The definitive version of this manuscript is available at http://www.blackwell-synergy.com/doi/abs/10.1111/j.1460-9568.2006.04921.x Early life modulators and predictors of adult synaptic plasticity. Katherine G. Akers1, Masato Nakazawa1, Russell D. Romeo3, John A. Connor2, Bruce S. McEwen3, & Akaysha C. Tang1,2* 1 Department of Psychology Department of Neurosciences University of New Mexico Albuquerque, NM 87131 2 3 Laboratory of Neuroendocrinology Rockefeller University New York, NY 10021 *Corresponding author: Akaysha C. Tang MSC032220 Department of Psychology 1 University of New Mexico Albuquerque, NM 87131 Tel: (505) 277-4025 Cell: (505) 610-9077 Fax: (505) 277-1394 E-mail: firstname.lastname@example.org Keywords: neonatal handling; hypoxia; open field; rat; social recognition memory. Early experience and synaptic plasticity Akers et al. 2 ABSTRACT Early life experience can induce long-lasting changes in brain and behavior that are opposite in direction, such as enhancement or impairment in regulation of stress response, structural and functional integrity of the hippocampus, and learning and memory. To explore how multiple early life events jointly determine developmental outcome, we investigated the combined effects of neonatal trauma (anoxia on postnatal day 1) and neonatal novelty exposure (postnatal day 221) on adult social recognition memory (3 months of age) and synaptic plasticity in the CA1 of the rat hippocampus (4.5-8 months of age). While neonatal anoxia selectively reduced posttetanic potentiation (PTP), neonatal novel exposure selectively increased long-term potentiation (LTP). No interaction between anoxia and novelty exposure was found on either PTP or LTP. These findings suggest that the two contrasting neonatal events have selective and distinct effects on two different forms of synaptic plasticity. At the level of behavior, the effect of novelty exposure on LTP was associated with increased social memory, and the effect of anoxia on PTP was not accompanied by changes in social memory. Such a finding suggests a bias toward the involvement of LTP over PTP in social memory. Finally, we report a surprising finding that an early behavioral measure of emotional response to a novel environment obtained at 25 days of age can predict adult synaptic plasticity measured several months later. Therefore, individual differences in emotional responses present during the juvenile stage may contribute to adult individual differences in cellular mechanisms that underlie learning and memory. Early experience and synaptic plasticity Akers et al. 3 INTRODUCTION Early life experience can have detrimental or beneficial impact on individual brain and behavioral development (Harlow, 1959; Levine, 1960; Bowlby, 1962; Denenberg, 1964; Rutter, 1972; Meaney et al., 1996; Chapillon et al., 2002; Pryce & Feldon, 2003). Early aversive events such as prolonged maternal separation or disrupted maternal care impair hypothalamic-pituitaryadrenal (HPA) axis function (e.g. Ladd et al., 2004; Knuth & Etgen, 2005; Neumann et al., 2005) and reduce hippocampal synaptic plasticity (Brunson et al., 2005) and memory function (e.g. Lehmann et al., 1999; Huot et al., 2002; Sandstrom & Hart, 2005). In contrast, mild early stimulation such as neonatal handling, brief isolation, or increased maternal care enhance HPA function (e.g. Francis et al., 1999; Macri et al., 2004; Panagiotaropoulos et al., 2004), hippocampal synaptic plasticity (Kehoe & Bronzino, 1999), and memory function (e.g. Aguilar et al., 2002; Lehmann et al., 2002; Garoflos et al., 2005). A growing body of evidence further indicates that early aversive events and mild stimulation interact to determine the outcome of development (Wakshlak & Weinstock, 1990; Escorihuela et al., 1994; Maccari et al., 1995; Francis et al., 2002; Morley-Fletcher et al., 2003; Bredy et al., 2004; Hellemans et al., 2004). One specific early aversive event that has a profound impact on development is neonatal anoxia, a form of birth trauma that increases hyperactivity in the open field during juvenility (e.g. Nyakas et al., 1991; Iuvone et al., 1996), impairs performance in various memory tasks during adulthood (e.g. Buwalda et al., 1995; Mishima et al., 2005), and increases synaptic plasticity immediately or shortly after the trauma (Jensen et al., 1998; Zhang et al., 2005) with long-term effects on synaptic plasticity unknown. In contrast to these effects of neonatal anoxia, an early mild stimulation paradigm called neonatal novelty exposure decreases behavioral reactivity in the open field (Tang, 2001), enhances performance in memory tasks (Tang, 2001; Tang et al., Early experience and synaptic plasticity