Neurobiology of Learning and Memory

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1 Neurobiology of Learning and Memory 1 (213) Contents lists available at SciVerse ScienceDirect Neurobiology of Learning and Memory journal homepage: Pre-learning stress that is temporally removed from acquisition exerts sex-specific effects on long-term memory Phillip R. Zoladz a,, Ashlee J. Warnecke a, Sarah A. Woelke a, Hanna M. Burke a, Rachael M. Frigo a, Julia M. Pisansky a, Sarah M. Lyle a, Jeffery N. Talbot b a Department of Psychology, Sociology, & Criminal Justice, Ohio Northern University, Ada, OH 4581, USA b Department of Pharmaceutical & Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern University, Ada, OH 4581, USA article info abstract Article history: Received 8 October 212 Revised 13 December 212 Accepted 15 December 212 Available online 22 December 212 Keywords: Amygdala Cortisol Hippocampus Learning Memory Stress We have examined the influence of sex and the perceived emotional nature of learned information on pre-learning stress-induced alterations of long-term memory. Participants submerged their dominant hand in ice cold (stress) or warm (no stress) water for 3 min. Thirty minutes later, they studied 3 words, rated the words for their levels of emotional valence and arousal and were then given an immediate free recall test. Twenty-four hours later, participants memory for the word list was assessed via delayed free recall and recognition assessments. The resulting memory data were analyzed after categorizing the studied words (i.e., distributing them to positive-arousing, positive-non-arousing, negative-arousing, etc. categories) according to participants valence and arousal ratings of the words. The results revealed that participants exhibiting a robust cortisol response to stress exhibited significantly impaired recognition memory for neutral words. More interestingly, however, males displaying a robust cortisol response to stress demonstrated significantly impaired recall, overall, and a marginally significant impairment of overall recognition memory, while females exhibiting a blunted cortisol response to stress demonstrated a marginally significant impairment of overall recognition memory. These findings support the notion that a brief stressor that is temporally separated from learning can exert deleterious effects on long-term memory. However, they also suggest that such effects depend on the sex of the organism, the emotional salience of the learned information and the degree to which stress increases corticosteroid levels. Ó 212 Elsevier Inc. All rights reserved. 1. Introduction Stress can enhance, impair or have no effect on learning and memory. One factor that plays an important role in dictating whether stress enhances or impairs learning is the stage of learning and memory that is affected by the stress. Perhaps the most consistent finding in this area of work is that stress administered after learning enhances consolidation (Beckner, Tucker, Delville, & Mohr, 26; Cahill, Gorski, & Le, 23; Hui, Hui, Roozendaal, McGaugh, & Weinberger, 26; Preuss & Wolf, 29; Smeets, Otgaar, Candel, & Wolf, 28). The effects of stress on encoding and retrieval, however, have been less clear. Although a great deal of work has shown that stress impairs retrieval (Buchanan & Tranel, 28; Buchanan, Tranel, & Adolphs, 26; Kuhlmann, Piel, & Wolf, 25; Park, Zoladz, Conrad, Fleshner, & Diamond, 28; Smeets et al., 28; Tollenaar, Elzinga, Spinhoven, & Everaerd, 28), there Corresponding author. Address: Department of Psychology, Sociology, & Criminal Justice, Ohio Northern University, 525 S. Main St. Hill 13, Ada, OH 4581, USA. Fax: address: p-zoladz@onu.edu (P.R. Zoladz). are discrepancies in the literature, with some studies reporting stress enhancing or having no effect on such processes (Beckner et al., 26; Buchanan & Tranel, 28; Schwabe et al., 29; Yang et al., 23). Investigations examining pre-learning stress have also been inconclusive and have revealed enhancements, impairments or no effects at all (Diamond et al., 26; Duncko, Johnson, Merikangas, & Grillon, 29; Elzinga, Bakker, & Bremner, 25; Jelicic, Geraerts, Merckelbach, & Guerrieri, 24; Kim, Koo, Lee, & Han, 25; Kim, Lee, Han, & Packard, 21; Nater et al., 27; Park et al., 28; Payne et al., 26, 27; Schwabe, Bohringer, Chatterjee, & Schachinger, 28a; Smeets et al., 28; Zoladz et al., 211a). Researchers have claimed that the timing of stress plays a critical role in the stress-induced modulation of learning and memory (Diamond, Campbell, Park, Halonen, & Zoladz, 27; Joels, Fernandez, & Roozendaal, 211; Joels, Pu, Wiegert, Oitzl, & Krugers, 26; Schwabe, Joels, Roozendaal, Wolf, & Oitzl, 212). Diamond and colleagues suggested that stress exerts a biphasic effect on hippocampal function, which consequentially results in differential effects on long-term memory depending on when the stress is administered relative to learning (Diamond et al., 27). According to these investigators, stress activates the amygdala, which produces a ra /$ - see front matter Ó 212 Elsevier Inc. All rights reserved.

