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Chronic administration of fluoxetine impairs inhibitory avoidance in male but not female mice
Behavioural Brain Research 136 (2002) 483 /488 www.elsevier.com/locate/bbr
Research report
Chronic administration of fluoxetine impairs inhibitory avoidance in male but not female mice Santiago Monleo´n, Adoracio´n Urquiza, M. Carmen Arenas, Concepcio´n VinaderCaerols, Andre´s Parra * ´ rea de Psicobiologı´a, Facultad de Psicologı´a, Universitat de Vale`ncia, Blasco Iba´n˜ez 21, 46010-Valencia, Spain A Received 18 March 2002; received in revised form 24 June 2002; accepted 24 June 2002
Abstract The effects of chronic administration of fluoxetine (20 mg/kg/day i.p.) on a one-trial step-through inhibitory avoidance task were investigated in male and female CD1 mice. In Experiment 1, treatment was administered for 21 days before the training session, whereas in Experiment 2, other subjects were subjected to the same treatment starting 24 h after the training session. The comparison of test versus training latencies showed memory deterioration with pre-training administration of fluoxetine (Experiment 1), which affected males but not females. Sex differences in this task were also observed in Experiment 1, with females showing a better performance. Sex differences were evident in controls as well as in treated animals. The locomotor activity of the animals was also tested in Experiment 1. Due to the absence of sex differences in the drug effects on this measure, the sex differences in the effects of fluoxetine on inhibitory avoidance were hardly attributable to non specific effects on locomotor activity. The lack of effect of post-training administration of fluoxetine (Experiment 2) constitutes additional support of the idea that the observed effect on inhibitory avoidance in Experiment 1 is specifically related to learning and memory. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fluoxetine; Antidepressants; Memory; Inhibitory avoidance; Sex differences; Mice
1. Introduction Depressed patients usually show cognitive problems such as a diminished capacity for thinking and concentration [2] and, as a possible consequence, loss of memory. Thus, a significant association between depression and memory impairment has been observed [7]. In addition to these memory deficits in depression, many antidepressant drugs used in clinical practice have anticholinergic properties. Since cholinergic transmission at muscarinic receptors has been implicated in higher brain functions such as learning and memory [21], the anticholinergic actions of antidepressants appear responsible for memory impairment [1,9,12,39]. Therefore, these anticholinergic effects can be considered as unwanted and preventable characteristics of antidepressant drugs [39]. All this suggests that a wider
* Corresponding author E-mail address: [email protected] (A. Parra).
knowledge of the cognitive profile of antidepressants is necessary to be able to choose the most appropriate treatment of depressed patients with memory problems. The pharmacology of memory in animals is commonly studied using inhibitory avoidance (also called passive avoidance) (e.g. [16]). This behavioural paradigm has also been widely used for studying memory formation [18]. In the step-through version, the animal must inhibit crossing to the dark compartment of a box to avoid a footshock [6]. In previous studies using this technique, impairment of memory by the antidepressants amitriptyline [10,36] and maprotiline [37] was observed. Both drugs have strong anticholinergic properties. The antidepressant selective serotonin reuptake inhibitor fluoxetine has little affinity for muscarinic acetylcholine receptors. Consequently, its effects on memory appear less harmful than those of tricyclic antidepressants [12,20,39]. Besides the treatment of depression and associated anxiety [26], fluoxetine is also used in other mental illnesses, such as obsessivecompulsive disorder [4], nervous bulimia [13], obesity
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[17], aggression [40] and posttraumatic stress [23]. This drug has greatly prescribed during the last decade as a consequence of its profile with fewer adverse side effects compared to classical antidepressants. The effects of acute administration of fluoxetine (5, 10, and 20 mg/kg) on memory consolidation have been investigated in male and female OF1 mice in this laboratory. Acute treatment with fluoxetine did not impair inhibitory avoidance learning, and the highest dose even increased response latencies compared to lowest dose and control groups [30]. The present study was designed to investigate the chronic effects of fluoxetine on inhibitory avoidance learning in mice. Male and female mice were included because of the wellknown gender differences in the epidemiology of depression (women /men) [2,11,19], their different responses to antidepressant treatment [11], and because sex differences in inhibitory avoidance have been reported [3,14,15].