Akers et al. 4 2003), and increases synaptic plasticity (Tang & Zou, 2002) long after the initial neonatal stimulations. Neonatal anoxia and neonatal novelty exposure, therefore, appear to produce opposite effects, at least on measures of open field behavior and memory. Such opposing actions have been confirmed for the open field measure in a recent study (Tang & Nakazawa, 2005). Given that the two early events have opposing effects on memory when studied separately, we hypothesize that anoxia may lead to long-term deficits in both memory and synaptic plasticity, which may underlie learning and memory (Bliss & Collingridge, 1993; Stevens, 1998), and that these potential detrimental effects might be reversible by subsequent neonatal novelty exposure. Taking advantage of the fact that the present study was conducted using the same animals whose open field behaviors were previously characterized during the juvenile stage (Tang & Nakazawa, 2005), we test another hypothesis that open field activity early in life may serve as a long range marker for adult synaptic plasticity. METHODS AND MATERIALS Animals. All procedures were in accordance with the Institutional Animal Care and Use Committee at the University of New Mexico. Eleven pregnant Long-Evans hooded dams (Charles River, Raleigh, NC) arrived at the vivarium 16 days prior to giving birth. Litter size at birth ranged from 8 to 18 pups. Pups were housed with their dams until weaning on postnatal day 22. Thereafter, rats were housed individually in plastic cages (51 x 25 x 22 cm) with a 12-h light/dark cycle (lights on at 7:00 am) and food and water ad libitum. Experimental design. A 2 x 2 factorial design was used with neonatal anoxia (Anoxia vs. Control) as a between-litter factor and neonatal novelty exposure (Novel vs. Home) as a within-litter factor. Specifically, entire litters were randomly assigned to either the Control or the Anoxic condition, and within each litter approximately half of the pups were assigned to the Early experience and synaptic plasticity Akers et al. 5 Novel group and the other half to the Home group. The same groups of animals were tested in the open field, a social recognition test, and synaptic plasticity experiment in the order shown in Fig. 1. The social recognition test was chosen as a memory test because we were interested in the potential involvement of the hippocampus in social memory. The order each litter experienced anoxia treatment, group membership marking, and neonatal novelty exposure was counterbalanced among the four experimental conditions. Furthermore, within a specific test, the order in which each rat was assessed was also counterbalanced. All testing was performed by experimenters who were blind to the animals’ group identity. Neonatal anoxia and neonatal novelty exposure. On postnatal day 1, after the dam was removed from the home cage and placed into a holding cage, pups were transferred from the home cage to a plastic airtight chamber (25 x 20 x 13 cm) equipped with an air inlet and outlet. Pups were exposed for 25 min to a continuous flow of either 100% nitrogen gas (Anoxic condition) or room air (21% O2/79% N2, Control condition) at a flow rate of 3 liters/min. The chamber temperature was held at 31° C by partial immersion in water heated by a submersible heater (Whisper, Tetra, VA). Immediately after anoxia treatment, litters were culled to 8-10 pups. Next, pups were pseudorandomly assigned to Novel and Home groups and group membership was marked by tattooing the ventral surface of the hind paws before pups were returned to the home cage. Neonatal novelty exposure (Tang, 2001) was performed daily from P2-21 by an experimenter blind to the Anoxic versus Control litter conditions. First, the dam was removed from the home cage and placed into a separate holding cage. Next, the Novel pups were identified and individually transferred to small non-home cages lined with fresh sawdust. After spending 3 min in the non-home cages, Novel pups were returned to the home cage in which the Early experience and synaptic plasticity Akers et al. 6 Home pups remained. After the Novel and Home pups were reunited, the dam was returned to the home cage. During the pup transfer, every time a Novel pup was touched, a yoked Home pup was touched in a similar manner and for a similar duration. Thus, both experimenter contact and maternal separation were matched between Novel and Home groups. This ensured that the only difference in treatment was the brief, repeated exposures to a novel environment. Detailed description of these behavioral manipulations can be found elsewhere (Tang & Nakazawa, 2005). Open field testing. Initial open field