2 78 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) pid enhancement of hippocampal synaptic plasticity that initially facilitates the storage of information; however, as times passes, the hippocampus descends into a refractory state for producing new plasticity, and the storage of information is impaired. Part of the basis for this theory was electrophysiological work indicating that glucocorticoids, as well as electrical stimulation of the amygdala, could exert immediate excitatory, but delayed inhibitory, effects on hippocampal synaptic plasticity (Akirav & Richter-Levin, 1999; Frey, Bergado-Rosado, Seidenbecher, Pape, & Frey, 21; Karst et al., 25; Wiegert, Joels, & Krugers, 26). Indeed, a general consensus has begun to emerge suggesting that if the stress converges in time with the learning experience and activates neurobiological mechanisms that are in common with the learning experience, then long-term memory can be enhanced (Joels et al., 26; Sandi, 211). Such stress-induced enhancements are thought to be associated with the rapid effects of noradrenergic activity, non-genomic actions of corticosteroids and increased glutamatergic transmission. Stress-induced impairments of hippocampal function, on the other hand, are thought to be associated with delayed, gene-dependent activity of corticosteroids and NMDA receptor desensitization (Akirav & Richter-Levin, 22; Diamond et al., 27; Halonen, Zoladz, Park, & Diamond, 27; Joels, Velzing, Nair, Verkuyl, & Karst, 23). Much of the support for this temporal dynamics model has come from non-human animal work. For instance, Diamond et al. (27) found that brief stress administered immediately prior to learning enhanced long-term spatial memory in rats; however, if the same stress was administered 3 min before learning, no enhancement was observed. The stress-induced enhancement of long-term spatial memory was mimicked by epinephrine and blocked by propranolol, a b-adrenergic receptor antagonist (Halonen et al., 27). In contrast, the pre-learning stress-induced impairment of long-term spatial memory, which was induced by prolonged (i.e., 3 min) stress prior to training, was not prevented by propranolol. The findings of human work in this area have been mixed. Some studies examining pre-learning stress effects on longterm memory have been consistent with the temporal dynamics model (e.g., Schwabe et al., 28a), while others have observed stress-induced enhancements or impairments of memory in situations that do not coincide with predictions that would arise from this theory (e.g., Smeets et al., 29). Recently, we found support for the temporal dynamics model by showing that brief stress administered immediately before learning enhanced long-term memory in healthy young adults, while the same brief stressor administered 3 min before learning impaired long-term memory (Zoladz et al., 211a). Importantly, the stress-induced enhancement of memory was associated with participants heart rate response to the stress, an indication of autonomic nervous system and noradrenergic activity, while the stress-induced impairment of memory was associated with participants corticosteroid and blood pressure responses to the stress. Combined with the above evidence from rodent research, these findings supported the possibility that noradrenergic mechanisms are responsible for prelearning stress-induced enhancements of long-term memory, but they are eventually suppressed by the delayed increase in corticosteroids, which results in memory impairment (Schwabe et al., 212). Most, but not all, of the research examining stress effects on learning and memory has reported greater effects for information that is emotional in nature (Schwabe, Wolf, & Oitzl, 21; Wolf, 29). This finding makes sense considering the amygdala, an area of the brain that is highly involved in emotional memory formation (McGaugh, 24), is significantly activated by emotional material (Dolcos, LaBar, & Cabeza, 24; Strange & Dolan, 24). One limitation of studies of this type, however, is that investigators have frequently confounded the emotional valence (e.g., positive, negative, and neutral) of the words with the arousal level (arousing, non-arousing) of the words. That is to say, few, if any, studies have systematically manipulated both the emotional valence and arousal level of the learned information in order to examine how such manipulations influence stress-induced alterations of learning and memory (see Schwabe et al., 28a for a consideration of this point). In the present experiment, we examined the influence of pre-learning stress on memory for information that varied in both emotional valence and arousal by systematically varying the levels of each based on subjective ratings provided by participants. Sex differences also abound in the stress memory literature, and a clear agreement on how sex mediates such effects has not been reached. Investigators have contended that sex differences depend on the type of learning that occurs (Conrad et al., 24), the stage of learning that is affected by the stress (Andreano & Cahill, 29) and the hormonal response of the participant to the stress (Andreano & Cahill, 26; Andreano & Cahill, 29). Perhaps one relatively consistent finding in this area is that corticosteroidmediated modulation of learning and memory has been observed more in males than in females. Several studies have reported cortisol- or stress-induced alterations of learning and memory or significant relationships between cortisol and memory in males, but not females (Andreano & Cahill, 26; Jackson, Payne, Nadel, & Jacobs, 26; Wolf, Schommer, Hellhammer, McEwen, & Kirschbaum, 21; Wood, Beylin, & Shors, 21; Zorawski, Blanding, Kuhn, & LaBar, 26). Ultimately, additional work is needed to assess the influence of sex on stress memory interactions, especially in humans. The purpose of the present study was to examine the influence of sex and the perceived emotional nature of the learned information on pre-learning stress effects on long-term memory. Given the aforementioned findings, we predicted that a brief stressor administered 3 min before learning would impair long-term memory, particularly for emotional information (Zoladz et al., 211a). We also expected that, since pre-learning stress-induced impairments of learning may be more dependent on corticosteroid mechanisms, males might be more adversely affected by the stress than females. 2. Method 2.1. Participants Ninety-seven healthy men and women (38 men, 59 women; age: M = 19.18, SD = 1.15) from Ohio Northern University volunteered to participate in the present experiment. Individuals were excluded from participating if they met any of the following conditions: diagnosis of Raynaud s disease or peripheral vascular disease; presence of skin diseases, such as severe psoriasis, eczema or scleroderma; history of severe head injury; current treatment with psychotropic medications, narcotics, beta-blockers, steroids or any other medication that was deemed to significantly affect central nervous or endocrine system function; mental or substance use disorder; regular nightshift work. Individuals who smoked were allowed to participate in the study; information regarding individuals smoking habits was collected prior to the experiments via a short demographic survey. There was only one participant who reported smoking on a regular basis, and inclusion of the data from this participant in the statistical analyses did not alter the results. Females who took birth control on a regular basis were also allowed to participate in the study; prior to participation, we asked female participants if they took birth control via a short demographic survey. Females who reportedly took birth control were not significantly different from naturally cycling females on any physiological or behavioral measure, nor did stress significantly interact with birth control in these analyses. Therefore, we treated