2. Materials and methods 2.1. Subjects Male and female CD1 mice (CRIFFA, Lyon, France) were used as experimental subjects. They arrived at the laboratory at 42 days of age and were housed in groups of 4 in translucent plastic cages (27 x 27 x 14.5 cm3), with tops made of stainless steel bars (Panlab S.L., Barcelona, Spain). Subjects were maintained at a constant temperature (219/2 8C), under a modified light schedule (white lights on 19:30 /07:30 h), with food and water available ad libitum. Each mouse was tested only once. The tests were always carried out during the dark phase of the light-dark cycle, and took place after 11/19 days of acclimatisation to the animal house. 2.2. Drugs Fluoxetine hydrochloride (Eli Lilly, Indianapolis, U.S.A.) was dissolved in physiological saline (0.9% NaCl) and injected intraperitoneally at a volume of 0.01 ml/g body weight. Doses of drug were calculated as the respective salts. The control groups received the same volume of physiological saline. 2.3. Apparatus An inhibitory avoidance apparatus for mice (Ugo Basile, Comerio /Varese, Italy) was used. The cage, constructed from Perspex sheets, was divided into two sections (each of 15 x 9.5 x 16.5 cm3). These chambers were separated by a flat-box partition, including an automatically-operated sliding door at floor level. A light (24 V, 10 W) was always on in the ceiling of the starting
side, with the other side remaining dark. The starting side was white and the other black. The floor was made of stainless steel bars, 0.7 mm in diameter and 8 mm apart. The computerised equipment for locomotor activity measurement in rodents ‘Actisystem II’ with ‘DAS 16’ software (both from Panlab S.L., Barcelona, Spain) were also used in Experiment 1. Basically, this system records the changes produced in the magnetic field of its sensor unit (35 x 35 cm2) by the spontaneous locomotor activity of the animals and the registration is converted into digital counts.
3. Procedure 3.1. Experiment 1: Chronic pre-training treatment Twenty-four CD1 mice of each sex were randomly allocated to two groups and subjected to daily treatment for 21 days with saline (S) or 20 mg/kg of fluoxetine (F20). Subjects were randomly subjected to a one-trial step-through inhibitory avoidance task 24 h after the last injection. In the training session, each mouse was individually placed in the lighted compartment. After a 90 sec adaptation period in the safe chamber, the door between the compartments was opened and the time taken to enter the dark compartment was measured. As soon as the mouse entered the dark compartment, the sliding door was closed and a foot-shock of 0.7 mA for 5 sec was delivered through the grid floor. Crossing latencies longer than 300 sec resulted in the termination of the trial and a latency of 300 sec recorded. The locomotor activity of the animals was also monitored for 5 min immediately after the training session in order to control this possibly confounding factor. Following the schedule used in a previous study [30], the test session was carried out 4 days later with the same procedure as in the training session (but without shock delivery). 3.2. Experiment 2: Chronic post-training treatment Another 24 male and 24 female CD1 mice were randomly allocated to two groups and were subjected to chronic daily S or F20 treatments for 21 days after the training phase of the inhibitory avoidance procedure (starting 24h later). Subjects were subjected to the test phase 4 days after the final injection. Otherwise, the same parameters as in the previous experiment were used. All testing of both experiments was run during the dark phase of the light cycle. 3.3. Data analysis The inhibitory avoidance data were subjected to the nonparametric Mann-Whitney U -tests for comparisons
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between pairs of groups of treatment. Comparisons between training and test sessions within the same drug treatment were performed using the Wilcoxon test. As the locomotor activity data fulfilled the criteria for normality and homogeneity, the activity counts were subjected to analysis of variance (ANOVA), with Treatment and Sex (two levels each) as between-subject factors and Time (five min blocks) as within-subject factor. The Newman-Keuls test was used for post hoc comparisons. All analyses were performed using the ‘Statistica’ version 5.5 for Windows software package [45].
Only the main factor Sex was statistically significant (F(1,43) /8.23, P B/0.01), with males showing more activity than females (independently of treatment administered). The Treatment x Sex interaction was not statistically significant (F(1,43) /0.18, n.s.). Even so, the Newman-Keuls test for post-hoc comparisons was run with this interaction, checking that there were no significant differences between any pair of groups. The only statistically significant interaction was Treatment x Time (F(4,172) /4.35, P B/0.01) and the post-hoc comparisons showed greater motor activity in saline animals in min 2 (P B/0.01) and 5 (P B/0.05) compared with min 1.
4. Results
4.2. Experiment 2: Chronic post-training treatment
4.1. Experiment 1: Chronic pre-training treatment
As in Experiment 1, the comparison of latencies of treated animals versus controls was not statistically significant, in males (U /65.5, n.s.) or females (U / 66, n.s.). The comparison of training latencies versus test latencies revealed longer latencies in the test phase than in the training phase in males (S: T /4, P B/0.01; F20: T /7, P B/0.05) and females (S: T /1, P B/0.01; F20: T /1, P B/0.01). Finally, no sex differences were observed in this experiment (S: U /64.5, n.s.; F20: U / 65.5, n.s.) (see Fig. 2).