activity was measured when rats were in the juvenile stage (P25), a time in development at which perinatal anoxia/hypoxia-induced changes in open field behavior are particularly apparent (e.g. Dell'Anna et al., 1991; Nyakas et al., 1991; Iuvone et al., 1996). Specifically, rats (N = 74 rats) were tested in a novel open field (60 x 60 x 60 cm) during four 20-s trials with an intertrial interval of ~5 min. This short trial duration was chosen to capture individual differences in the initial response upon entering a novel environment rather than to obtain a more prolonged activity measure. Initial activity was measured by the average number of squares crossed across the four trials. Novelty and Anoxia treatment effects on this open field activity measure, as well as additional details of open field testing, have been documented in a previous publication (Tang & Nakazawa, 2005). Social recognition memory testing. On P100-101, habituation of social investigative behavior was measured within Novel-Home pairs of rats over four 5-min sessions on two consecutive days (day 1: S1-S3; day 2: S1). Pairs of rats (N = 24 pairs) were formed from the same Anoxic vs. Control condition, matched in weight, and non-siblings. To allow group identification, rats were marked on both sides of the body with red or green food coloring, and colors were counterbalanced among experimental conditions. Social sessions occurred in a Early experience and synaptic plasticity Akers et al. 7 neutral testing cage. On each day, social sessions were preceded by a 5-min cage habituation (Hab) session, during which rats were separated from each other by a partition placed in the middle of the cage. During inter-session intervals (10 min between S1 and S2; 2 min between S2 and S3), rats were returned to their home cages located in the testing room. Social sessions were videotaped for offline analysis. Social investigative behavior was defined as being oriented toward (tip of nose within ~1 cm) or in direct contact with the conspecific while sniffing or grooming. If social investigation was present at any time during a 5-s interval, an occurrence of one was counted. As a large percentage of social investigation was mutual (occurring simultaneously in both rats), only unidirectional investigation was included in the analysis. Long-term memory was assessed by a long-term habituation (LTH) score, defined as (S1– D2S1)/S1*100. Additional details of social recognition testing can be found elsewhere (Tang et al., 2003). Electrophysiology. In vitro brain slice experiments were performed when rats were between 4.5-8 months of age (N = 21 rats). After rats were deeply anesthetized with halothane and decapitated, the brain was quickly extracted and immediately placed into ice-cold cutting solution (in mM: 220 sucrose, 3 KCl, 6 MgSO4, 0.2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 0.43 ketamine) that was oxygenated with 95% O2/5% CO2. Because neonatal novelty exposure preferentially affects hippocampal synaptic plasticity in the right hemisphere (Tang, 2003), only the right hippocampus was used. The hippocampus was sliced (350μm transverse slices) using a Vibroslice (Campden Instruments) and incubated in oxygenated artificial cerebrospinal fluid (ACSF, in mM: 126 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgSO4, 10 glucose, 2 CaCl2) at 34° C for one hour. After incubation, slices were held at room temperature until recording. All recordings were made within 10 hours of decapitation. Early experience and synaptic plasticity Akers et al. 8 During recording, slices were submerged in a recording chamber and continuously perfused (2 ml/min) with oxygenated ACSF at 34° C. Field excitatory postsynaptic potentials (EPSPs) were recorded from the CA1 stratum radiatum using glass microelectrodes (2-3 MΩ) filled with ACSF. Evoked EPSPs were elicited by stimulating the Schaeffer collateral fibers with a bipolar electrode. Test pulses (70 μs duration, 0.1 Hz) were set at an intensity that evoked 50% of maximum EPSP amplitude before intrusion of a population spike (0.2-0.3 mA). LTP was induced by a high frequency stimulation (HFS) protocol consisting of 3 trains of 100 test pulses delivered at 100 Hz with a 1-min interval between trains, an induction protocol optimized for both early and delayed LTP (Behnisch & Reymann, 1995; Wilsch et al., 1998). Note that test pulses were given at the same rate during the 1-min intervals between the HFS trains (6 test pulses between each train) to allow measures of PTP. Signals were amplified 2000 times (NeuroData IR283, Cygnus Technology, Inc.), filtered with a bandpass filter of 1 Hz-5 kHz, and digitized at a rate of 5 kHz (Digidata 