3 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) all females as a single group in the statistical analyses for this study. Participants were asked to refrain from using recreational drugs (e.g., marijuana) for 3 days prior to the experimental sessions; to refrain from drinking alcohol or exercising extensively for 24 h prior to the experimental sessions; and, to refrain from eating or drinking anything but water for 2 h prior to the experimental sessions. Participants were awarded class credit upon completion of the study. All of the methods for the experiment were approved by the Institutional Review Board at Ohio Northern University Experimental procedures The experimental timeline for the present experiment is presented in Fig. 1. To control for diurnal variations in cortisol levels, all testing was carried out between 12 and 18 h Socially Evaluated Cold Pressor Test (SECPT) Participants were asked to submerge their dominant hand, up to and including the wrist, in a bath of water for 3 min. Those participants who had been randomly assigned to the stress condition (N = 49; 2 males and 29 females) placed their hand in a bath of ice cold ( 2 C) water, while participants who had been randomly assigned to the control condition (N = 48; 18 males and 3 females) placed their hand in a bath of warm (35 37 C) water. The water was maintained at the appropriate temperature by a Lauda Brinkmann RMT6 circulating water bath. To maximize the stress response, participants in each experiment were encouraged to keep their hand in the water bath for the entire 3-min period. However, if a participant found the water bath too painful, he or she was allowed to remove his or her hand from the water and continue with the experiment. Only five participants from the stress condition removed their hand from the water prior to 3 min elapsing (M water time = s, SD = 4.99), and all participants from the no stress condition kept their hand in the water for the entire 3-min period. Based on previous work (Schwabe, Haddad, & Schachinger, 28b), a social evaluative component was added to the cold pressor manipulation. Participants in the stress condition were misleadingly informed that they were being videotaped during the procedure for subsequent evaluation of their facial expressions, and throughout the water bath manipulation, they were asked to keep their eyes on a camera that was located on the wall of the laboratory Subjective pain and stress ratings All participants were asked to rate the painfulness and stressfulness of the water bath manipulation at 1-min intervals on 11- point scales ranging from to 1, with indicating a complete lack of pain or stress and 1 indicating unbearable pain or stress. If a participant removed his or her hand from the water before 3 min had elapsed, the remaining data points were automatically scored as 1 s for each measure Word presentation Thirty minutes following exposure to the water bath manipulation, participants were presented with a list of 3 words, which were selected from the Affective Norms for English Words (Bradley & Lang, 1999). Based on standardized valence and arousal ratings, we chose 1 neutral (5 arousing and 5 non-arousing), 1 positive (5 arousing and 5 non-arousing) and 1 negative (5 arousing and 5 non-arousing) words, which, across emotional valence and arousal categories, were balanced for word length and word frequency. As per the methods employed by Schwabe et al. (28a), participants were instructed to read each word aloud and rate its emotional valence on a scale from 3 (very negative) to +3 (very positive) and its arousal level on a scale of 1 (not arousing) to 7 (very highly arousing) on a sheet of paper containing the list of words. These manipulations were performed to promote encoding of the words, and they allowed us to analyze the final memory data based on participants own ratings of the words (see Section 2.3). Two versions of the word list were used in the experiments. According to the Affective Norms for English Words (Bradley & Lang, 1999), the mean (±SEM) standardized valence and arousal ratings for the words that made up these lists were as follows: word list 1 (positive arousing words (e.g., erotic): valence = 7.77 ±.16, arousal = 6.84 ±.29; positive non-arousing words (e.g., butterfly): valence = 7.41 ±.13, arousal = 3.23 ±.8; negative arousing words (e.g., poison): valence = 2.32 ±.2, arousal = 6.71 ±.37; negative non-arousing words (e.g., bored): valence = 2.5 ±.2, arousal = 3.75 ±.24; neutral arousing words (e.g., lightning): valence = 4.61 ±.25, arousal = 6.53 ±.12; neutral non-arousing words (e.g., hairpin): valence = 5. ±.18, arousal = 3.54 ±.13) and word list 2 (positive arousing words (e.g., lust): valence = 7.69 ±.26, arousal = 6.86 ±.22; positive non-arousing words (e.g., secure): valence = 7.4 ±.9, arousal = 3.46 ±.21; negative arousing words (e.g., bloody): valence = 2.3 ±.2, arousal = 6.74 ±.17; negative non-arousing words (e.g., trash): va- Fig. 1. Timeline for the methodology employed in the present study. Participants were exposed to the water bath manipulation at time point. Participants in the stress condition placed their dominant hand in ice cold ( 2 C) water while believing that they were being videotaped [Socially Evaluated Cold Pressor Test (SECPT)]; participants in the no stress condition placed their dominant hand in warm (35 37 C) water. Thirty minutes following the initiation of the water bath manipulation, participants were presented with a list of 3 words. They were asked to read each word aloud and to rate each word s emotional valence and arousal level. Immediately following word list encoding, participants were given a free recall assessment. To verify the induction of a stress response, saliva samples (S in the figure) and cardiovascular measurements (C in the figure) were obtained from participants throughout the experimental session. Twenty-four hours later, participants returned to the laboratory to complete free recall and recognition tests regarding the word list that was studied on the previous day. Recognition memory was assessed 15 min following the free recall assessment.