The comparison of latencies of treated animals versus controls was not statistically significant in males (U / 56, P /0.05) or females (U /58.5, P /0.05). Conversely, the comparison of training latencies versus test latencies was statistically significant in control and treated females (S: T /0, P B/0.01; F20: T /0, P B/ 0.01) as well as in saline males (S: T /11, P B/0.05), but not in males treated with fluoxetine (F20: T /12, P /0.05). Sex differences were also observed, females showing longer response latencies than males (S: U /30, P B/0.05; F20: U /29, P B/0.05), in test latencies (see Fig. 1). The locomotor activity analysis showed no statistically significant differences with respect to Treatment (F(1,43) /0.02, n.s.) and Time (F(4,172) /1.60, n.s.).
5. Discussion In Experiment 1 (pre-training treatment) the comparison of training latencies versus test latencies showed performance deterioration in the group of males treated
Fig. 1. Effects of daily intraperitoneal pre-training treatment (S /saline, F20 /20 mg/kg fluoxetine) for 21 days on an inhibitory avoidance task in male and female CD1 mice (n /12 in each group). Results are expressed as medians (9/ interquartile range). # P B/0.05, ## P B/0.01 vs training session (Wilcoxon tests); * P B/0.05 vs males (Mann-Whitney tests).
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Fig. 2. Effects of daily intraperitoneal post-training treatment (S /saline, F20 /20 mg/kg fluoxetine) for 21 days on an inhibitory avoidance task in male and female CD1 mice (n /12 in each group). Results are expressed as medians (9/ interquartile range). # P B/0.05, ## P B/0.01 vs training session (Wilcoxon tests).
with fluoxetine (i.e., no significant difference between training and test latencies). This impairing effect of the drug was not observed in females. More marked drug effects have been found in this laboratory in males than females both with the antidepressant amitriptyline in inhibitory avoidance [10,36], and with several neuroleptics in active avoidance (see [35] for a review). The possible explanations for this observation can have either a central or a peripheral origin. With respect to the first possibility, numerous studies have suggested that central neurotransmission is modulated by oestrogens and progesterone. Thus, for example, sex steroids modulate serotonin neurotransmission [27] as well as dopamine and GABAA receptors [5]. With respect to the second, there are sex differences in drug-metabolizing enzyme activities, these being 40-100% higher in female than in male mice [41], which would result in less drug availability in the CNS of females as compared with males. Nevertheless, although gender differences in pharmacokinetics and pharmacodynamics have been recognised in animals and humans, this aspect has not received much attention in experimental animal studies [34]. The presence of drug effect in males but not in females may be explained by control females seeming to have learned the task better than their counterpart males (see Fig. 1). The drug could have had greater influence depending on how well the behaviour has been learned. If learning has been strong, as is the case of females (i.e., high median in the test phase) the effect will be weak, not reaching statistical significance, although data from treated animals seem to be more disperse than those of controls. In males with weaker learning (i.e., low median
in the test phase), the treatment abolished the differences in performance between training and test. Sex differences in inhibitory avoidance observed in control subjects in Experiment 1, females showing better performance than males, has also been previously reported [3,14,15,30]. It has been suggested that these sex differences could be related to the central serotonergic system [14]. A purely behavioural explanation for such sex differences could also be argued. The sex differences in locomotor activity observed in Experiment 1, males showing more activity than females, that are in agreement [8] and in disagreement [43] with other observations, could favour the poorer performance of males in a task where the correct response is to inhibit responding. But it is important to note that the sex differences in locomotor activity were only significant taking saline and treated animals together. As stated above, the Treatment x Sex interaction was not statistically significant and even when the post-hoc comparisons were performed there were no significant differences in locomotor activity between males and females, in saline or treated animals. Thus the sex differences in inhibitory avoidance observed in the present study should not be attributed to sex differences in locomotor activity. Motor effects of fluoxetine have been observed under a variety of experimental conditions; in particular, a drug-induced increase in locomotor activity has been found with this antidepressant [8]. This may produce a behavioural desinhibition, which could lead to an impairment of inhibitory avoidance responding. Thus, before a change in performance can be attributed to learning and memory processes, this possibly confound-
