Pre-training administration of tianeptine, but not propranolol, protects hippocampus-dependent memory from being impaired by predator stress

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1 European Neuropsychopharmacology (2008) 18, Pre-training administration of tianeptine, but not propranolol, protects hippocampus-dependent memory from being impaired by predator stress Adam M. Campbell a,b, Collin R. Park a,b, Phillip R. Zoladz a,b, Carmen Muñoz d, Monika Fleshner e, David M. Diamond a,b,c, a Medical Research, VA Hospital, Bruce B. Downs Blvd., Tampa, FL, USA b Department of Psychology, University of South Florida, 4202 E. Fowler, Ave., PCD 4118G, Tampa, FL, USA c Department of Molecular Pharmacology and Physiology, University of South Florida, 4202 E. Fowler, Ave., PCD 4118G, Tampa, FL, USA d I. R. I. Servier, Courbevoie, France e Center for Neuroscience and Department of Kinesiology and Applied Physiology, University of Colorado, Boulder, CO, USA Received 20 November 2006; received in revised form 8 March 2007; accepted 24 April 2007 KEYWORDS Predator stress; Rat; Memory; Amnesia; Antidepressant; Animal model Abstract Extensive research has shown that the antidepressant tianeptine blocks the adverse effects of chronic stress on hippocampal functioning. The current series of experiments extended this area of investigation by examining the influence of tianeptine on acute stress-induced impairments of spatial (hippocampus-dependent) memory. Tianeptine (10 mg/kg, ip) administered to adult male rats before, but not after, water maze training blocked the amnestic effects of predator stress (occurring between training and retrieval) on memory. The protective effects of tianeptine on memory occurred in rats which had extensive pre-stress training, as well as in rats which had only a single day of training. Tianeptine blocked stress effects on memory without altering the stressinduced increase in corticosterone levels. Propranolol, a β-adrenergic receptor antagonist (5 and 10 mg/kg, ip), in contrast, did not block stress-induced amnesia. These findings indicate that treatment with tianeptine, unlike propanolol, provides an effective means with which to block the adverse effects of stress on cognitive functions of the hippocampus Elsevier B.V. and ECNP. All rights reserved. 1. Introduction Corresponding author. University of South Florida, Department of Psychology, 4202 E. Fowler Ave. (PCD 4118G), Tampa FL 33620, USA. Tel.: ; fax: address: ddiamond@mail.usf.edu (D.M. Diamond). A thorough understanding of the etiology of mood and anxiety disorders and the mechanisms by which pharmacological treatments relieve depression and anxiety symptoms has not yet been accomplished. Nevertheless, two factors that are common to diverse mental disorders are stressful life events X/$ - see front matter 2007 Elsevier B.V. and ECNP. All rights reserved. doi: /j.euroneuro

2 88 A.M. Campbell et al. and hippocampal dysfunction (Esch et al., 2002; Geuze et al., 2005; Moghaddam and Jackson, 2004; Nemeroff et al., 2006; Smith et al., 2003). Extensive research has shown that stress exerts a profound influence on hippocampal functioning (Bremner, 2005; Diamond et al., 2004a; Eriksson and Wallin, 2004; Joels et al., 2004; Kim et al., 2006; Kim and Diamond, 2002; Lupien and Lepage, 2001; Roozendaal, 2002). For example, studies in rodents have shown that acute or chronic stress impairs hippocampus-dependent memory (Conrad et al., 1996; Conrad, 2005; Diamond et al., 1996, 1999, 2006; Park et al., 2006; Sandi et al., 2005; Woodson et al., 2003; Wright and Conrad, 2005) and blocks the induction of long-term (LTP) (Garcia, 2001; Huang et al., 2005; Lynch, 2004; McEwen and Chattarji, 2004; McEwen and Magarinos, 2001; Pavlides et al., 1996, 2002; Yamada et al., 2003) and primed (theta) burst potentiation (Alfarez et al., 2002, 2003; Diamond et al., 1994; Krugers et al., 2006; Mesches et al., 1999; Vouimba et al., 2006; Wiegert et al., 2006), two forms of synaptic plasticity which have been theorized to underlie memory formation (Diamond et al., 1988; Lisman, 2003; Lynch, 2004; Malenka and Bear, 2004). Other works in rodents, as well as non-human primates, have shown that chronic stress produces dendritic retraction, suppresses neurogenesis, cell proliferation and survival, and disturbs neurochemical markers of hippocampal functioning, such as glutamate transporter expression and neuronal growth factors (Buwalda et al., 2005; Karten et al., 2005; McEwen, 2000, 2004). Studies on people have provided findings consistent with the preclinical research, with evidence of glucocorticoid- or stress-induced impairments in cognitive measures of hippocampal functioning in control populations (Elzinga et al., 2005; Kirschbaum et al., 1996; Payne et al., 2002; Payne et al., 2006; Wolf et al., 2004), as well as diminished hippocampal volume and activation in people with anxiety, mood and neuroendocrine, i.e., glucocorticoid, disorders (Bremner, 2005; McEwen, 2005; Nemeroff et al., 2006; Starkman et al., 1999, 2003; Vythilingam et al., 2004). Therefore, preclinical research which focuses on alleviating or blocking the adverse effects of stress on hippocampal functioning is potentially relevant toward the development of more effective pharmacotherapy for mood disorders and anxiety. Our group has previously shown that inescapable exposure of rats to a cat (predator stress) suppressed