4 8 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) lence = 2.39 ±.25, arousal = 4.35 ±.24; neutral arousing words (e.g., volcano): valence = 4.71 ±.14, arousal = 6.58 ±.33; neutral non-arousing words (e.g., bland): valence = 5.1 ±.24, arousal = 3.48 ±.13) Memory testing Immediately following word list encoding, participants were given 5 min to write down as many words as they could remember from the list of words they just studied (i.e., immediate free recall). One day following the first experimental session, participants returned to the laboratory to have their memory for the list of words assessed. Participants were again given 5 min to write down as many words as they could remember from the list of words that they studied on the previous day (i.e., delayed free recall). Then, participants sat quietly and completed school work that they had brought to the laboratory. After 15 min had elapsed, participants were given a recognition test. They were presented with a list of words containing 3 old words (i.e., words that were presented on the previous day) and 3 new words (i.e., words that were not presented on the previous day) and were instructed to label each word as old or new. The new words were matched to the old words on emotional valence, arousal level, word length and word frequency, according to the ratings obtained from the Affective Norms for English Words (Bradley & Lang, 1999). To assess participants ability to discriminate between old and new words, we calculated a sensitivity index (d = z[p(hit) p(false alarm)]) for each category of word (i.e., positive-arousing, positive-non-arousing, negative-arousing, etc.) to be used for statistical analysis (Wickens, 22) Cardiovascular analysis Heart rate (HR) and blood pressure (BP) measurements were taken 2 min before (baseline), halfway through and 1 min after the water bath manipulation. Cardiovascular activity was measured with a vital signs monitor (Mark of Fitness WS-82 Automatic Wrist Blood Pressure Monitor) placed on the wrist of each participant s non-dominant hand Cortisol analysis Saliva samples were collected from participants 2 min before (baseline) and 28 min following exposure to the water bath manipulation to analyze salivary cortisol concentrations. The samples were collected in a Salivette saliva collection device (Sarstedt, Inc., Newton, NC). Participants were asked to place a swab of cotton in their mouths and chew on it so that it would easily absorb their saliva. Following 1 min of chewing, the swab was collected and placed in the Salivette conical tube and kept at room temperature until the experimental session was completed. The samples were subsequently stored at 2 C until assayed for cortisol. Saliva samples were thawed and extracted by low-speed centrifugation. Salivary cortisol levels were determined by enzyme immuno assay (Caymen Chemical Co., Ann Arbor, MI) according to the manufacturer s protocol. The minimum detectable concentration of cortisol was approximately 8 pg/ml, and the average interand intra-assay percent coefficients of variation were less than 6.9% and 6.8%, respectively Statistical analyses Prior to performing any statistical analyses, we conducted correlations to verify that participants valence and arousal ratings of the studied words were not significantly correlated; our analysis confirmed this prediction. Therefore, mixed-model ANOVAs were used to analyze all data; the between-subjects factors utilized in these analyses were stress and sex, and the within-subjects factors were word valence and arousal (for recall and recognition analyses) or time (for physiological and pain/stress ratings analyses). Participants in the stress condition were divided into and based on their cortisol responses to the SECPT. Those participants exhibiting a cortisol increase of at least 1.5 nmol/l following the SECPT were considered ; all other participants were considered. The cutoff for dividing participants into Responder and Non-Responder groups was based on previous work using a similar criterion (Schwabe et al., 28a; Zoladz et al., 211a). The analyses of participants valence and arousal ratings and memory for the words (i.e., immediate free recall, delayed free recall, and recognition) were performed based on categorizing the words (i.e., distributing the words to positive-arousing, positive-non-arousing, negative-arousing, etc. groups) according to participants subjective valence and arousal ratings that were obtained during the study (see Section 2.2.3). For each analysis of memory data, we conducted a mixed-model ANOVA analyzing the effects of stress, overall (i.e., stress vs. no stress), on memory performance, and then we conducted a subsequent mixed-model ANOVA to assess the influence of cortisol response to stress (i.e., vs. ) on memory performance. For memory measures affected by stress, we conducted additional ANCOVAs to control for the influence of participants valence and arousal ratings of the studied words on such effects. We also conducted bivariate correlations (Pearson s r) to explore possible relationships between participants physiological responses to stress and cognitive performance. To limit the inflation of Type I error rates in these analyses, the correlations were performed only for memory measures affected by stress. Alpha was set at.5 for all analyses, and Bonferroni-corrected post hoc tests were employed when necessary. Outlier data points that were at least 3 standard deviation units beyond the exclusive group means were eliminated from the analyses; less than 1% of all data were outliers. SPSS (version 18.; SPSS, Inc.) was used to perform all statistical analyses. 3. Results 3.1. Physiological responses Heart rate (see Table 1) The stress manipulation had no effect on HR (no significant effect of condition: F(2,88) = 1.43, p >.5, g 2 =.3). However, females exhibited an overall greater HR than males (significant effect of sex: F(1,88) = 4.53, p <.5, g 2 =.5), and HR decreased over time across all participants (significant effect of time: F(2,176) = 6.92, p <.1, g 2 =.7). No other significant effects were observed Systolic blood pressure (see Table 1) Stressed participants, independent of cortisol response to the stressor, exhibited greater systolic BP than non-stressed participants during the water bath manipulation (significant effect of condition: F(2,88) = 8.36, p <.1, g 2 =.16; significant effect of time: F(2,176) = , p <.1, g 2 =.62; significant Condition Time interaction: F(4,176) = 8.54, p <.1, g 2 =.16). Male participants also exhibited greater systolic BP than female participants, particularly in response to the water bath manipulation (significant effect of sex: F(1,88) = 31.16, p <.1, g 2 =.26; significant Sex Time interaction: F(2,176) = 4.43, p <.5, g 2 =.5). No other significant effects were observed Diastolic blood pressure (see Table 1) Stressed participants, independent of cortisol response to the stressor, exhibited greater diastolic BP than non-stressed partici-