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ing factor must be checked. For this reason, the locomotor activity of the animals was also tested in Experiment 1. The results showed no significant differences in this measure between the treated and nontreated groups, which may indicate a phenomenon of tolerance in this particular effect of fluoxetine. These results, along with the lack of differences in latencies of the training phase between treated and non-treated subjects, both in males and females, lead one to think that interfering motor effects of fluoxetine can be discarded in this study. Taking into account these results, locomotor activity was not evaluated in Experiment 2. The absence of effect of chronic treatment when the drug was administered between training and test (Experiment 2) constitutes an additional support to the idea that the observed effect in Experiment 1 is specifically related to learning and memory. It is important to remember that the injection series started 24 h after training took place, a period of time long enough to think that the consolidation phase of memory was already completed [29]. Considering that fluoxetine has a very weak anticholinergic effect, its impairing effects on inhibitory avoidance must be due to another mechanism of action. Other authors and ourselves have claimed that the anticholinergic actions of antidepressants are responsible for memory impairment [1,9,10,12,36,37,39]. Nevertheless, the effects of fluoxetine and other antidepressants on memory processes could be mediated by additional cerebral mechanisms, besides their anticholinergic actions. In this way, an important property of some antidepressants is that their chronic administration increases neurogenesis in the hippocampus [22,24]. It has been confirmed that fluoxetine particularly increases the content of neurotrophic factors in this region [25]. Considering that neurogenesis subserves long-term potentiation [44] and learning [42], it could be argued that antidepressants, instead of impairing, could actually favour memory. Nevertheless, the impairing effects of antidepressants on memory can still hold because the experimental extinction of previously learned behaviours seems to derive from the learning of new behaviours that are not compatible with the old ones [38]. Furthermore, recent studies show that extinction involves the synthesis of new proteins [31,47]. It could be the case that the role of antidepressants is to impair old memories by favouring the creation of new ones. Comparing our results with others, and taking into account only studies with sub-chronic and chronic administration (being those that administered fluoxetine acutely discarded), it is important to note that to our knowledge there are no studies on the effects of this drug on inhibitory avoidance, therefore the comparisons must be established with observations in which other
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learning tasks were involved. These tasks were spatial memory tests [33,46,48] and active avoidance [28,32]. The results obtained by these authors can be grouped into two, one with no or improving effects on spatial memory, and the other with impairing effects on active avoidance. Perhaps the sense of the effects depends on the kind of task. ‘‘The inhibitory avoidance task used in the present study shares a high degree of emotionality with active avoidance as both use electric shock’’. In summary, we have found that fluoxetine, in spite of being a drug with non significant anticholinergic activity, has an important impairing effect on inhibitory avoidance given before training. This effect was not attributable to non specific effects on motor activity, and was restricted to male subjects. Despite the fact that our study does not contribute to clarify the reasons for these gender differences in drug effects on behaviour, more studies including females can be needed in basic research in Behavioural Pharmacology. The great majority of basic research has focused on male rodents as animal models [34], perhaps females as experimental subjects are often neglected because they are thought to be too ‘variable’, although this aspect has not been categorically confirmed [35].
Acknowledgements The work reported herein was supported by the grant ‘Efecto de los antidepresivos sobre la memoria’ (Code GV 97-SH-22-89) from Generalitat Valenciana, the regional government of Valencia (Spain), and by the University of Valencia.
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[29] McGaugh JL, Izquierdo I. The contribution of pharmacology to research on the mechanisms of memory formation. Trends Pharmacol Sci 2000;21:208 /10. [30] Monleo´n S, Casino A, Vinader-Caerols C, Arenas MC. Acute effects of fluoxetine on inhibitory avoidance consolidation in male and female OF1 mice. Neurosci Res Commun 2001;28:123 /30. [31] Nader K, Schafe GE, Le Doux JE. Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature 2000;406:722 /6. [32] Nelson CJ, Jordan WP, Bohan RT. Daily fluoxetine administration impairs avoidance learning in the rat without altering sensory thresholds. Prog Neuropsychopharmacol Biol Psychiatry 1997;21:1043 /57. [33] Nowakowska E, Kus K, Chodera A, Rybakowski J. Behavioural effects of fluoxetine and tianeptine, two antidepressants with opposite action mechanisms, in rats. Arzneimittelforschung 2000;50:5 /10. [34] Palanza P. Animal models of anxiety and depression: how are females different? Neurosci Biobehav Rev 2001;25:219 /33. [35] Parra A, Arenas MC, Monleo´n S, Vinader-Caerols C, Simo´n VM. Sex differences in the effects of neuroleptics on escape-avoidance behavior in mice: a review. Pharmacol Biochem Behav 1999;64:813 /20. [36] Parra A, Everss E, Arenas MC, Vinader-Caerols C, Monleo´n S. Effects of acute amitriptyline administration on inhibitory avoidance, anxiety and activity in male and female mice. Pharmacol Biochem Behav (submitted). [37] Parra A, Monleo´n S, Arenas MC, Vinader-Caerols C. Effects of acute and chronic administration of maprotiline in an inhibitory avoidance task. Behav Brain Res 2000;109:1 /7. [38] Rescorla RA. Experimental extinction. In: Mowrer RR, Klein SB, editors. Handbook of contemporary learning theories. Mahwah: LEA, 2001:119 /54. [39] Riedel WJ, Van Praag HM. Avoiding and managing anticholinergic effects of antidepressants. CNS Drugs 1995;3:245 /59. [40] Scahill L, Koenig K. Pharmacotherapy in children and adolescents with pervasive developmental disorders. J Child Adolesc Psychiatr Nurs 1999;12:141 /3. [41] Shapiro BH, Agrawal AK, Pampori NA. Gender differences in drug metabolism regulated by growth hormone. Int J Biochem Cell Biol 1995;27:9 /20. [42] Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E. Neurogenesis in the adult is involved in the formation of trace memories. Nature 2001;410:372 /6. [43] Simo´n VM, Parra A, Min˜arro J, Arenas MC, Vinader-Caerols C, Aguilar MA. Predicting how equipotent doses of chlorpromazine, haloperidol, sulpiride, raclopride and clozapine reduce locomotor activity in mice. European Neuropsychopharmacol 2000;10:159 / 64. [44] Snyder JS, Kee N, Wojtowicz JM. Effects of adult neurogenesis on synaptic plasticity in the rat dentate gyrus. J Neurophysiol 2001;85:2423 /31. [45] StatSoft, Inc. (2000). STATISTICA for Windows [Computer program manual]. Tulsa, OK: StatSoft, Inc. [46] Stewart CA, Reid IC. Repeated ECS and fluoxetine administration have equivalent effects on hippocampal synaptic plasticity. Psychopharmacology 2000;148:217 /23. [47] Vianna MRM, Szapiro G, McGaugh JL, Medina JH, Izquierdo I. Retrieval of memory for fear-motivated training initiates extinction requiring protein synthesis in the rat hippocampus. Proc Nat Acad Sci 2001;98:12251 /4. [48] Yau JL, Noble J, Hibberd C, Seckl JR. Short-term administration of fluoxetine and venlafaxine decreases corticosteroid receptor mRNA expression in the rat hippocampus. Neurosci Lett 2001;306:161 /4.
Research report
Chronic administration of fluoxetine impairs inhibitory avoidance in male but not female mice Santiago Monleo´n, Adoracio´n Urquiza, M. Carmen Arenas, Concepcio´n VinaderCaerols, Andre´s Parra * ´ rea de Psicobiologı´a, Facultad de Psicologı´a, Universitat de Vale`ncia, Blasco Iba´n˜ez 21, 46010-Valencia, Spain A Received 18 March 2002; received in revised form 24 June 2002; accepted 24 June 2002
Abstract The effects of chronic administration of fluoxetine (20 mg/kg/day i.p.) on a one-trial step-through inhibitory avoidance task were investigated in male and female CD1 mice. In Experiment 1, treatment was administered for 21 days before the training session, whereas in Experiment 2, other subjects were subjected to the same treatment starting 24 h after the training session. The comparison of test versus training latencies showed memory deterioration with pre-training administration of fluoxetine (Experiment 1), which affected males but not females. Sex differences in this task were also observed in Experiment 1, with females showing a better performance. Sex differences were evident in controls as well as in treated animals. The locomotor activity of the animals was also tested in Experiment 1. Due to the absence of sex differences in the drug effects on this measure, the sex differences in the effects of fluoxetine on inhibitory avoidance were hardly attributable to non specific effects on locomotor activity. The lack of effect of post-training administration of fluoxetine (Experiment 2) constitutes additional support of the idea that the observed effect on inhibitory avoidance in Experiment 1 is specifically related to learning and memory. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fluoxetine; Antidepressants; Memory; Inhibitory avoidance; Sex differences; Mice
1. Introduction Depressed patients usually show cognitive problems such as a diminished capacity for thinking and concentration [2] and, as a possible consequence, loss of memory. Thus, a significant association between depression and memory impairment has been observed [7]. In addition to these memory deficits in depression, many antidepressant drugs used in clinical practice have anticholinergic properties. Since cholinergic transmission at muscarinic receptors has been implicated in higher brain functions such as learning and memory [21], the anticholinergic actions of antidepressants appear responsible for memory impairment [1,9,12,39]. Therefore, these anticholinergic effects can be considered as unwanted and preventable characteristics of antidepressant drugs [39]. All this suggests that a wider
* Corresponding author E-mail address: [email protected] (A. Parra).