hippocampal primed burst (PB) potentiation (a form of LTP), impaired spatial memory, and suppressed molecular and structural forms of plasticity (see Sandi, 2004; Diamond et al., 2004a,b, 2005; Diamond and Park, 2000; Kim and Diamond, 2002 for recent reviews). In a related work, we reported that the antidepressant tianeptine blocked the predator stress-induced suppression of hippocampal PB potentiation (Vouimba et al., 2006). Tianeptine is as effective as other agents, such as selective serotonin reuptake inhibitors (SSRIs), in the treatment of depression (Costa e Silva, 2004; McEwen and Olie, 2005; Nickel et al., 2003; Onder et al., 2005; Szadoczky and Furedi, 2002). Tianeptine has been shown to block the adverse effects of stress on cognitive (Conrad et al., 1996; Diamond et al., 2004a; File and Mabbutt, 1991; Jaffard et al., 1991; McEwen and Olie, 2005), electrophysiological (Jay et al., 2004; Rocher et al., 2004; Shakesby et al., 2002; Vouimba et al., 2006) and morphological/molecular (Conrad et al., 1999; Czeh et al., 2001; Fuchs et al., 2005; Kuroda and McEwen, 1998; McEwen et al., 1997; McEwen and Chattarji, 2004; Reagan et al., 2004; Watanabe et al., 1992) measures of hippocampal functioning. Our finding in which tianeptine blocked predator stress effects on hippocampal PB potentiation extended observations from other laboratories which have shown that tianeptine blocked the inhibitory effects of acute stress on LTP in the hippocampus (Shakesby et al., 2002) and prefrontal cortex (Jay et al., 2004; Rocher et al., 2004). Because tianeptine has been shown to block the stressinduced suppression of hippocampal PB potentiation and LTP, we hypothesized in the current work that tianeptine would block the stress-induced impairment of hippocampus-dependent memory. One additional observation illustrates the complexity of the influence of emotionality on memory. We have emphasized how stress can impair memory, but strong emotionality can also strengthen memory formation (Loftus and Bernstein, 2004; McGaugh, 2004; McGaugh and Roozendaal, 2002). Stimuli that evoke an increase in emotionality attract greater attentional resources, which can result in a great enhancement of the intensity and durability of memories for the arousing event (Burke et al., 1992; Detterman, 1975; Mackay and Ahmetzanov, 2005; Nielson et al., 2005). A major component of the neurobiology of the enhancement of memory by emotionality involves activation of β-adrenergic receptors. Administration of propranolol, a β-adrenergic receptor antagonist, blocks the arousal-induced enhancement of memory (Maheu et al., 2004a; McGaugh and Roozendaal, 2002; O'Carroll et al., 1999). There is also evidence that β- adrenergic receptors are not only involved in the enhancement of memory by emotionality, but also in the emotion-induced impairment of memory (Cahill, 2003; Hurlemann et al., 2005; Strange et al., 2003). Therefore, one component of the current series of experiments addressed the possibility that pretreatment of rats with propranolol would block the amnestic effects of predator stress on spatial memory. In summary, this series of experiments addressed the hypothesis that spatial memory would be protected from the amnestic effects of predator stress by pre-training administration of either an antidepressant (tianeptine) or an anxiolytic (propranolol). Preliminary findings from this research have been discussed previously (Diamond et al., 2004a). 2. Experimental procedures 2.1. Animals The subjects were male Sprague Dawley rats which weighed g at the time of arrival. Rats were pair-housed in Plexiglas cages with food and water available ad libitum. Water maze behavioral training began two weeks after the rats arrived. All rats were handled for 2 min each in the maze testing room for 3 days at the end of the two-week acclimation period Water maze training procedures All rats were trained in the radial arm water maze (RAWM) following general procedures we have described previously (Diamond et al., 1999, 2006; Park et al., 2006; Sandi et al., 2005; Woodson et al., 2003), with specific modifications described below. The RAWM is a black galvanized round tank (168 cm diameter, 56 cm height, 43 cm deep) filled with clear water (22 23 C). The tank contained 6 V- shaped inserts (54 cm height, 56 cm length) which formed six swim arms that radiated out of a central open area. A black platform

3 Tianeptine block stress induced amnesia 89 Figure 1 Timeline diagrams of the sequence of events occurring on the day of stress and drug testing in Experiments 1 3. All timelines are normalized to the drug injection occurring at time 0. In Experiments 1a and 3 the rats were given extensive preliminary training prior to the stress/drug testing days, and in Experiments 1b and 2 the rats were given no preliminary training prior to the stress/drug testing days. The sequence of events on the stress/drug testing days in Experiments 1a, b and 2 were identical: rats were injected with tianeptine, vehicle or propranolol, followed 30 min later by training (T1 T4 or T1 12 indicates 4 or 12 training trials), followed by a 30 min period in which the rats spent the entire time in their home cage (No Stress Condition) or 20 min of cat exposure (illustrated by the cat icon) followed by 10 min their home cage (Stress Condition), which terminated with the memory test trial. In Experiment 3, training (T1 T12) occurred