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Neonatal tactile stimulation enhances spatial working memory, prefrontal long-term potentiation, and D1 receptor activation in adult rats
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Neurobiology of Learning and Memory 89 (2008) 397–406 www.elsevier.com/locate/ynlme
Neonatal tactile stimulation enhances spatial working memory, prefrontal long-term potentiation, and D1 receptor activation in adult rats Ming Zhang, Jing-Xia Cai
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Division of Brain and Behavior, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang East Road, Kunming, Yunnan 650223, China Received 20 March 2007; revised 8 October 2007; accepted 31 October 2007 Available online 20 February 2008
Abstract Environmental stimuli during neonatal periods play an important role in the development of cognitive function. In this study, we examined the long-term effects of neonatal tactile stimulation (TS) on spatial working memory (SWM) and related mechanisms. We also investigated whether TS-induced effects could be counteracted by repeated short periods of maternal separation (MS). Wistar rat pups submitted to TS were handled and marked transiently per day during postnatal days 2–9 or 10–17. TS/MS pups were stimulated in the same way as TS pups and then individually separated from their mother for 1 h/day. Their nontactile stimulated (NTS) siblings served as controls. In adulthood, TS and TS/MS rats showed better performance in two versions of the delayed alternation task and superior in vivo long-term potentiation of the hippocampo–prefrontal cortical pathway when compared with controls. Furthermore, there were more doses of A77636 (a selective dopamine D1 agonist) to significantly improve SWM performance in TS and TS/MS rats than in NTS rats, suggesting that activation of prefrontal D1 receptors in TS and TS/MS rats is more optimal for SWM function than in NTS rats. MS did not counteract TS-induced effects because no significant difference was found between TS/MS and TS animals. These data indicate that in early life, external tactile stimulation leads to long-term facilitative effects in SWM-related neural function. 2007 Elsevier Inc. All rights reserved. Keywords: Tactile stimulation; Maternal separation; Working memory; Synaptic plasticity; Dopamine
1. Introduction During the neonatal period, the immature nervous system can be highly susceptible to even mild environmental interventions. For instance, it has been widely shown that repetitive brief early handling leads to improved development of the neuroendocrine system and enhanced capacities in dealing with stressful events in later stages of development (Levine, 1957; Levine, Alpert, & Levis, 1957; Meaney, Aiken, Bhatnager, Vanberkel, & Sapolsky, 1988). Previously, some studies reported that postnatal tactile stimulation (TS) as one kind of external sensory stimuli effectively accelerated the maturation of cortical pyramidal
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Corresponding author. Fax: +86 871 5191823. E-mail address: [email protected] (J.-X. Cai).
1074-7427/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2007.10.010
neurons (Schapiro & Vukovich, 1970), prevented maternal deprivation-induced alteration of neural stress markers (e.g., adrenocorticotropin hormone (ACTH), and corticotropin-releasing hormone (CRH) mRNA) (van Oers, de Kloet, Whelan, & Levine, 1998), pain sensitivity (Stephan, Helfritz, Pabst, & von Ho¨rsten, 2002), and improved passive avoidance response in adulthood (Zhang & Cai, 2006), suggesting that postnatal TS is also an effective way to influence functions of the developing brain. In the present study, we investigated whether postnatal TS experience could produce long-term beneficial effects on spatial working memory (SWM) in adulthood. SWM is an important cognitive function depending on the integrity of the prefrontal cortex (PFC) and hippocampus (Becker, Walker, & Olton, 1980; Brito & Brito, 1990; Brito, Thomas, Davis, & Gingold, 1982; Meck, Church, & Olton, 1984).