1322A, Axon Instruments). Data were collected and analyzed using pCLAMP 8.1 software (Axon Instruments). EPSP initial slope was averaged over six successive traces to obtain minute-byminute measures for PTP and LTP. PTP and LTP were defined as the percentage change from baseline for the minutes immediately after each of the 3 HFS trains and for the last 10 minutes of the 30-min post-HFS recording period, respectively. Corticosterone assay. Neonatal novelty exposure was previously found to lower basal levels of corticosterone (CORT) (Tang et al., 2003), a stress hormone known to affect synaptic plasticity (De Kloet et al., 1999; McEwen, 1999). Because blood samples were easily collected at the time of sacrifice immediately before LTP experiments, we also measured basal CORT concentration from the blood serum. Animals were sacrificed at approximately 10:00am. Blood Early experience and synaptic plasticity Akers et al. 9 samples were collected in EDTA-coated tubes and centrifuged. Serum was removed and stored at -20° C until radioimmunoassay was performed. Serum CORT concentrations were measured in duplicate in a single assay using a Coat-A-Count Corticosterone Kit (Diagnostic Products, Los Angeles, CA). The lower limit of detectibility was 10.90 ng/ml, and the intra-assay coefficient of variation was 5.2%. Statistical analysis. For statistical testing of synaptic plasticity measures, animal was the unit of analysis (i.e. multiple slice measures from each animal were averaged together). For analysis of PTP, repeated measures ANOVA was applied to the first 3 post-baseline minutes of data with HFS Train as a within-subject factor and Novelty and Anoxia as between-subject factors. For analysis of LTP, ANOVA was applied to the average of the last 10 minutes of the 30-min post-HFS recording period with Novelty and Anoxia as between-subject factors. For analysis of the social recognition memory measures, a pair of rats was the unit of analysis, with Novelty as a within-pair factor and Anoxia as a between-pair factor. Pearson’s correlation coefficient was used to correlate synaptic plasticity measures with a previously reported measure of open field activity obtained from the same rats (Tang & Nakazawa, 2005). To test whether correlations differed among treatment groups, we used univariate ANCOVA with Novelty and Anoxia as between-subject factors and Open Field Activity as a covariate. For analysis of stress hormone measures, univariate ANOVA was used with Novelty and Anoxia as between-subject factors. Outliers greater than 1.5 times the interquartile range were removed from analysis (Agresti & Finlay, 1997). Based on prior experience (Tang & Zou, 2002; Tang et al., 2003), directional tests were used to test Novelty effects. Effect size was expressed as f and d for Ftests and t-tests, respectively (f: large: > 0.4, medium: > 0.25, small: < 0.1; d: large: > 0.8, medium: > 0.5, small: < 0.2) (Rosenthal & Rosnow, 1984). Early experience and synaptic plasticity Akers et al. 10 RESULTS Effects of neonatal anoxia and neonatal novelty exposure on synaptic plasticity. Approximately 4.5-8 months after the neonatal anoxia treatment and subsequent novelty exposures, synaptic plasticity was examined in hippocampal slices from adult rats using a LTP induction protocol comprised of 3 HFS trains. EPSP slope prior to HFS did not differ among Novelty and Anoxic conditions (p > 0.2; Table 1; representative traces shown in Fig. 3ac). The 3 HFS trains produced successively greater increases in PTP of EPSP slope (Fig. 2). Temporal patterns across the 3 HFS trains revealed that the rate of PTP increase was lower in Anoxic than in Control rats (F(2,32) = 4.45, p = 0.020, f = 0.53; Fig. 2 inset). This PTP difference between Anoxia and Control rats increased across the 3 HFS trains, with pair-wise comparisons showing a significant difference after the 3rd train (t(18) = 2.25, p = 0.037, d = 0.99; Fig. 3b). The Novelty effect and Novelty by Anoxia interaction effect on PTP were not significant (p > 0.2). A different pattern of treatment effects was found for LTP (Fig. 2). Overall, LTP in Novel rats was significantly greater than in Home rats (F(1,17) = 3.65, p = 0.037, f = 0.46). Although the Novelty by Anoxia interaction effect on LTP did not reach statistical significance (p > 0.2), pair-wise comparisons revealed that it was only within the Control condition that Novel rats had greater LTP than Home rats (t(7) = 1.92, p = 0.048, d = 1.30, Fig. 3d-left), with no difference found between Novel and Home rats within the Anoxic condition (p > 0.2; Fig. 3dright). The effect of Anoxia on LTP was not significant (p > 0.2). This lack of Anoxia effect cannot be attributed