5 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) Table 1 Cardiovascular activity before, during and after the water bath manipulation. DV/condition Pre During Post Heart rate (bpm) Male (5.14) (3.86) 64.5 (3.4) Female (4.29) (4.94) 7.42 (2.9) Male (5.8) (4.4) (5.44) Female (3.52) (2.7) (3.18) No stress Male (1.86) (2.65) (2.2) Female 81.1 (2.5) (1.96) 8.37 (1.8) Systolic blood pressure (mm Hg) Male (4.33) (8.54) * (5.48) Female (2.63) (4.4) * (3.38) Male (3.8) (5.9) * (2.5) Female (4.16) (4.47) * (2.57) No stress Male (2.86) (3.11) (3.51) Female (2.6) (1.8) (1.93) Diastolic blood pressure (mm Hg) Male (2.93) (3.4) * 78.5 (3.44) Female (1.9) (3.44) * 74.8 (2.41) Male (2.53) (5.19) * 8. (2.1) Female 8.63 (2.26) (4.9) * (1.95) No stress Male (2.31) (2.55) (2.69) Female (1.9) 83.6 (1.4) (1.17) Data are presented as means ± SEM. * p <.5 relative to the no stress group. pants during the water bath manipulation (significant effect of condition: F(2,87) = 1.49, p <.1, g 2 =.19; significant effect of time: F(2,174) = , p <.1, g 2 =.72; significant Condition Time interaction: F(4,174) = 18.43, p <.1, g 2 =.3). Male participants also exhibited greater diastolic BP than female participants, particularly in response to the water bath manipulation (significant effect of sex: F(1,87) = 9.21, p <.1, g 2 =.1; significant Sex Time interaction: F(2,174) = 7.38, p <.1, g 2 =.8). No other significant effects were observed Salivary cortisol (see Fig. 2) Based on the criteria employed to divide participants into and, we ended up with the following sample sizes for each group: 2 (8 male, 12 female) and 29 (12 male, 17 female). As expected, exhibited greater salivary cortisol levels than nonstressed participants after the water bath manipulation (significant effect of condition: F(2,91) = 4.71, p <.5, g 2 =.9; significant effect of time: F(1,91) = 41.7, p <.1, g 2 =.31; significant Condition Time interaction: F(2,91) = 26.9, p <.1, g 2 =.36). No other significant effects were observed Subjective ratings of water bath manipulation (see Table 2) Pain ratings Stressed participants, independent of cortisol response to the stressor, reported greater pain ratings than non-stressed participants throughout the water bath manipulation, and these ratings increased over time (significant effect of condition: F(2,88) = 232.5, p <.1, g 2 =.84; significant effect of time: F(2,176) = 8., p <.1, g 2 =.8; Condition Time interaction trending toward significance: F(4,176) = 2.26, p =.6, g 2 =.5). No other significant effects were observed Stress ratings Stressed participants, independent of cortisol response to the stressor, reported greater stress ratings than non-stressed participants throughout the water bath manipulation (significant effect of condition: F(2,88) = 89.49, p <.1, g 2 =.67; significant effect of time: F(2,176) = 3.12, p <.5, g 2 =.3). No other significant effects were observed Word list ratings Valence ratings words were given more positive ratings than neutral words, which were given more positive ratings than negative words (significant effect of valence: F(2,18) = , p <.1, g 2 =.9). Non-arousing words were also given more positive ratings than arousing words (significant effect of arousal: F(1,9) = 5.61, p <.5, g 2 =.6). The latter effect seemed to be driven by neutral words being rated more negatively if they were arousing (significant Valence Arousal interaction: F(2,18) = 14.8, p <.1, g 2 =.14). No other significant effects were observed Arousal ratings words were rated as more arousing than non-arousing words (significant effect of arousal: F(1,9) = 19.49, p <.1, Cortisol (nmol/l) Water Bath Males * Learning Time (min) Time (min) Cortisol (nmol/l) Females Fig. 2. Salivary cortisol concentrations before and after the water bath manipulation in males (left) and females (right). In both sexes, were the only participants to exhibit significantly increased cortisol concentrations following the water bath manipulation. Data are presented as means ± SEM; = p <.5 relative to the no stress group. Water Bath * Learning

6 82 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) Table 2 Pain and stress ratings of the water bath manipulation. DV/Condition Minute 1 Minute 2 Minute 3 Painfulness (scale of 1) Male 6.88 (.64) * 6.75 (.88) * 7.25 (.88) * Female 5.75 (.6) * 6.25 (.66) * 6.75 (.88) * Male 6.42 (.65) * 6.67 (.62) * 6.92 (.56) * Female 6.18 (.6) * 6.65 (.49) * 7.18 (.46) * No stress Male.28 (.14).17 (.9).18 (.1) Female.1 (.8).11 (.6).1 (.6) Stressfulness (scale of 1) Male 6. (.94) * 5.75 (1.1) * 5.75 (1.3) * Female 5.25 (.72) * 5.33 (.7) * 5.67 (.71) * Male 5. (.84) * 5.42 (.92) * 5.67 (.85) * Female 5.35 (.7) * 5.76 (.63) * 6.6 (.58) * No stress Male.24 (.11).53 (.19).41 (.15) Female.1 (.6).27 (.1).3 (.1) Data are presented as means ± SEM. * p <.5 relative to the no stress group. g 2 =.68). Interestingly, however, males rated the words, overall, as less arousing than females (significant effect of sex: F(1,9) = 4.82, p <.5, g 2 =.5). In addition, positive words were rated as more arousing than negative words, which were rated as more arousing than neutral words (significant effect of valence: F(2,18) = 13.16, p <.1, g 2 =.53). This effect was dependent on both the sex of the participant and the arousal level of the words, as males, in general, rated negative, arousing words as less arousing than females did (significant Valence Arousal interaction: F(2,18) = 7.63, p <.1, g 2 =.8; significant Sex Valence Arousal interaction: F(2,18) = 3.52, p <.5, g 2 =.4). No other significant effects were observed Memory testing Immediate free recall (see Fig. 3) The analysis of stress effects, overall, revealed that the stress manipulation had no effect on short-term memory (no significant effect of stress: F(1,92) =.1, p >.5, g 2 =.). We then analyzed for the involvement of cortisol response in the effects of stress on immediate free recall; this analysis again revealed that the stress manipulation had no effect on short-term memory (no significant effect of condition: F(2,9) = 1.36, p >.5, g 2 =.3). However, participants recalled more arousing words than nonarousing words (significant effect of arousal: F(1,9) = 5.93, p <.1, g 2 =.36). This effect was dependent on word valence, as participants recalled more arousing words than non-arousing words only when the words were neutral or positive in valence (significant Valence Arousal interaction: F(2,18) = 9.61, p <.1, g 2 =.1). No other significant effects were observed Delayed free recall (see Fig. 4) The analysis of the effects of stress, overall, revealed that the stress manipulation had no effect on delayed free recall (no significant effect of stress: F(1,9) =.5, p >.5, g 2 =.). We then analyzed for the involvement of cortisol response in