first, followed by 30 min in the home cage or with the cat, followed by administration of tianeptine or vehicle. The rats then spent 70 min in their home cage, followed by the memory test trial. (12 cm diameter) hidden 1 cm below the surface of the water was at the end of one of the arms. The arm that contained the platform is referred to as the goal arm. In Experiments 1a and 3, rats were trained to learn a new location of the hidden platform on each of the multiple days of training (Multi- Day Training). In Experiments 1b and 2, all learning and memory testing was conducted in only one day of training (Single-Day Training). The following sections describe in detail the two types of RAWM training procedures. The timing and sequence of events on stress-memory training days are illustrated in Fig Multi-day water maze training (Experiments 1a and 3) On each day of training, rats were given 4 acquisition trials to learn the location of the goal arm, followed 30 (Experiment 1a) or 90 (Experiment 3) min later by the memory test trial (Fig. 1). Rats were given a maximum search time of 1 min per trial, after whichtheywereallowedtoremainontheplatformfor15s.ifarat didn't find the platform within 1 min it was guided there by the experimenter. The platform was always in the same arm on each trial within each day, and in a different arm across days. The rats, therefore, needed to learn a new goal arm location on each day of training. All rats were run 5 6 days/week, for up to 3 weeks, until they performed at a high rate of accuracy on the memory test trial on three consecutive days of training. Specifically, the rats needed to meet a performance criterion of committing a total of no more than one error on the memory test trial over three consecutive days of training, as we have described previously (Diamond et al., 1999; Park et al., 2006; Woodson et al.,

4 90 A.M. Campbell et al. 2003). This restrictive criterion ensured that all rats were well-trained and performed at a high degree of accuracy in their within-day spatial learning and memory performance prior to the initiation of the drug and stress testing. Once rats met the performance criterion, they were given two more (post-criterion) days of training. All drug and stress manipulations were performed during the two post-criterion days of training. Blood samples were obtained within 2 min after the memory test trial on the second post-criterion day, which was the final manipulation performed on the animals. The blood was allowed to clot and then the serum was collected and kept frozen until it was assayed for corticosterone (CORT) by radioimmunoassay (RIA) Single-day water maze training (Experiments 1b and 2) In Experiments 1b and 2, water maze training was modified to allow for the entire assessment of learning and memory testing, complete with stress and drug manipulations, in a single day of training. One week after arrival at the university facility, rats were given 3 days of handling, followed the next day by a single day of water maze testing. All rats were given RAWM training consisting of one acquisition session, which was composed of twelve training trials in succession (a maximum of 1 min/trial). After each trial the rats were allowed to spend 15 s on the platform. After the twelfth trial, the rats were given a 30 min delay period during which they were either returned to the home cage (No Stress) or they were given cat exposure (predator stress; described below). For all groups, the 30 min delay period was terminated with a single memory test trial Blood sampling and corticosterone assay Blood was obtained in less than 2 min from a small (1 mm) nick in the tip of the tail immediately after the memory test trial on the final day of testing. The blood was centrifuged and then the serum was extracted and frozen for RIA analysis of CORT levels Pharmacological manipulations Tianeptine (TIA; 10 mg/kg, ip) or 0.9% saline (SAL; 1 ml/kg) was administered 30 min prior to training in Experiments 1a, 1b and 2, or immediately after stress in Experiment 3. This dose of tianeptine was chosen because previous work has shown that it blocks the adverse effects of stress on cognitive (Conrad et al., 1996; Diamond et al., 2004a; File and Mabbutt, 1991; Jaffard et al., 1991; McEwen and Olie, 2005), electrophysiological (Jay et al., 2004; Rocher et al., 2004; Vouimba et al., 2006) and morphological/molecular (Conrad et al., 1999; Fuchs et al., 2005; Kuroda and McEwen, 1998; McEwen et al., 1997; Reagan et al., 2004) measures of hippocampal functioning. Propranolol (PROP; 5 or 10 mg/kg, ip) was administered 30 min before training and stress in Experiment 2 (Fig. 1). We chose these doses of propranolol because previous work has shown that they have anxiolytic effects on rat behavior (Angrini et al., 1998; Cole and Koob, 1988; Lucki et al., 1989). We hypothesized that these doses of propranolol would block anxiogenic effects of cat exposure and therefore prevent its impairing effects on water maze performance Stress manipulation Predator stress always occurred immediately after the acquisition phase was completed (Fig. 1). The stress manipulation began with placement of the rats in a small clear Plexiglas box ( cm) which was ventilated with numerous small (1 cm diameter) holes. Then the rats were transported, in the box, to the cat housing room, where they were placed in a large ventilated chamber (750 cm 570 cm 