to a general failure in the anoxia treatment, which clearly affected both PTP (present study), body weight and open field activity (Tang & Nakazawa, 2005). Effects of neonatal anoxia and neonatal novelty exposure on social memory. Social recognition memory testing was conducted approximately 1.5-5 months prior to synaptic Early experience and synaptic plasticity Akers et al. 11 plasticity experiments. Raw frequencies of social investigation during each social session and decreases in investigation across sessions (habituation) for all four treatment groups are shown in Fig. 4abcd. Novel rats exhibited greater LTH than Home rats (F(1,20) = 10.96, p = 0.002, f = 0.74, Fig. 4e), which replicates previous findings obtained from rats in the absence of neonatal trauma (Tang et al., 2003). Although the Novelty by Anoxia interaction effect on LTH did not reach statistical significance (p > 0.2), pair-wise comparisons revealed that it was only within the Control condition that Novel rats had greater LTH than Home rats (t(9) = 2.05, p = 0.035, d = 1.44, Fig. 4e-left), with no difference found between Novel and Home rats within the Anoxic condition (p = 0.189, Fig. 4e-right). Thus, the pattern of treatment effects on LTH is similar to that on LTP, in that neonatal novelty exposure increased the dependent measure only in Control but not in Anoxic rats (compare pattern of treatment effects in Fig. 4e with that in Fig. 3d). Correlations between LTH and measures of LTP and PTP were not significant (p > 0.2) Relationship between early life response to a novel environment and adult synaptic plasticity. We measured early life response to a novel environment using ambulatory activity during four 20-s exposures to an open field on P25. Detailed analysis and discussion of this open field data were published elsewhere (Tang & Nakazawa, 2005). Here, we computed correlations between average open field activity across the 4 trials and measures of adult synaptic plasticity. A significant correlation between open field activity and LTP was found within Novel Anoxic rats (r(5) = -0.830, p = 0.041); correlations within the other three treatment groups were not significant (p > 0.1). Differences in correlations between treatment groups were tested using ANCOVA. Novel and Home rats showed a significant difference in this correlation (F(1,13) = 5.02, p = 0.043, f = 0.62), with a large negative correlation found in Novel rats (r(10) = -0.802, p = 0.003, Fig. 5a) but no correlation found in Home rats (r(9) = 0.052, p > 0.2, Fig. 5b). Anoxic Early experience and synaptic plasticity Akers et al. 12 and Control rats did not differ in this correlation (p > 0.2; Fig 5ab, Anoxic: filled squares, Control: open triangles). Correlations between open field activity and PTP were not significant in any of the four treatment groups, which showed no differences from each other (p > 0.2). Basal CORT concentration did not differ among treatment groups (p > 0.2; in ng/ml: Control Novel: 58.0±16.2, Control Home: 44.9±10.6, Anoxic Novel: 54.4±16.3, Anoxic Home: 62.4±13.6). This should not be taken as evidence that there was no difference in HPA function among groups, as both neonatal trauma and mild neonatal stimulation are known to result in changes in HPA function (e.g. Meaney et al., 1988; Boksa et al., 1996) that may be more readily detected in evoked than in basal CORT measures. Furthermore, it has been shown that the effect of early mild stimulation on basal CORT may not be expressed until animals have reached senescence (Meaney et al., 1988; Tang et al., 2003). The correlations between basal CORT concentration and PTP or LTP measures were not significant (p > 0.2). DISCUSSION We investigated the combined effects of neonatal anoxia and neonatal novelty exposure on adult hippocampal synaptic plasticity (PTP and LTP) and social memory. We found that anoxia selectively reduced PTP, and novelty exposure selectively increased LTP. No significant interaction between anoxia and novelty exposure was found on either PTP or LTP. These findings suggest that short- and long-term synaptic plasticity are selectively and differentially modulated by neonatal trauma and neonatal mild stimulation, perhaps via separate cellular mechanisms. Furthermore, we found that the novelty exposure-induced increase in LTP was accompanied by an enhancement in social memory. In contrast, the anoxia-induced reduction in PTP was not accompanied by a change in social memory. These findings therefore suggest a bias toward the involvement of LTP over PTP in social memory. Finally, we evaluated open Early experience and synaptic plasticity Akers et al. 13 field activity as a predictor of adult synaptic