli, A., 1988. Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology 94, 147–160. Borsini, F., Volterra, G., Meli, A., 1986. Does the behavioural “despair” test measure “despair”? Physiology and Behavior 38 (3), 385–386. Brown, P.L., Hurley, C., Repucci, N., Drugan, R.C., 2001. Behavior analysis of stress controllability effects in a new swim stress paradigm. Pharmacology, Biochemistry and Behavior 68, 263–272.
C.M. Calil, F.K. Marcondes / Life Sciences 79 (2006) 1712–1719 Bruner, C.A., Vargas, I., 1994. The activity of rats in a swimming situation as a function of water temperature. Physiology and Behavior 5, 21–28. Calil, C.M., Bianchi, F.J., Tanno, A.P., Cunha, T.S., Marcondes, F.K., 2002. Analysis of the meaning of the immobility time in swimming experimental models. Brazilian Journal of Pharmaceutical Sciences 38 (4), 479–485. Cunha, T.S., Tanno, A.P., Costa Sampaio Moura, M.J., Marcondes, F.K., 2005. Influence of high-intensity exercise training and anabolic androgenic steroid treatment on rat tissue glycogen content. Life Sciences 77 (9), 1030–1043. Dawson, C.A., Nadel, E.R., Horvath, S.M., 1968. Cardiac output in the coldstressed swimming rat. American Journal of Physiology 214, 320–325. De Boer, S.F., Koopmans, S.J., Slangen, J.L., Van der Gugten, J., 1990. Plasma catecholamines, corticosterone and glucose responses to repeated stress in rats: effect of interstressor interval length. Physiology and Behavior 47, 117–1124. De Pablo, J.M., Parra, A., Segovia, S., Guillamon, A., 1989. Learned immobility explains the behavior of rats in the forced swimming test. Physiology and Behavior 46 (2), 229–237. Drugan, R.C., Eren, S., Hazi, A., Silva, J., Christianson, J.P., Kent, S., 2005. Impact of water temperature and stressor controllability in swim stressinduced changes in body temperature, serum corticosterone, and immobility in rats. Pharmacology, Biochemistry and Behavior 82, 397–403. Genaro, G., Schmidekand, W.R., Franci, C.R., 2004. Social condition affects hormone secretion and exploratory behavior in rats. Exploration and hormones in grouped or isolated rats. Brazilian Journal of Medical and Biological Research 37, 833–840. Hilakivi, L.A., Hilakivi, I., 1987. Increased adult behavioral “despair” in rats neonatally exposed to desipramine or zimeldine: an animal model of depression? Pharmacology Biochemistry and Behavior 28 (3), 367–369. Hinojosa, F.R., Spricigo Jr., L., Izidio, G.S., Bruske, G.R., Lopes, D.M., Ramos, A., 2005. Evaluation of two genetic animal models in behavioral tests of anxiety and depression. Behavioral and Brain Research 168 (1), 127–136. Ivy, J.L., Kuo, C.H., 1998. Regulation of GLUT4 protein and glycogen synthase during muscle glycogen synthesis after exercise. Acta Physiologica Scandinavica 162 (3), 295–304. Kramer, K., Dijkstra, H., Bast, A., 1991. Control of physical exercise of rats in a swimming basin. Physiology and Behavior 58, 271–276. Lo, S., Russell, J.C., Taylor, A.W., 1970. Determination of glycogen in small tissue samples. Journal of Applied Physiology 28 (2), 234–236. Marcondes, F.K., Vanderlei, L.C.M., Lanza, L.L.B., Spadari-Bratfisch, R.C., 1996. Stress-induced subsensitivity to catecholamines depends on the estrous cycle. Canadian Journal of Physiology and Pharmacology 74, 663–669. Martí, J., Armario, A., 1993. Effects of diazepam and desipramine in the forced swimming test: influence of previous experience with the situation. European Journal of Pharmacology 236, 295–299. Martí, J., Armario, A., 1994. Forced swimming behavior is not related to the corticosterone levels achieved in the test: a study with four inbred rat strains. Physiology and Behavior 59 (2), 369–373. Marliss, E.B., Kreisman, S.H., Manzon, A., Halter, J.B., Vranic, M., Nessim, S., 2000. Gender differences in glucoregulatory responses to intense exercise. Journal of Applied Physiology 88 (2), 457–466.
1719
Nishimura, H., Tsuda, A., Oguchi, M., Tanaka, I., Tanaka, M., 1987. Is immobility of rats in the forced swim test “Behavioral despair”? Physiology and Behavior 42, 93–95. Niswender, G.D., Midgley Jr., A.R., Monroe, S.E., Reichert Jr., L.E., 1968. Radioimmunoassay for rat luteinizing hormone with antibovine LH serum and ovine LH 131I. Proceedings of the Society for Experimental Biology and Medicine 128, 807–811. Niswender, G.D., Chen, C.L., Midgley Jr., A.R., Meites, J., Ellis, E., 1969. Radioimmunoassay for rat prolactin. Proceedings of the Society for Experimental Biology and Medicine 130, 793–797. Ollenberger, G.P., Matte, G., Wilkinson, A., West, N.H., 1998. Relative distribution of blood flow in rats during surface and submerged swimming. Comparative Biochemistry and Physiology 119 (1), 271–277. Peeters, B.W.M.M., Smets, R.J.M., Broekkamp, C.L.E., 1991. The involvement of glicocorticoids in the acquired immobility response is dependent on the water temperature. Physiology and Behavior 51, 127–129. Porsolt, R.D., Le Pichon, M., Jalfre, M., 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266 (5604), 730–732. Porsolt, R.D., Bertin, A., Jalfre, M., 1978a. “Behavioural despair” in rats and mice: strain differences and the effects of imipramine. European Journal of Pharmacology 51 (3), 291–294. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978b. Behavioural despair in rats: a new model sensitive to antidepressant treatments. European Journal of Pharmacology 47 (4), 379–391. Shors, T.J., Pickett, J., Wood, G., Paczynski, M., 1999. Acute stress persistently enhances estrogen levels in the female rat. Stress 3 (2), 163–171. Smith, I.O., 1979. Adaptative changes in the sympathetic nervous system and some effector organs of the rat following long term exercise or cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hypertrophy. Acta Physiologica Scandinavica 477, 53–54. Spadari, R.C., De Moraes, S., 1988. Repeated swimming stress and responsiveness of the isolated rat pacemaker to the chronotropic effect of noradrenaline and isoprenaline: role of adrenal corticosteroids. General Pharmacology 19 (4), 553–557. Taltavull, J.F., Chefer, V.I., Shippenberg, T.S., Kiyatkin, E.A., 2003. Severe brain hypothermia as a factor underlying behavioral immobility during cold water forced swim. Brain Research 975, 244–247. Tanno, A.P., Bianchi, F.J., Moura, M.J.C.S., Marcondes, F.K., 2002. Atrial supersensitivity to noradrenaline in stressed female rats. Life Science 71 (25), 2973–2981. Thierry, B., Steru, L., Chermat, R., Simon, P., 1984. Searching-waiting strategy: a candidate for an evolutionary model of depression? Behavioral and Neural Biology 41 (2), 180–189. Vanderwolf, C.H., 1991. Effects of water temperature and core temperature on rat's performance in a swim-to-platform test. Behavioral and Brain Research 44, 105–106. Vecsei, P., 1979. Glucocorticoids: cortisol, corticosterone and compounds. In: Jaffe, M. (Ed.), Methods of Hormone Radioimmunoassay. Academic Press, San Diego, CA, USA, pp. 767–792.