580 cm) which contained an adult female cat. The Plexiglas box enabled the rats to see, hear and smell the cat, but did Figure 2 Tianeptine blocked stress effects on memory without affecting serum corticosterone levels in rats given extensive preliminary training to a performance criterion (see text), followed by post-criterion stress, drug and memory testing. Stress increased memory errors (top) and CORT levels (bottom) in vehicle-treated rats. Stress did not increase memory errors in tianeptine-treated rats (top), despite their stress-induced elevation of CORT levels (bottom). The indicates pb0.05 (ANOVA with Holm Sidak post-hoc tests) compared to the vehicle/no stress group. In this and in all subsequent figures, all data from No Stress groups are illustrated with diagonal bars and data from Stress groups are illustrated with solid bars. not allow for physical contact between them. The rats remained in the chamber with the cat for 20 min, and then they were returned to their home cages in the water maze training room. The rats were then given the memory test trial 10 (Experiments 1 and 2) or 70 (Experiment 3) min later. Therefore, the total time from the end of training to the memory test trial was 30 (Experiments 1 and 2) or 90 (Experiment 3) min Statistical analysis and data presentation A two-way repeated-measures ANOVA was performed on water maze errors committed in a Group by Trial analysis during the 4 (Experiments 1a and 3) or 12 (Experiments 1b and 2) acquisition trials. A one-way ANOVA was performed on CORT levels, as well as errors committed on the memory test trial, followed by post-hoc Holm Sidak test (SPSS, SigmaStat), when necessary. 3. Results There were no significant effects of drug administration on learning, which is confirmed below in the statistical analyses for each experiment. Any effects of the drug and stress manipulations were solely on performance on the memory test trial, which are illustrated in Figs. 2 5.

5 Tianeptine block stress induced amnesia 91 There was a significant main effect of stress for CORT levels (F[3, 25]=47.928, pb.001), which was unaffected by tianeptine treatment. Tianeptine treatment did not affect CORT levels in the non-stressed group, nor did tianeptine affect CORT levels in the stressed group (Fig. 2). In summary, we have found that: 1) post-training stress impaired spatial memory; 2) the stress effect on memory was blocked by pre-training treatment with tianeptine; and 3) tianeptine treatment had no effect on CORT levels under stress, as well as non-stress, conditions. Figure 3 Tianeptine blocked stress effects on memory without affecting serum corticosterone levels in rats which had no preliminary water maze training. Stress increased memory errors (top) and CORT levels (bottom) in vehicle-treated rats. Stress did not increase memory errors in tianeptine-treated rats (top), despite their stress-induced elevation of CORT levels (bottom). The indicates pb 0.05 (ANOVA with Holm Sidak post-hoc tests) compared to the vehicle/no stress group Experiment 1a: Influence of pre-training tianeptine and stress on spatial memory (Multi-Day Training) Four groups of rats were tested in Experiment 1a: Home Cage/ Saline (HC/SAL; n=15), Home Cage/Tianeptine (HC/TIA; n=14), Stress/Saline (STR/SAL; n = 15), Stress/Tianeptine (STR/TIA; n=12). There was a main effect of trials (F[4, 204] =43.932, pb 0.001) for the acquisition phase. All other analyses and interactions were not significant. Therefore, all 4 groups exhibited comparable performance during the acquisition phase of training. On the memory test trial there was a significant main effect of stress (F[1, 52] =22.704, pb ), a significant main effect of drug (F[1, 52] =11.144, pb 0.002), and significant Stress Drug interaction (F[1, 52] =16.440, pb.0001). Post-hoc comparisons (Holm Sidak test) revealed significant difference in the number of errors between STR/SAL and HC/SAL groups (pb 0.001), which confirms that cat exposure in saline-injected rats increased the number of memory errors (Fig. 2). The effects of stress on memory were blocked by pre-treatment of rats with tianeptine, as the STR/SAL group made significantly more memory errors than the STR/TIA group (pb0.001). Tianeptine, administered to the home cage (non-stress) group had no effect on performance; there was no significant difference in the number of errors committed by the HC/TIA versus HC/SAL groups (t=.523, pb0.604). Figure 4 Comparison of tianeptine (TIA) and propranolol (P5/ P10; 5 and 10 mg/kg) effects on the stress-induced modulation of memory and CORT. Tianeptine blocked stress effects on memory without affecting serum corticosterone levels. Stress increased memory errors (top) and CORT levels (bottom) in vehicle (VEH)-treated rats. Stress did not increase memory errors in tianeptine-treated rats (top), despite their stressinduced elevation of CORT levels (bottom). Propranolol had no effects on memory under control (No Stress) conditions, but stressed rats treated with either dose of propranolol (P5/P10) exhibited impaired memory. Stress increased CORT in propranoltreated no stress groups, with a greater elevation of CORT levels in stressed rats treated with 10 mg/kg. The indicates pb0.05 (ANOVA with Holm Sidak post-hoc tests) compared to the vehicle/no stress group. The β indicates that CORT levels in the stress/p10 group were greater than those in the stress/ vehicle group.