plasticity. Among rats that experienced neonatal novelty exposure, initial activity in a novel open field during juvenility correlated with LTP during adulthood. Such a long-range correlation across developmental stages demonstrates that emotional response to novelty early in life may serve as a potential behavioral marker for adult synaptic plasticity. Neonatal anoxia effect on PTP. We found that neonatal anoxia selectively affected hippocampal PTP during adulthood. Specifically, anoxia reduced the rate of PTP over repeated HFS trains, and this effect was present regardless of whether rats experienced neonatal novelty exposure. That is, the anoxia effect was not reversed by subsequent novelty exposure. This finding contrasts with previous reports that mild post-trauma stimulation effectively blocked the impact of neonatal anoxia/hypoxia on open field activity, learning and memory, and hippocampal structure (Iuvone et al., 1996; Chou et al., 2001; Rodrigues et al., 2004; Tang & Nakazawa, 2005). Thus, it appears that mild stimulation may alleviate symptoms of neonatal trauma at the levels of behavior and gross brain morphology but not at the level of synaptic plasticity, perhaps due to the two early events having selective effects on distinct mechanisms underlying two different forms of synaptic plasticity (PTP and LTP). Because PTP is presynaptic in origin (Zucker & Regehr, 2002), the decreased PTP in Anoxic rats suggests that neonatal anoxia may produce an enduring impairment in some presynaptic mechanism underlying short-term plasticity, such as Ca2+ influx or activity of Ca2+-binding proteins. The inability of novelty exposure to counteract the effect of neonatal anoxia on PTP may be explained by a selective effect of this mild stimulation on mechanisms specific to LTP that are not involved in PTP. Further studies are required to elucidate the precise mechanisms by which Early experience and synaptic plasticity Akers et al. 14 neonatal anoxia and novelty exposure exert their selective effects on short- and long-term synaptic plasticity. A selective PTP deficit may have consequences for learning and memory. For example, mice lacking the presynaptic protein synapsin II showed a selective reduction in PTP but not LTP (Rosahl et al., 1995; Spillane et al., 1995). Despite the presence of normal LTP, these mice displayed no evidence of contextual fear conditioning following training, suggesting that PTP plays a role in learning and memory in this task independently of LTP (Silva et al., 1996). In another study, rats with poor performance in a water maze task exhibited less PTP than higherperforming rats, again despite having comparable LTP (Diana et al., 1994). The fact that the anoxia-induced PTP reduction in the present study was not accompanied by a decrease in social recognition memory suggests that a PTP-memory connection may be specific to test situations involving explicit stress, as in the case of water maze and fear conditioning tasks but not in tests involving brief interactions with a conspecific. Alternatively, it is possible that the lack of PTPsocial memory connection is specific to the induction protocol used in the present study. Nevertheless, the finding that neonatal anoxia reduced PTP may offer a possible mediating mechanism for previously reported perinatal anoxia/hypoxia-induced learning and memory deficits in water maze and fear conditioning tests (e.g. Nyakas et al., 1989; Dell'Anna et al., 1991; Buwalda et al., 1995; Simonova et al., 2003; Yang et al., 2004; Mikati et al., 2005; Mishima et al., 2005). It should be noted that because PTP is indicative of presynaptic neurotransmitter release (Zucker & Regehr, 2002), a PTP deficit may have a broad range of effects on basic sensory/motor processes apart from learning and memory. Perinatal anoxia/hypoxia has been thought to have little or no effect on many cellular measures at adulthood. For example, while neonatal anoxia on the day of birth led to significant Early experience and synaptic plasticity Akers et al. 15 CA1 cell loss between P3-P13 compared to controls, this difference disappeared by P20 due to generation of new neurons (Daval et al., 2004). The neonatal anoxia effect on hippocampal LTP seems to follow a similar pattern, with an increase in LTP relative to controls observed after a post-trauma delay of 10 min (Jensen et al., 1998) and 8 days (Zhang et al., 2005) but a lack of effect when the delay was a few months (Jensen et al., 1998). Perhaps for this reason, the majority of perinatal anoxia/hypoxia studies have predominantly been limited to functional assessment immediately or shortly after the traumatic episode, ranging from a few minutes