knowledge of the cognitive profile of antidepressants is necessary to be able to choose the most appropriate treatment of depressed patients with memory problems. The pharmacology of memory in animals is commonly studied using inhibitory avoidance (also called passive avoidance) (e.g. [16]). This behavioural paradigm has also been widely used for studying memory formation [18]. In the step-through version, the animal must inhibit crossing to the dark compartment of a box to avoid a footshock [6]. In previous studies using this technique, impairment of memory by the antidepressants amitriptyline [10,36] and maprotiline [37] was observed. Both drugs have strong anticholinergic properties. The antidepressant selective serotonin reuptake inhibitor fluoxetine has little affinity for muscarinic acetylcholine receptors. Consequently, its effects on memory appear less harmful than those of tricyclic antidepressants [12,20,39]. Besides the treatment of depression and associated anxiety [26], fluoxetine is also used in other mental illnesses, such as obsessivecompulsive disorder [4], nervous bulimia [13], obesity
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 1 9 4 - 8
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[17], aggression [40] and posttraumatic stress [23]. This drug has greatly prescribed during the last decade as a consequence of its profile with fewer adverse side effects compared to classical antidepressants. The effects of acute administration of fluoxetine (5, 10, and 20 mg/kg) on memory consolidation have been investigated in male and female OF1 mice in this laboratory. Acute treatment with fluoxetine did not impair inhibitory avoidance learning, and the highest dose even increased response latencies compared to lowest dose and control groups [30]. The present study was designed to investigate the chronic effects of fluoxetine on inhibitory avoidance learning in mice. Male and female mice were included because of the wellknown gender differences in the epidemiology of depression (women /men) [2,11,19], their different responses to antidepressant treatment [11], and because sex differences in inhibitory avoidance have been reported [3,14,15].
2. Materials and methods 2.1. Subjects Male and female CD1 mice (CRIFFA, Lyon, France) were used as experimental subjects. They arrived at the laboratory at 42 days of age and were housed in groups of 4 in translucent plastic cages (27 x 27 x 14.5 cm3), with tops made of stainless steel bars (Panlab S.L., Barcelona, Spain). Subjects were maintained at a constant temperature (219/2 8C), under a modified light schedule (white lights on 19:30 /07:30 h), with food and water available ad libitum. Each mouse was tested only once. The tests were always carried out during the dark phase of the light-dark cycle, and took place after 11/19 days of acclimatisation to the animal house. 2.2. Drugs Fluoxetine hydrochloride (Eli Lilly, Indianapolis, U.S.A.) was dissolved in physiological saline (0.9% NaCl) and injected intraperitoneally at a volume of 0.01 ml/g body weight. Doses of drug were calculated as the respective salts. The control groups received the same volume of physiological saline. 2.3. Apparatus An inhibitory avoidance apparatus for mice (Ugo Basile, Comerio /Varese, Italy) was used. The cage, constructed from Perspex sheets, was divided into two sections (each of 15 x 9.5 x 16.5 cm3). These chambers were separated by a flat-box partition, including an automatically-operated sliding door at floor level. A light (24 V, 10 W) was always on in the ceiling of the starting
side, with the other side remaining dark. The starting side was white and the other black. The floor was made of stainless steel bars, 0.7 mm in diameter and 8 mm apart. The computerised equipment for locomotor activity measurement in rodents ‘Actisystem II’ with ‘DAS 16’ software (both from Panlab S.L., Barcelona, Spain) were also used in Experiment 1. Basically, this system records the changes produced in the magnetic field of its sensor unit (35 x 35 cm2) by the spontaneous locomotor activity of the animals and the registration is converted into digital counts.
3. Procedure 3.1. Experiment 1: Chronic pre-training treatment Twenty-four CD1 mice of each sex were randomly allocated to two groups and subjected to daily treatment for 21 days with saline (S) or 20 mg/kg of fluoxetine (F20). Subjects were randomly subjected to a one-trial step-through inhibitory avoidance task 24 h after the last injection. In the training session, each mouse was individually placed in the lighted compartment. After a 90 sec adaptation period in the safe chamber, the door between the compartments was opened and the time taken to enter the dark compartment was measured. As soon as the mouse entered the dark compartment, the sliding door was closed and a foot-shock of 0.7 mA for 5 sec was delivered through the grid floor. Crossing latencies longer than 300 sec resulted in the termination of the trial and a latency of 300 sec recorded. The locomotor activity of the animals was also monitored for 5 min immediately after the training session in order to control this possibly confounding factor. Following the schedule used in a previous study [30], the test session was carried out 4 days later with the same procedure as in the training session (but without shock delivery). 3.2. Experiment 2: Chronic post-training treatment Another 24 male and 24 female CD1 mice were randomly allocated to two groups and were subjected to chronic daily S or F20 treatments for 21 days after the training phase of the inhibitory avoidance procedure (starting 24h later). Subjects were subjected to the test phase 4 days after the final injection. Otherwise, the same parameters as in the previous experiment were used. All testing of both experiments was run during the dark phase of the light cycle. 3.3. Data analysis The inhibitory avoidance data were subjected to the nonparametric Mann-Whitney U -tests for comparisons
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between pairs of groups of treatment. Comparisons between training and test sessions within the same drug treatment were performed using the Wilcoxon test. As the locomotor activity data fulfilled the criteria for normality and homogeneity, the activity counts were subjected to analysis of variance (ANOVA), with Treatment and Sex (two levels each) as between-subject factors and Time (five min blocks) as within-subject factor. The Newman-Keuls test was used for post hoc comparisons. All analyses were performed using the ‘Statistica’ version 5.5 for Windows software package [45].