6 92 A.M. Campbell et al Experiment 1b: Influence of pre-training tianeptine and stress on spatial memory (Single-Day Training) Four groups of rats were tested in Experiment 1b: HC/SAL (n=8), HC/TIA (n=8), STR/SAL (n=8), STR/TIA (n=8). There was a main effect of trials (F[6, 168]=20.636, pb0.0001) for the acquisition phase. All other analyses and interactions were not significant. The 4 groups, therefore, exhibited comparable performance during the acquisition phase of training. On the memory test trial there were significant main effects of stress (F[1, 28]=22.828, pb.0001) and drug (F[1, 28]=15.070, pb 0.001), as well as a significant Stress Drug interaction (F[1, 28]= , pb.0001). Post-hoc analyses (Holm Sidak) revealed significant differences in errors between the STR/SAL and HC/SAL groups (pb 0.001), which confirmed that cat exposure significantly increased errors on the memory test trial in saline-injected rats. The STR/TIA versus HC/TIA groups, by contrast, showed no difference in errors on the memory test trial (p=0.7), and there was no significant difference between HC/SAL and HC/TIA groups (p=0.8). Therefore, tianeptine did not affect already strong memory performance under non-stress conditions, and tianeptine blocked the impairing effect of predator stress on memory (Fig. 3). There was a significant main effect of stress on CORT levels (F[1, 36]= , pb0.0001), but no main effect of drug and no significant Stress Drug interaction. As with Experiment 1a, CORT levels were elevated in both stress groups compared to home cage groups, and tianeptine did not affect CORT levels under control, as well as stress, conditions (Fig. 3). In summary, Experiments 1a and 1b produced similar findings, despite differences in methodology between the two experiments. Tianeptine blocked the stress-induced impairment of spatial memory without producing any effect on the stressinduced increase in CORT levels (Figs. 2 and 3), independent of whether the rats were well-trained prior to stress and drug testing (Experiment 1a) or were naïve when all manipulations (training, stress and drug administration) occurred in a single day of behavioral testing (Experiment 1b) Experiment 2: Influence of pre-training treatment with tianeptine versus propranolol (PROP) on the stress-induced impairment of memory Eight groups of rats were tested in Experiment 2: HC/SAL (n=8); HC/5 PROP (n=8, 5 mg/kg of PROP); HC/10 mg PROP (n=8, 10 mg/kg of PROP); HC/TIA (n=8); STR/SAL (n=8); STR/5 PROP (n = 8, 5 mg/kg of PROP), STR/10 PROP (n = 8, 10 mg/kg of PROP); STR/TIA (n=8). All rats were run in the RAWM using single-day water maze training (Fig. 1). There was no effect of drug treatment on performance of the groups on the acquisition trials (ANOVA, pn0.1). On the memory test trial there was a significant main effect of stress (F[1, 56] =41.636, pb 0.001) and Drug (F[3, 56] =5.309, pb 0.003), and a significant Stress Drug interaction (F[3, 56] = 4.025, pb 0.012). In the post-hoc analysis, there was no influence of the drug manipulations on memory for the non-stress groups (p=0.8), but there were significant differences among the stress groups: the STR/TIA group differed from the STR/SAL (pb 0.001) and both STR/PROP (5 and 10 mg/kg) groups (pb and pb 0.001, respectively). These findings indicate that post-training Fig. 5 Tianeptine administered post-stress did not block the stress-induced increase in errors on the memory test trial. The vehicle- and tianeptine-treated groups exhibited significant and equivalent increases in errors on the memory test trial. The indicates pb 0.05 (ANOVA with Holm Sidak post-hoc tests) compared to the vehicle/no stress group. stress increased memory errors in the saline- and propranololinjected groups, but not in the tianeptine-injected group (Fig. 4). Additional post-hoc tests revealed significant differences within drug between the stressed and non-stressed groups. Specifically, there was a significant effect of stress on memory for both doses of propranolol (both p's b 0.001). Therefore, both doses of propranolol were ineffective at blocking the increase in errors produced by predator stress. There was no significant difference in memory errors between the STR/TIA and HC/TIA groups (p b 0.748), indicating that tianeptine blocked the stressinduced increase in errors on the memory test trial. There were main effects of stress (F[1, 56]=75.053, pb0.001) and drug (F[1, 56]=42.418, pb0.001), as well as a significant Stress Drug interaction (F[3, 56] =12.550, pb 0.001) for CORT levels. Specific post-hoc tests revealed that there was no difference in CORT levels between STR/SAL and STR/TIA groups, as well as between the HC/SAL and HC/TIA groups. There were significant differences in CORT levels between the HC/SAL and both the HC/PROP 5 and HC/PROP 10 groups (p b 0.001). Thus, under non-stress conditions, propranolol produced an increase in CORT levels. In addition, CORT levels in the STR/VEH, STR/TIA and STR/PROP 5 groups were all greater than the HC/VEH group, with a further increase in CORT levels in the STR/PROP 10 group, relative to the STR/VEH group (pb0.001). Overall, these findings indicate that stress increased CORT levels equivalently in saline- and tianeptine-treated groups, and that CORT levels were further elevated in the stressed propranolol-treated groups (Fig. 4) Experiment 3: Influence of post-training and post-stress administration of tianeptine on memory Experiments 1 and 2 showed that tianeptine, administered prior to training and stress, blocked the stress-induced impairment of memory. Experiment 3 was designed to test the possibility that post-stress administration of tianeptine would block the stressinduced impairment of memory. In Experiments 1a, 1b and 2, tianeptine was administered 70 min before the memory test trial (see Fig. 1 for the timeline). In Experiment 3, tianeptine was administered after training and stress, followed 70 min