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Several lines of evidence have indicated that the prelimbic subarea of the medial PFC (mPFC) receives glutamatergic projection from the CA1/subicular region of the ventral hippocampus (vHIP) in rodents (Jay, Thierry, Wiklund, & Glowinski, 1992; Jay & Witter, 1991; Swanson, 1981). This vHIP–mPFC neural pathway has been shown to be involved in information encoding processes, behavioral inhibition, and SWM function (Floresco, Seamans, & Phillips, 1997; Izaki, Hori, & Nomura, 2000; Izaki, Maruki, Hori, & Nomura, 2001; Wang & Cai, 2006). Interestingly, both SWM performance (Murphy, Arnsten, Goldman-Rakic, & Roth, 1996; Murphy, Arnsten, Jentsch, & Roth, 1996; Seamans, Floresco, & Phillips, 1998; Zahrt, Taylor, Mathew, & Arnsten, 1997) and vHIP– mPFC long-term potentiation (LTP) (Gurden, Takita, & Jay, 2000; Gurden, Tassin, & Jay, 1999) can be modulated by the mPFC dopaminergic D1 receptor with a narrow range of optimal activation. The development of the mesocorticolimbic dopamine system is susceptible to early life environmental variations, such as early handling and maternal care (Brake, Zhang, Diorio, Meaney, & Gratton, 2004; Meaney, Brake, & Gratton, 2002; Zhang, Chretien, Meaney, & Gratton, 2005). Therefore, we also explored TS-induced effects on vHIP–mPFC LTP and prefrontal D1 receptor activation. Another type of postnatal manipulation, maternal separation (MS), may serve as an adverse stressful event for CNS development and yield behavioral disorders opposite to those caused by early handling and TS, such as increased anxiety behavior (Biagini, Pich, Carani, Marrama, & Agnati, 1998; Huot, Thrivikraman, Meaney, & Plotsky, 2001; Kalinichev, Easterling, & Holtzman, 2002; Kalinichev, Easterling, Plotsky, & Holtzman, 2002) and impaired spatial learning and memory (Huot, Plotsky, Lenox, & McNamara, 2002; Huot et al., 2001; Oitzl, Workel, Fluttert, Frosch, & De Kloet, 2000). Reports of several previous studies indicated that MS-induced stress response in rat pups was effectively reversed by stoking treatment (Suchecki, Rosenfeld, & Levine, 1993; van Oers et al., 1998). On the other hand, whether TS-induced effects could be counteracted by additional MS treatment remains unknown. Considering both psychological (MS per se) and physiological (e.g., hunger and thirst) stressful factors could be involved in a long-term (e.g., 3–24 h) MS procedure, we employed a repeated 1 h/d MS paradigm involving less physiological stress to test whether MS as a psychological factor could be stressful enough to cause adverse response against TS-induced neurobehavioral effects. 2. Materials and methods 2.1. Animals Juvenile Wistar rats were obtained from Kunming General Hospital and housed in a laboratory colony. During 3–4 months of age, female rats were mated with breeder males (3:1 or 2:1) and housed individually after pregnancy. Both sexes of offspring were housed with their mothers until
weaning, and only male offspring were used in this study. Food and water were available ad libitum. The colony room was maintained at 22 ± 1 C on a 12 h light/dark cycle (light on: 7:00–19:00). All experiments were conducted during the light phase. The animal experimental protocols were compatible with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health in 1996.
2.2. TS and MS procedure The pup birthday was defined as postnatal day 0 (PND 0). Each litter was culled to 9–12 pups with an effort to balance the ratio of sexes. A splitlitter design was introduced (Fig. 1), so there was generally only one male pup per litter for each group. In the same litter, male pups were randomly located to one of the following groups: the nontactile stimulated group (NTS), PND 2–9 TS, PND 10–17 TS, PND 2–9 TS/MS, and PND 10– 17 TS/MS. The two neonatal periods (PND 2–9, 10–17) were chosen for consideration to compare which period was more critical for external stimulus during the stress-hyporesponsive period (Sapolsky & Meaney, 1986). The TS and TS/MS manipulations were conducted between 09:00 and 13:00 once daily from PND 2 through PND 9, or from PND 10 through PND 17. The dam was removed for 3–4 min, during which TS and TS/ MS pups were handled and marked. The experimenter would grab some sawdust from the nest with a gloved hand to reduce the possibility of transient novelty exposure that has been shown to affect the development of cognitive functions (Tang, 2001). The TS pups were picked up individually and the pup’s back or rear area was stained regularly with a brush dipped in liquid dye (0.1% picric acid) 10 times for approximately 30 s. The dye remnant was absorbed with soft paper and then the pup was placed back in the home cage. Pups allotted to TS/MS were also handled and marked in the same way as the TS pups, but on the contralateral skin to distinguish them from siblings with different treatment in the same nest, and then individually placed into a circular cup (11 cm diameter) bedded with sawdust taken from its own nest. The cups were immediately put into an incubator (30 ± 0.5 C) for 1 h daily. Pups assigned to NTS were left undisturbed and served as controls. Cage cleaning was performed only once a week in an effort not to touch pups any more. At PND 22, dams were taken away and 3–5 male siblings of different groups were housed in one Plexiglas cage until testing in adulthood. As body weight is an indication of general nutritional development, all subjects were weighed at PND 30, 60, and 90.
2.3. Working memory protocol The apparatus was a wooden and brown T-maze with a start arm (90 cm · 13 cm · 25 cm) and two choice arms (65 cm · 13 cm · 25 cm). A start box (25 cm · 13 cm · 25 cm) was separated by a removable door
Fig. 1. Illustration of the split-litter design used in the present study. This figure shows an example of a litter including a dam and five male pups (female pups are not shown). T1, T2: pups received tactile stimulation (TS) during PND 2–9 or 10–17, respectively. M1, M2: pups received TS and then were separated from their mother (i.e., tactile stimulation and maternal separation, TS/MS) for 1 h per day during PND 2–9 or 10–17, respectively. N: pups received nontactile stimulation (NTS) and served as controls.