to a maximum of several days (for reviews, see Nyakas et al., 1996; Calvert & Zhang, 2005). Our finding that PTP was reduced among anoxic rats aged 4.5-8 months clearly indicates that neonatal anoxia has long-lasting impact on hippocampal synaptic plasticity. Neonatal novelty exposure effect on LTP. We found that neonatal novelty exposure produced a selective enhancement of LTP during adulthood, a finding that provides non-trivial extension and replication of our previous study (Tang & Zou, 2002). First, the effect of experimental manipulation (ex. exposure to stress) on LTP is known to differ depending on the specific LTP induction protocol used (Mesches et al., 1999). Second, experimental manipulations (ex. brain trauma) can differentially affect animals of the same strain but from different suppliers (Oliff et al., 1995). In the present study, we replicated the novelty exposureinduced enhancement of LTP despite using a different LTP induction protocol and animal supplier as those used in our previous study. These two extensions, therefore, increase the validity of the finding that adult LTP can be enhanced by brief neonatal exposures to novelty. The observation that the neonatal novelty exposure-induced increase in LTP was significant only in control rats but not in anoxic rats might hint at a blocking of the novelty effect on LTP by anoxia. However, as the interaction effect between the two treatments was not Early experience and synaptic plasticity Akers et al. 16 statistically significant, one does not have evidence for such a conclusion. Interestingly, however, the pattern of LTP across the four treatment groups mirrored that of LTH, a measure of social recognition memory. Specifically, both LTP and LTH were increased by neonatal novelty exposure only in control rats (compare Fig. 3d to Fig. 4e). This association between a novelty exposure effect on LTP and social memory contrasts with the dissociation between an anoxia effect on PTP and a lack of anoxia effect on social memory, suggesting that LTP may more likely be involved in social recognition memory than PTP. The lack of individual-level correlation between LTP and social memory is not surprising as there was a large and varying time delay between the two measures (from several days to several months apart). Thus, this should not be taken as evidence that hippocampus is not involved in social memory. Early emotional response to novelty as a long-range predictor for adult synaptic plasticity. We found that neonatal novelty exposure but not neonatal anoxia had a significant effect on the correlation between open field activity at the juvenile stage (P25) and LTP at adulthood (4.5-8 months). Specifically, a large correlation was found only in Novel but not in Home rats. Ample evidence suggests that open field activity is related to circulating CORT, the end product of the HPA axis (see brief summary in Tang & Nakazawa, 2005). For example, open field activity has been shown to correlate with circulating CORT level (Piazza et al., 1991; Vallee et al., 1997). Therefore, the presence of a correlation between open field activity and LTP within Novel rats may reflect an increased sensitivity of LTP to circulating CORT in stressful situations, such as exposure to a novel environment (e.g. open field). Such an increase in sensitivity between Novel and Home rats has been demonstrated by a greater modulation of LTP by stress levels of CORT in Novel rats (Zou et al., 2001). Early experience and synaptic plasticity Akers et al. 17 The present finding of a correlation between open field activity and LTP is in agreement with that reported by Schulz et al. (2002; 2004) in that individual differences in open field activity can predict individual differences in measures of synaptic plasticity. While the direction of correlation in the present study was opposite to that reported in Schulz et al.’s studies, this most likely reflects several differences between studies that could change the direction of correlation, including the duration of activity measure, age of animals, and the difference in delays between measures. Importantly, in the present study, the delay between the measure of open field activity—often considered a measure of emotional response to novelty (Denenberg, 1969; Chapillon et al., 2002)—and the measure of synaptic plasticity spanned two developmental stages: juvenility and adulthood. Thus, the present correlation indicates that individual differences in emotional response to novelty present early in life may reflect stable underlying differences in neural mechanisms that influence adult synaptic plasticity. This longrange predictive power mirrors the