7 Tianeptine block stress induced amnesia 93 later by the memory test trial. In this manner, the time between tianeptine injection and the memory test trial was kept constant at 70 min in all 3 experiments. Twenty-six rats in four groups were given RAWM training in Experiment 3: HC/SAL (n=6), HC/TIA (n=6), STR/SAL (n=7) and STR/TIA (n=7). There was a significant main effect of stress (F [1, 22]=18.639, pb.0001) on the memory test trial, with no main effect of drug (F[1, 22]=.279, pb0.602) and no significant Stress Drug interaction (F[1, 22] =0.279, p b0.602). Post-hoc analysis verified that there was a significant increase in errors for both of the stress groups (TIA/STR and SAL/STR) compared to the non-stress groups (HC/TIA and HC/SAL) (all significant comparisons, pb 0.001). Therefore, post-stress administration of tianeptine did not block the stress-induced impairment of memory (Fig. 5). 4. Discussion This series of experiments demonstrated that tianeptine blocked the amnestic effect of acute predator stress on spatial memory. Specifically, a single administration of tianeptine prior to water maze training protected rats' memory of the platform location from being impaired by post-training predator stress. Blockade of the predator stress-induced impairment of spatial (hippocampus-dependent) memory by tianeptine occurred in rats which were given extensive preliminary water maze training (Experiment 1a), as well as in rats given only a single day of training (Experiment 1b). Pretreatment with propranolol, unlike tianeptine, did not block predator stress-induced amnesia (Experiment 2). The memory-protective effects of tianeptine were evident when treatment was given before (Experiments 1 and 2), but not after (Experiment 3), the training and stress had occurred. We have also provided evidence that tianeptine does not block stress effects on memory by altering the predator stressinduced responsivity of the hypothalamic pituitary adrenal (HPA) axis. Indeed, tianeptine-treated stressed rats demonstrated intact memory despite exhibiting a normal stressinduced increase in levels of serum CORT Comparison of tianeptine and propranolol in protecting memory from stress Extensive research has investigated the neurobiology and neuropharmacology of memory enhancement by strong emotionality (Cahill, 2000; LeDoux, 2000; McGaugh, 2002; Sandi, 2003). The findings clearly implicate the noradrenergic system, acting in conjunction with corticosteroids, in processes underlying the arousal-related enhancement of memory (Roozendaal et al., 2004a,b). Thus, activation of central β-adrenergic receptors is an important component of the strengthening (Maheu et al., 2004a; McGaugh, 2002; McGaugh and Roozendaal, 2002; O'Carroll et al., 1999), as well as the weakening (Cahill, 2003; Hurlemann et al., 2005; Strange et al., 2003), of memory by strong emotionality in rats and people. Findings from one of our studies may be relevant toward understanding how stress can both enhance and impair memory. We studied rats that were given shock avoidance conditioning immediately after they had learned the location of a hidden platform in the RAWM (Diamond et al., 2004b). These rats formed a strong memory of the fear-provoking experience, as indicated by their subsequent avoidance of the place where they were shocked. We also found that the shock impaired their memory for the location of the hidden platform in the water maze. These findings were consistent with decades of conceptual and empirical research on the idea that the allocation of attentional resources toward one stimulus can result in reduced processing of other stimuli (Burke et al., 1992; Detterman, 1975; Mackay and Ahmetzanov, 2005; Nielson et al., 2005). It was therefore possible that processes mediating the arousal-related enhancement of memory were involved in the arousal-related impairment of memory as well. Therefore, as propranolol has been shown to block the arousalinduced enhancement of memory, and the arousal-induced enhancement and impairment of memory may have mechanisms in common, we hypothesized that propranolol should block the stress-induced impairment of memory. The findings from Experiment 2, however, did not support this hypothesis. Pre-treatment with propranolol, unlike tianeptine, did not block predator stress-induced amnesia. The absence of an effect of propranolol on the stressinduced impairment of memory in the present study may be based on differences in methodology between the current work and other work on people and rodents. Most studies on arousal and memory typically measure the enhancement of memory for stimuli