the effects of stress on delayed free recall; this analysis revealed that male recalled fewer words than all other groups (significant Condition Sex interaction: F(2,88) = 3.3, p <.5, g 2 =.7). Also, arousing words were better recalled than non-arousing words, particularly when they were neutral or positive words (significant effect of arousal: F(1,88) = 75.6, p <.1, g 2 =.46; significant Valence Arousal interaction: F(2,176) = 5.25, p <.1, g 2 =.6). No other significant effects were observed. We then conducted an ANCOVA on the delayed free recall data, using the valence and arousal ratings as covariates. This analysis revealed that when controlling for such word ratings, the aforementioned stress effects on memory remained significant (significant Condition Sex interaction: F(2,86) = 3.16, p <.5, g 2 =.7) Delayed recognition (see Fig. 5) The analysis of the effects of stress, overall, on recognition revealed that stressed participants recognized fewer words than non-stressed participants, particularly when they were positive and non-arousing (effect of condition trending toward significance: F(1,86) = 3.89, p =.52, g 2 =.4; significant Stress Valence Arousal interaction: F(2,172) = 3.1, p <.5, g 2 =.4). We then analyzed for the involvement of cortisol response in the effects of stress on recognition; this analysis revealed that neutral words were better recognized than negative words (significant effect of valence: F(2,168) = 3.92, p <.5, g 2 =.5). This effect may have been more evident in and non-stressed participants because recognized fewer neutral words than all other groups (significant Condition Valence interaction: F(4,168) = 2.55, p <.5, g 2 =.6). Male and female also tended to recognize fewer words, overall, than their respective no stress group, but this effect was only approaching significance (Condition Sex interaction trending toward significance: F(2,84) = 2.7, p =.73, g 2 =.6). words were also better recognized than nonarousing words, particularly when they were positive in valence (significant effect of arousal interaction: F(1,84) = 4.65, p <.5, g 2 =.5; significant Valence Arousal interaction: F(2,168) = 9.55, p <.1, g 2 =.1). No other significant effects were observed. We then conducted an ANCOVA on the recognition data, using the valence and arousal ratings as covariates. This analysis revealed that when controlling for such word ratings, the aforementioned stress effects on memory were only approaching significance (Condition Valence interaction trending toward significance: F(4,164) = 2.3, p =.93, g 2 =.5; Condition Sex interaction trending toward significance: F(2,82) = 2.82, p =.65, g 2 =.6) Associations between physiological stress response and memory There were significant negative correlations between cortisol and (1) overall delayed free recall, r(19) =.46, and (2) emotional word (i.e., positive, negative, neutral) delayed free recall, r(19) =.48, which were observed in stressed males (i.e., and combined) only (p s <.5). 4. Discussion We have found that brief stress administered 3 min before learning exerts deleterious effects on long-term (24-h) memory in human participants. Importantly, however, the magnitude of these effects and the type of influence that corticosteroids had on such effects appeared to differ across the sexes. We did find that, independent of sex, exhibited impaired recognition of emotionally neutral information, but this effect seemed to be visibly more pronounced in males. In addition, males exhibiting a robust stress-induced increase in cortisol displayed significantly impaired recall and a marginally significant impairment of recognition memory, while females exhibiting a blunted stress-induced

7 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) Males 8 Females Free Recall (% of total) Free Recall (% of total) Non- Non- Non- Non- Non- Non- Fig. 3. Immediate free recall performance in males (left) and females (right). No statistically significant group differences were observed, suggesting that stress exposure did not impact short-term memory. Data are presented as means ± SEM. 6 5 Males 6 5 Females Free Recall (% of total) Non- * Non- Non- * Free Recall (% of total) Non- Non- Non- Fig. 4. Long-term (24-h) free recall performance in males (left) and females (right). Male recalled fewer words, overall, than all other groups. This effect seemed to be driven by a large reduction in their recall of non-arousing words (verified via planned comparisons; see Section 4); however, the Condition Sex Arousal interaction was not significant. Data are presented as means ± SEM; = p <.5 relative to the Non-Responder and no stress groups. increase in cortisol exhibited a marginally significant impairment of recognition memory. In general, our findings support the temporal dynamics model of stress memory interactions by revealing that even a brief stressor that is temporally separated from the learning experience can impair long-term memory, hypothetically via amygdala-induced inhibition of hippocampal processing. However, the differences observed across male and female participants draw attention to the fact that sex differences must be integrated into this model for it to be a more comprehensive approach to understanding emotional memory processing Neurobiological mechanisms Extensive work has shown that stress exerts differential effects on various stages of learning and memory. For instance, stress can enhance, impair or have no effect on encoding/acquisition (e.g., Diamond et al., 26; Payne, Nadel, Allen, Thomas, & Jacobs, 22; Schwabe et al., 28a). On the other hand, stress generally facilitates consolidation (Beckner et al., 26; Cahill et al., 23; Hui et al., 26; Preuss & Wolf, 29; Smeets et al., 28), while impairing retrieval (Buchanan & Tranel, 28; Buchanan et al., 26; de Quervain, Roozendaal, & McGaugh, 1998; Diamond et al., 26; Kuhlmann et al., 25; Smeets et al., 28; Tollenaar et al., 28), effects that have both been associated with stress-induced increases in glucocorticoids. In the present study, we observed significant effects of stress on delayed, but not immediate, memory testing, suggesting that stress largely affected the consolidation of information. Since we observed significant stress-induced impairments of long-term memory in both males and females, it might appear that our results are in conflict with the prevailing view that stress enhances consolidation. However, it is important to emphasize that a significant portion of studies demonstrating stress-induced enhancements of memory consolidation have administered the stress after learning (Beckner et al., 26; Cahill et al., 23; Hui et al., 26; Preuss & Wolf, 29; Smeets et al., 28). The effects of pre-learning stress on memory consolidation are less clear and often depend on the timing of the stress relative to learning (Diamond et al., 27; Zoladz et al., 211a). For