well-established finding in humans that behavioral response to novelty at childhood can predict a range of individual differences in adult psychological and brain function (Schwartz et al., 2003). Early experience and synaptic plasticity Akers et al. 18 ACKNOWLEDGEMENTS We thank Elise McHugh for editorial assistance. This publication was made possible by Grant Number #5P20-RR015636-04 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Early experience and synaptic plasticity Akers et al. 19 ABBREVIATIONS CA, cornu Ammonis; CORT, corticosterone; EPSP, excitatory post-synaptic potential; HFS, high frequency stimulation; HPA, hypothalamic-pituitary-adrenal; LTH, long-term habituation; LTP, long-term potentiation; P, postnatal day; PTP, post-tetanic potentiation Early experience and synaptic plasticity Akers et al. 20 REFERENCES Agresti, A. & Finlay, B. (1997) Introduction to multivariate relationships. In Finlay, B. (ed.) Statistical methods for the social sciences. Prentice-Hall, Upper Saddle River, NJ, pp. 356-372. Aguilar, R., Escorihuela, R.M., Gil, L., Tobena, A. & Fernandez-Teruel, A. (2002) Differences between two psychogenetically selected lines of rats in a swimming pool matching-toplace task: long-term effects of infantile stimulation. Behav Genet, 32, 127-134. Behnisch, T. & Reymann, K.G. 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(2001) Neonatal exposure to a novel environment enhances the effects of corticosterone on neuronal excitability and plasticity in adult hippocampus. Brain Res Dev Brain Res, 130, 1-7. Zucker, R.S. & Regehr, W.G. (2002) Short-term synaptic plasticity. Annu Rev Physiol, 64, 355405. Early experience and synaptic plasticity Figure 1 Akers et al. 30 Early experience and synaptic plasticity Figure 2 Akers et al. 31 Early experience and synaptic plasticity Figure 3 Akers et al. 32 Early experience and synaptic plasticity Figure 4 Akers et al. 33 Early experience and synaptic plasticity Figure 5 Akers et al. 34 Early experience and synaptic plasticity Akers et al. Table 1. Baseline EPSP slope did not differ among treatment groups (N: number of rats; M: number of slices). Control Anoxia Novel N/M Slope (mV/ms) (5/15) -0.77±0.10 Home (4/9) -0.76±0.10 Novel (6/17) -0.77±0.08 Home (6/19) -0.79±0.10 35 Early experience and synaptic plasticity Akers et al. 36 FIGURE LEGENDS Figure 1. Experimental timeline. Neonatal anoxia: on postnatal day 1 (P1), half the rat litters were exposed to 100% N2 gas for 25 min (Anoxia), while the other half were exposed to room air (Control). Neonatal novelty exposure: from P2-21, half the pups within each litter were exposed to a novel cage for 3 min a day (Novel), while the other half remained in the home cage (Home). Open field activity: initial activity within a novel open field was assessed during the juvenile stage (P25). Social recognition memory: upon entering adulthood (P100-101), social recognition memory was assessed by habituation of social investigation across repeated sessions with a conspecific. Synaptic plasticity: in vitro hippocampal PTP and LTP were measured between 4.5-8 months of age, long after the initial neonatal events. Figure 2. High frequency stimulation (HFS)-induced potentiation of EPSPs in the CA1 of the hippocampus. Slope of EPSPs (% of baseline) before and after HFS (three 1-s trains at 100 Hz, 1-min inter-train interval). HFS trains indicated by arrows, and rates of potentiation across trains between Anoxic and Control rats are shown in inset. For all figures, data are displayed as mean ± sem; * p< 0.05. Figure 3. Neonatal anoxia and neonatal novelty exposure selectively modulated short- and longterm synaptic plasticity. (ac) Representative EPSP traces for PTP and LTP, with black and gray lines for before and after HFS, respectively. (b) Neonatal anoxia reduced PTP after the 3rd HFS train across Novel and Home groups. (d) Neonatal novelty exposure increased LTP only in the Control group. Early experience and synaptic plasticity Akers et al. 37 Figure 4. Effects of neonatal novelty exposure on long-term (24-hr) social recognition memory. (a-d) Raw frequencies of social investigation across 4 sessions (Day1: S1-3; Day2: D2S1) for each treatment group. (e) Neonatal novelty exposure increased long-term habituation (LTH) of social investigative behaviors in the Control but not in the Anoxic group. Figure 5. Emotional response to novelty during the juvenile stage (P25) as a predictor of adult synaptic plasticity (4.5-8 months of age). The correlation between open field activity (# of squares crossed) and adult LTP was significant in the Novel group (a) but not in the Home group (b). Anoxic rats: filled squares; Control rats: open triangles.