which are intrinsically arousing. For example, studies on people typically employ words or images that are intrinsically arousing versus those that are intrinsically neutral, e.g., taboo versus neutral words or images of carnage (Bush and Geer, 2001; Kensinger and Corkin, 2004; Kensinger and Schacter, 2006; Mackay and Ahmetzanov, 2005). It is the arousing word or image that is remembered better than the emotionally neutral word, and propranolol has been shown to block the influence of this intrinsic arousal on memory. Similarly, in rodent work, propranolol has been shown to impair memory-related processes in tasks that are intrinsically arousing, such as shock avoidance, conditioned taste aversion and water maze (Cahill et al., 2000; Debiec and LeDoux, 2004; Ji et al., 2003; Miranda et al., 2003; Przybyslawski et al., 1999; Roozendaal et al., 2004a). In the current study, predator exposure was an extrinsic stressor which interfered with the rat's memory of the platform location. Extrinsic stress has been used in a small number of animal and human studies of emotion and memory (Kim and Diamond, 2002; Kirschbaum et al., 1996; Payne et al., 2002, 2006; Thompson et al., 2001), but we are not aware of any studies that have examined the capacity for noradrenergic modulators, such as propranolol, to block the effects of extrinsic stress on memory. Work conducted by Roozendaal (2002) and others has shown that the post-training administration of CORT enhances memory, while pre-retrieval stress or the administration of CORT impairs memory. In addition, these investigators have found that the effects of CORT administration on the consolidation or retrieval of memory can be blocked by treatment with propranolol. The present set of experiments differs from that of Roozendaal's work in that we examined whether propranolol or tianeptine would block the stress-induced impairment of spatial memory, in general. Thus, we cannot distinguish between predator stress effects on the consolidation versus retrieval phases of memory because the stress occurred in the delay period

8 94 A.M. Campbell et al. between the training and retrieval phases of testing in a single day of training. Nevertheless, the findings reveal that tianeptine, but not propranolol, can block the stressinduced impairment of memory. Whether tianeptine acts specifically to protect consolidation versus retrieval mechanisms from being impaired by stress remains to be determined. The precise explanation for why propranolol was ineffective at blocking predator stress-induced amnesia is not known. We have suggested that one factor may be whether the stimulus to be remembered is intrinsically arousing or if the arousing stimulus is extrinsic to the training task, and another factor may be whether stress affects consolidation or retrieval phases of memory. We also discovered what at first appeared to be an anomalous finding that is, the administration of propranolol led to significantly elevated CORT levels in the non-cat-exposed rats and produced even greater stress-induced elevations of CORT in rats that had been exposed to the cat (see bottom of Fig. 3). Comparable findings have been reported in human studies, in which the administration of propranolol led to greater baseline (Kizildere et al., 2003) and stress-induced increases in CORT (Williams et al., 1988), as well as greater stress-induced increases in ACTH (Oberbeck et al., 1998). In the present set of experiments, all of the rats were exposed to some stress because of the clear stress component (i.e., immersion in cold water) associated with water maze training. Our findings are therefore analogous to human studies reporting greater stress-induced increases in CORT and ACTH following treatment with propranolol. Thus, despite propranolol's anxiolytic properties, it appears to augment the stress-induced secretion of glucocorticoids. It is also known that stress can alter the physiological responses of amygdala neurons to adrenergic stimulation (Braga et al., 2004). The stress-induced perturbation of amygdala physiology can therefore potentially influence how adrenergic receptor modulators, such as propranolol, will interact with the stress-induced modulation of memory. These findings reinforce the notion that stress activates multiple neurotransmitter systems and alters physiological activity in different brain structures. Our finding that tianeptine, but not propranolol, blocked predator stress effects on memory provides further support for the idea that there are different mechanisms which underlie the emotioninduced enhancement versus impairment of memory Mechanisms underlying the tianeptinemediated protection of memory and LTP Recent studies have investigated the modulation of stress effects on LTP in the hippocampus, prefrontal cortex (PFC) and amygdala by tianeptine (Jay et al., 2004; Rocher et al., 2004; Shakesby et al., 2002; Vouimba et al., 2006). The findings on LTP in the hippocampus and PFC are consistent with the broader literature which has shown that tianeptine blocks the adverse effects of acute and chronic stress on these two structures (Costa e Silva, 2004; Diamond et al., 2004a; Fuchs, 2005; Jay et al., 2004; McEwen and Olie, 2005; Rocher et al., 2004). For example, Shakesby et al. (2002) found