Only the main factor Sex was statistically significant (F(1,43) /8.23, P B/0.01), with males showing more activity than females (independently of treatment administered). The Treatment x Sex interaction was not statistically significant (F(1,43) /0.18, n.s.). Even so, the Newman-Keuls test for post-hoc comparisons was run with this interaction, checking that there were no significant differences between any pair of groups. The only statistically significant interaction was Treatment x Time (F(4,172) /4.35, P B/0.01) and the post-hoc comparisons showed greater motor activity in saline animals in min 2 (P B/0.01) and 5 (P B/0.05) compared with min 1.
4. Results
4.2. Experiment 2: Chronic post-training treatment
4.1. Experiment 1: Chronic pre-training treatment
As in Experiment 1, the comparison of latencies of treated animals versus controls was not statistically significant, in males (U /65.5, n.s.) or females (U / 66, n.s.). The comparison of training latencies versus test latencies revealed longer latencies in the test phase than in the training phase in males (S: T /4, P B/0.01; F20: T /7, P B/0.05) and females (S: T /1, P B/0.01; F20: T /1, P B/0.01). Finally, no sex differences were observed in this experiment (S: U /64.5, n.s.; F20: U / 65.5, n.s.) (see Fig. 2).
The comparison of latencies of treated animals versus controls was not statistically significant in males (U / 56, P /0.05) or females (U /58.5, P /0.05). Conversely, the comparison of training latencies versus test latencies was statistically significant in control and treated females (S: T /0, P B/0.01; F20: T /0, P B/ 0.01) as well as in saline males (S: T /11, P B/0.05), but not in males treated with fluoxetine (F20: T /12, P /0.05). Sex differences were also observed, females showing longer response latencies than males (S: U /30, P B/0.05; F20: U /29, P B/0.05), in test latencies (see Fig. 1). The locomotor activity analysis showed no statistically significant differences with respect to Treatment (F(1,43) /0.02, n.s.) and Time (F(4,172) /1.60, n.s.).
5. Discussion In Experiment 1 (pre-training treatment) the comparison of training latencies versus test latencies showed performance deterioration in the group of males treated
Fig. 1. Effects of daily intraperitoneal pre-training treatment (S /saline, F20 /20 mg/kg fluoxetine) for 21 days on an inhibitory avoidance task in male and female CD1 mice (n /12 in each group). Results are expressed as medians (9/ interquartile range). # P B/0.05, ## P B/0.01 vs training session (Wilcoxon tests); * P B/0.05 vs males (Mann-Whitney tests).
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Fig. 2. Effects of daily intraperitoneal post-training treatment (S /saline, F20 /20 mg/kg fluoxetine) for 21 days on an inhibitory avoidance task in male and female CD1 mice (n /12 in each group). Results are expressed as medians (9/ interquartile range). # P B/0.05, ## P B/0.01 vs training session (Wilcoxon tests).