8 84 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) Discrimination Index (d') Males β Discrimination Index (d') Females β. Non- Non- Non-. Non- Non- Non- Fig. 5. Long-term (24-h) recognition performance in males (left) and females (right). recognized fewer neutral words than all other groups. The Condition Sex interaction was approaching significance, suggesting that male and female tended to recognize fewer words, overall, than their respective no stress group. The observed effect in male seemed to be driven by a large reduction in their recognition of arousing words, while the observed effect in female Non- seemed to be driven by a large reduction in their recognition of non-arousing words (verified via planned comparisons; see Section 4); however, the Condition Sex Arousal interaction was not significant. Data are presented as means ± SEM; b = p =.73 relative to the no stress group. instance, according to the temporal dynamics model, pre-learning stress would enhance consolidation only if the stressor was brief and presented in close temporal proximity to the learning experience. If, on the other hand, the stressor was long-lasting or was a brief stressor that was temporally separated from the learning experience, then consolidation would hypothetically be impaired (see Diamond et al., 26 for an example). Research has also shown that pre-learning stress effects on long-term memory depend on the sex of the organism and the type of information being acquired. For instance, studies have reported that pre-learning stress often facilitates memory for emotional information, while impairing memory for neutral information (Jelicic et al., 24; Payne et al., 26; Payne et al., 27). Moreover, the stress-induced enhancement of emotional memory has been reported to be stronger in females, while the stress-induced impairment of neutral memory has been reported to be stronger in males (Payne et al., 26). Along similar lines, we found that stress impaired the recognition of neutral words in, an effect that seemed to be more pronounced in males, and that the stress-induced impairment of recall in males and recognition in females was largely driven by reduced memory for non-arousing information (discussed further below). Thus, how stress affects consolidation depends on several factors related to the timing of the stressor, the type of information being learned and the sex of the organism. Research has accumulated over the past decade to suggest that corticosteroids act as two-stage rockets, exerting immediate excitatory, but delayed inhibitory, effects on cellular function and synaptic plasticity (Joels et al., 26; Joels et al., 211). In relation to the temporal dynamics model of emotional memory processing, the initial excitatory phase of hippocampal function that immediately follows stress onset is thought to depend, at least in part, on noradrenergic activity coupled with non-genomic activity of corticosteroids and an ensuing increase in glutamatergic transmission (Diamond et al., 27; Karst et al., 25). The inhibitory phase that follows shortly thereafter is thought to depend on NMDA receptor desensitization and delayed, gene-dependent activity of corticosteroids (Diamond et al., 27; Joels et al., 23). The present findings support this model, at least to a degree, in that, overall, exhibited impaired recognition memory for neutral words, while male exhibited impaired recall and a marginally significant impairment of recognition memory. However, the predictions based on the temporal dynamics model were less evident in female participants. That is to say, despite the finding of impaired recognition memory for neutral information in, this effect seemed to be driven largely by deficits in males. Moreover, stressed females demonstrated no significant alteration of recall performance, and the marginally significant impairment that was observed on recognition memory in females was observed in, not. There was also a significant negative correlation between cortisol and memory in stressed males, but no such association was observed in females. In fact, if anything, our findings would suggest that cortisol had some sort of protective effect on memory, at least for that of recognition memory overall, in females. Ultimately, these results support the temporal dynamics model by showing that brief stress that was temporally separated from the learning experience impaired long-term memory in both male and female participants. However, the sex-dependent influence of corticosteroids on these effects indicate that there is a need to update the model, which was based largely on research conducted in males, by considering how sex differences influence the relationship between timing, stress memory interactions and their neurobiological basis. Another, fairly complementary, approach to the biological basis of stress memory interactions takes into account general differences in the effects of stress on amygdala and hippocampus function. Extensive work has shown that an intact amygdala is essential for enhanced memory of emotional information (Adolphs, Cahill, Schul, & Babinsky, 1997; Cahill et al., 1996; Canli, Zhao, Brewer, Gabrieli, & Cahill, 2), as well as for the stress-induced effects on hippocampus-dependent learning and synaptic plasticity (Kim et al., 21, 25; Zoladz, Park, & Diamond, 211b). There is also considerable evidence for the direct modulation of hippocampal function via amygdala activation (Akirav & Richter-Levin, 1999, 22; Richter-Levin & Akirav, 23; Vouimba, Yaniv, & Richter-Levin, 27), which provides a potential mechanism to explain stress-induced effects on information storage. It has been speculated that amygdala activation underlies the stress-induced enhancement of emotional memory, while hippocampal inhibition underlies the stress-induced impairment of neutral memory (Diamond et al., 27; Jelicic et al., 24; Payne et al., 27). Theoret-