that treatment of rats with tianeptine blocked the stress-induced inhibition of LTP in the hippocampus. We recently replicated the findings of Shakesby et al. with our observations that tianeptine blocked the stress-induced suppression of PB potentiation in CA1, without blocking the stress-induced enhancement of LTP in the amygdala (Vouimba et al., 2006). While the precise mechanisms underlying the tianeptinemediated protection of memory from stress are not fully understood, it is clear that tianeptine does not accomplish its LTP and memory-protective effects via the modulation of glucocorticoid levels. We have found here that tianeptine blocked stress effects on memory without altering the stressinduced increase in CORT levels. This finding is consistent with findings from other studies which have shown that elevated CORT levels, alone, do not impair memory or LTP. For example, we have shown previously that male rats exposed to a female rat exhibited intact spatial memory, despite the fact that female-exposed rats had increased CORT levels which were equivalent to those in cat-exposed rats (Woodson et al., 2003). In addition, we have found that rats given an injection of CORT, which produced stress levels of serum CORT, were without effect on spatial memory (Park et al., 2006). Similarly, Kim et al. (2001) demonstrated that amygdala damage blocked the effects of stress on LTP and spatial memory without altering the stress-induced increase in CORT levels. Additional evidence that there is no relation between CORT levels and short-term memory performance is our finding that propranolol, administered under non-stress conditions, increased CORT levels without affecting memory performance (Fig. 4). Finally, Shakesby et al. (2002) found that tianeptine blocked stress effects on hippocampal LTP without affecting the stress-induced increase in CORT levels. Thus, CORTserves as an indicator of a heightened stress state and is clearly involved in the modulation of LTP and memory (de Quervain et al., 2000; Lupien et al., 1999, 2002; Lupien and McEwen, 1997; Maheu et al., 2004b; Okuda et al., 2004; Roozendaal, 2003; Roozendaal et al., 2004b), but other factors, besides increased levels of CORT, appear to be necessary for the acute stress-induced modulation of hippocampus-dependent memory. Our findings are consistent with the hypothesis that stress interferes with the capacity for the hippocampus to generate new (learning-related) plasticity (Diamond et al., 2004b, 2005), and that tianeptine reverses this effect via a CORTindependent process. When tianeptine was administered before learning and stress, as in Experiments 1 and 2, the memory appears to have been stored in a manner in which its retrieval was not impaired by stress. This perspective is consistent with our recent findings which indicate that when tianeptine was administered prior to training, the resultant memory was retrieved successfully even when the stress occurred 24 h later (unpublished observations). Thus, tianeptine appears to enable information to be stored in a manner which protects it from being impaired by later occurring stress. Tianeptine may accomplish this feat by normalizing stressinduced changes in NMDA receptor, i.e., calcium, currents (Kole et al., 2002) Summary and conclusions The current experiments found, with two different training procedures, that acute predator stress significantly impaired hippocampus-dependent memory. Whether rats were given multiple days or only a single day of training, acute stress

9 Tianeptine block stress induced amnesia 95 occurring immediately after learning impaired within-day spatial memory. In both forms of training, tianeptine completely blocked the acute stress-induced impairment of memory without affecting the stress-induced increase in CORT levels. In contrast, the administration of propranolol, a β-adrenergic receptor antagonist, had no effect on the impairment of memory produced by acute stress. These findings, in conjunction with complementary work on the tianeptinemediated reversal of the stress-induced suppression of LTP in the hippocampus (Shakesby et al., 2002; Vouimba et al., 2006), indicate that treatment with tianeptine provides an effective means with which to block the adverse effects of stress on cognitive functions of the hippocampus. Role of the funding sources Funding for this study was provided by the Veterans Administration and Servier, which was included in the design, data collection, analysis, interpretation of the findings and in the writing of the manuscript. Contributors Adam Campbell performed the training of the animals and wrote the first draft of the manuscript as a part of his dissertation research. Collin Park contributed to the statistical analysis of the research. Phillip Zoladz contributed to the writing of the revised manuscript. Carmen Munoz contributed to the design of the study and to the writing of the manuscript. Monika Fleshner assayed the serum corticosterone levels and David Diamond was responsible for the writing of the manuscript and the overall design and implementation of the study. Conflict of interest David Diamond has received honoraria as well as research grants from Servier (Paris, France). 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