with fluoxetine (i.e., no significant difference between training and test latencies). This impairing effect of the drug was not observed in females. More marked drug effects have been found in this laboratory in males than females both with the antidepressant amitriptyline in inhibitory avoidance [10,36], and with several neuroleptics in active avoidance (see [35] for a review). The possible explanations for this observation can have either a central or a peripheral origin. With respect to the first possibility, numerous studies have suggested that central neurotransmission is modulated by oestrogens and progesterone. Thus, for example, sex steroids modulate serotonin neurotransmission [27] as well as dopamine and GABAA receptors [5]. With respect to the second, there are sex differences in drug-metabolizing enzyme activities, these being 40-100% higher in female than in male mice [41], which would result in less drug availability in the CNS of females as compared with males. Nevertheless, although gender differences in pharmacokinetics and pharmacodynamics have been recognised in animals and humans, this aspect has not received much attention in experimental animal studies [34]. The presence of drug effect in males but not in females may be explained by control females seeming to have learned the task better than their counterpart males (see Fig. 1). The drug could have had greater influence depending on how well the behaviour has been learned. If learning has been strong, as is the case of females (i.e., high median in the test phase) the effect will be weak, not reaching statistical significance, although data from treated animals seem to be more disperse than those of controls. In males with weaker learning (i.e., low median
in the test phase), the treatment abolished the differences in performance between training and test. Sex differences in inhibitory avoidance observed in control subjects in Experiment 1, females showing better performance than males, has also been previously reported [3,14,15,30]. It has been suggested that these sex differences could be related to the central serotonergic system [14]. A purely behavioural explanation for such sex differences could also be argued. The sex differences in locomotor activity observed in Experiment 1, males showing more activity than females, that are in agreement [8] and in disagreement [43] with other observations, could favour the poorer performance of males in a task where the correct response is to inhibit responding. But it is important to note that the sex differences in locomotor activity were only significant taking saline and treated animals together. As stated above, the Treatment x Sex interaction was not statistically significant and even when the post-hoc comparisons were performed there were no significant differences in locomotor activity between males and females, in saline or treated animals. Thus the sex differences in inhibitory avoidance observed in the present study should not be attributed to sex differences in locomotor activity. Motor effects of fluoxetine have been observed under a variety of experimental conditions; in particular, a drug-induced increase in locomotor activity has been found with this antidepressant [8]. This may produce a behavioural desinhibition, which could lead to an impairment of inhibitory avoidance responding. Thus, before a change in performance can be attributed to learning and memory processes, this possibly confound-
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ing factor must be checked. For this reason, the locomotor activity of the animals was also tested in Experiment 1. The results showed no significant differences in this measure between the treated and nontreated groups, which may indicate a phenomenon of tolerance in this particular effect of fluoxetine. These results, along with the lack of differences in latencies of the training phase between treated and non-treated subjects, both in males and females, lead one to think that interfering motor effects of fluoxetine can be discarded in this study. Taking into account these results, locomotor activity was not evaluated in Experiment 2. The absence of effect of chronic treatment when the drug was administered between training and test (Experiment 2) constitutes an additional support to the idea that the observed effect in Experiment 1 is specifically related to learning and memory. It is important to remember that the injection series started 24 h after training took place, a period of time long enough to think that the consolidation phase of memory was already completed [29]. Considering that fluoxetine has a very weak anticholinergic effect, its impairing effects on inhibitory avoidance must be due to another mechanism of action. Other authors and ourselves have claimed that the anticholinergic actions of antidepressants are responsible for memory impairment [1,9,10,12,36,37,39]. Nevertheless, the effects of fluoxetine and other antidepressants on memory processes could be mediated by additional cerebral mechanisms, besides their anticholinergic actions. In this way, an important property of some antidepressants is that their chronic administration increases neurogenesis in the hippocampus [22,24]. It has been confirmed that fluoxetine particularly increases the content of neurotrophic factors in this region [25]. Considering that neurogenesis subserves long-term potentiation [44] and learning [42], it could be argued that antidepressants, instead of impairing, could actually favour memory. Nevertheless, the impairing effects of antidepressants on memory can still hold because the experimental extinction of previously learned behaviours seems to derive from the learning of new behaviours that are not compatible with the old ones [38]. Furthermore, recent studies show that extinction involves the synthesis of new proteins [31,47]. It could be the case that the role of antidepressants is to impair old memories by favouring the creation of new ones. Comparing our results with others, and taking into account only studies with sub-chronic and chronic administration (being those that administered fluoxetine acutely discarded), it is important to note that to our knowledge there are no studies on the effects of this drug on inhibitory avoidance, therefore the comparisons must be established with observations in which other
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learning tasks were involved. These tasks were spatial memory tests [33,46,48] and active avoidance [28,32]. The results obtained by these authors can be grouped into two, one with no or improving effects on spatial memory, and the other with impairing effects on active avoidance. Perhaps the sense of the effects depends on the kind of task. ‘‘The inhibitory avoidance task used in the present study shares a high degree of emotionality with active avoidance as both use electric shock’’. In summary, we have found that fluoxetine, in spite of being a drug with non significant anticholinergic activity, has an important impairing effect on inhibitory avoidance given before training. This effect was not attributable to non specific effects on motor activity, and was restricted to male subjects. Despite the fact that our study does not contribute to clarify the reasons for these gender differences in drug effects on behaviour, more studies including females can be needed in basic research in Behavioural Pharmacology. The great majority of basic research has focused on male rodents as animal models [34], perhaps females as experimental subjects are often neglected because they are thought to be too ‘variable’, although this aspect has not been categorically confirmed [35].
Acknowledgements The work reported herein was supported by the grant ‘Efecto de los antidepresivos sobre la memoria’ (Code GV 97-SH-22-89) from Generalitat Valenciana, the regional government of Valencia (Spain), and by the University of Valencia.
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