9 P.R. Zoladz et al. / Neurobiology of Learning and Memory 1 (213) ically, in the face of stress, the processing of information with survival value (e.g., arousing) would take priority over the storage of other, less important (e.g., non-arousing) information (Diamond et al., 27). In the present study, it is possible that stress enhanced amygdala function while at the same time inhibited hippocampal function, which manifested as impaired memory, particularly for emotionally neutral information. Indeed, a close examination of the effects of stress on memory in the present study revealed that the stress-induced impairment of recall in males and the stress-induced marginally significant impairment of recognition in females seemed to be largely driven by reduced memory for non-arousing information. These speculations were corroborated by planned comparison analyses (p s <.5), despite the lack of statistically significant Condition Sex Arousal interactions. Also,, independent of sex, recognized significantly fewer neutral words than all other groups, even though this effect also seemed to be more pronounced in males. On the other hand, the marginally significant impairment of recognition memory observed in males seemed to be largely driven by reduced memory for arousing information, which according to the aforementioned perspective, might be expected to be enhanced, if anything, as a result of a stress-induced increase in amygdala activity. It is possible that stress does not always enhance one s memory for arousing information. Indeed, some research has shown that whether or not the information is related to the stressor is important in determining if memory is affected by the stress. Smeets et al. (29) found that stress enhanced participants memory for words only if the words were highly arousing and directly related to the stressor that was employed in the study. Thus, the traditional stress-induced enhancement of arousing information may not have taken place in the present study because the studied words were not related to the cold pressor stress Sex differences Our finding of greater deleterious effects of pre-learning stress on long-term memory in males, relative to females, is not uncommon. Others have reported significant effects of stress on memory in males, while finding no differences (e.g., Andreano & Cahill, 26) or an enhancement of memory (e.g., Payne et al., 26) in females. Comparable findings have also been reported in studies examining stress effects on working memory (e.g., Cornelisse, van Stegeren, & Joels, 211). It is possible that participants perceived arousal of the studied words influenced the observed sex differences in the present study. We found that males rated the studied words as less arousing than females did. Thus, it is possible that stress exerted greater deleterious effects on memory in males because the studied information, overall, was not perceived as emotionally arousing as it was in females. This speculation would relate to previous work that has shown greater stress-induced impairments of memory for neutral information in males (Payne et al., 26). The present findings are also markedly similar to those from investigators who have reported negative or curvilinear relationships between cortisol and memory in males, but not females (Andreano & Cahill, 26; Wolf et al., 21). We found that male exhibited impaired long-term free recall and a marginally significant impairment of long-term recognition memory. Female, on the other hand, displayed a marginally significant impairment of long-term recognition memory. Thus, as indicated above, cortisol was potentially associated with better memory, at least for that of recognition memory overall, in females, in contrast to males. A closer examination of the female recognition memory data, however, suggests that a strong cortisol response to the stress might have simply preserved their memory (Payne et al., 26), rather than facilitating it, since female performed better relative to female only, and female performed more poorly than the no stress group. Since we did not observe any significant sex differences in cortisol response to the stressor, these differential effects that we observed on measures of memory are not likely attributable to variations in hypothalamus pituitary adrenal axis response. Studies have shown that differences in the menstrual cycle play an important role in the stress-induced alteration of cognition. For instance, Andreano, Arjomandi, and Cahill (28) observed a positive correlation between stress-induced salivary cortisol and memory only when female participants were in the mid-luteal phase of the menstrual cycle, a time when progesterone levels are significantly elevated. Subsequent work substantiated these findings by reporting that females exhibiting high levels of progesterone (i.e., mid-luteal phase) demonstrate better memory for emotional information, greater stress-induced elevations of salivary cortisol and stronger stress-induced enhancements of emotional memory than women with low levels of progesterone (i.e., non-luteal phase) (Ertman, Andreano, & Cahill, 211; Felmingham, Fong, & Bryant, 212). Females also exhibit stronger responses of the amygdalahippocampus neural network to emotional stimuli when they are in the luteal phase (Andreano & Cahill, 21). Although we did not control for the menstrual cycle in the present study, it is possible that the differential effects observed in males vs. females could be attributable to such a factor. If female and were in different stages of the menstrual cycle, they could have exhibited differential sensitivity to stress hormones, which led to different effects on memory. Indeed, research has shown that the luteal phase of the menstrual cycle is associated with altered glucocorticoid sensitivity (Rohleder, Schommer, Hellhammer, Engel, & Kirschbaum, 21; Rohleder, Wolf, Kirschbaum, & Wolf, 29) and elevated norepinephrine levels (Minson, Halliwill, Young, & Joyner, 2). In order to verify this speculation, however, future work will need to control for this factor when examining pre-learning stress effects on memory. It is also possible that the female brain does not respond to emotion in the same manner that the male brain does. Some research has shown that the brain responses of males and females to emotional stimuli are hemisphere-specific. For instance, Cahill and others have reported that, when presented with emotional stimuli, males tend to exhibit greater activity in the right amygdala, while females tend to exhibit greater activity in the left amygdala (Cahill, Uncapher, Kilpatrick, Alkire, & Turner, 24; Cahill et al., 21; Canli, Desmond, Zhao, & Gabrieli, 22; Mackiewicz, Sarinopoulos, Cleven, & Nitschke, 26). Interestingly, the right amygdala has been associated with one s memory for gist, while the left amygdala has been associated with one s memory for details (Fink, Marshall, Halligan, & Dolan, 1999; Fink et al., 1996). Thus, it may not be surprising to note that, under emotionally arousing conditions, males exhibit greater memory for the central aspects of a scene, while females exhibit greater memory for the details (Cahill & van Stegeren, 23). This differential activation of brain areas in response to emotional stimuli could play an important role in sex influences on stress memory interactions. It could also be related to stress-induced switches in learning style that appear to differ across the sexes (Beck & Luine, 21) Limitations and caveats Although the present findings provide important insight into the factors that influence stress memory interactions, there were some limitations that deserve attention. First, when we divided the stressed participants into and, we ended up with a relatively small sample size of male (N = 8). Since a significant portion of our effects were evident in