M. Zhang, J.-X. Cai / Neurobiology of Learning and Memory 89 (2008) 397–406 from the start arm. There was a food well (3 cm diameter and 1 cm deep) located at the end of each choice arm. The T-maze was 70 cm above the ground in a behavioral testing room that was adjacent to the colony room. Beginning at PND 85, each rat was handled for 5 min per day for 5 days. During PND 90–92, each rat was allowed to freely explore the T-maze for 15 min daily. The floor of T-maze was divided into grids every 13 cm. The spontaneous activities (i.e., locomotion, erection, and grooming) were recorded. No food except water was provided following the last session of the spontaneous activity. After 2 days of food restriction, each rat was trained to run a T-maze from the start box to the food wells in which a sunflower seed was put to minimize the need for dietary restriction. They habituated to the T-maze, including the movement of the door. Each rat was immediately fed 12–15 g of chow after training. All rats reached the criterion of eating 16 seeds from food wells within 5 min for two consecutive days after 4–7 days training. This habituation procedure aimed at minimizing affective factors and ensuring the veracity of delays used in the delayed alternation task as following. Two versions of the delayed alternation task were used in the present study. In both versions, 16 trials made up a session per day. A rat was reinforced for entering either arm on the first trial, which was excluded from the session. In the subsequent 15 trials, it was reinforced only if it entered the maze arm that was not chosen on the immediately preceding trial. At first, a ‘‘correction’’ version was used to promote training and evaluate working memory capacity as much as possible (Izaki et al., 2001; Zhang & Cai, 2005). That is, if a rat made an error choice, the following correction trials were excluded from the error choice. The trained rat was placed in the start box during the intertrial delay, which was initially ‘‘0 s’’ (i.e., approximately 1–2 s, as true 0 s is not possible). When a rat’s choice accuracy at a delay of 0 s reached P86.7% for consecutive 2 days, it was further trained for 30 days. The delay was gradually increased or decreased at 5 s intervals depending on whether the choice accuracy reached P86.7%. If it was true, the delay was increased on the next day. Otherwise, the delay was decreased and 0 s served as the ‘‘floor’’. During intertrial delay, the start box was covered with transparent Plexiglas and the choice arms were cleaned with water. Another batch of naı¨ve animals were used in the second version of the delayed alternation task. That is, the correction trials were included in the total choice. Rats were trained at the delay 0 s for 10 days, and then were further trained at a delay of 40 s for 14 days. The 40 s delay was selected because we considered that 40 s would be suitable to distinguish the SWM performance between different groups depending on the training data of the first version of the delayed alternation task.
2.4. Spatial discrimination task Naı¨ve animals were trained in a spatial discrimination task as a control task with similar locomotive and motivational demands as those in the delayed alternation task. Animals of each group were trained to run the left or right choice arm of the T-maze. The intertrial interval was 40 s, and 16 trials made up one session daily. The criterion was to reach a choice accuracy P93.3% for two consecutive days.
2.5. Drug administration After the delayed alternation task, 8 litters were used in the behavioral pharmacological experiment. Investigators were blind to animal treatment and drug doses. Herein, all rats were tested in the second version of the delayed alternation task in which 11 trials made up one session daily and the first trial was excluded. The rats were initially adapted to intraperitoneal (i.p.) injections of saline. The sterile saline (1 ml/kg) was injected 30 min prior to cognitive testing. Once the total choice accuracy of saline treatments scored 70% for at least three consecutive training days, 0.001, 0.01, 0.1, or 1 mg/kg doses of A77636 (( )-(1R,3S)-3-adamantyl-1-(aminomethyl)-3,4-dihydro-5,6-dihydro-1H-2-benzopyran hydrochloride; Sigma, St. Louis, MO), a selective dopamine D1 receptor full agonist, was administered in a counterbalanced order. After injection of each dose
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of A77636, the saline injections were continued to ensure the choice accuracy baseline at 70%. A minimum of seven washing-out days were inserted between drug administrations.
2.6. Electrophysiological recording Since there was no obvious effect of infantile age on behavioral testing, pups were only manipulated during PND 2–9 in the electrophysiological studies. When at 3 months, naı¨ve male rats were anesthetized with sodium pentobarbital (50–55 mg/kg, i.p.) and placed in a stereotaxic frame (Model I; Jiangwan, China). An extracellular recording electrode and a stimulating electrode consisting of a pair of Teflon-coated wires (90% platinum and 10% iridium, 75 lm outer diameter), were respectively implanted into the PL subarea (coordinates from bregma: AP = 3.3–3.4 mm, ML = 0.7–0.9 mm, DV = 3.5–4.2 mm) and the ipsilateral CA1/subicular region (AP = 5.6–6.3 mm, ML = 5.2–5.5 mm, DV = 4.0–6.0 mm), according to the atlas of Paxinos and Watson (1986). Placements of the electrodes were adjusted to obtain the maximum amplitude of the field postsynaptic potential (fPSP). The test fPSPs were evoked with constant current pulses (0.1 ms duration) delivered every 30 s. The stimulation intensity (0.20–0.40 mA) was used to evoke fPSP amplitude at 70% of the maximum. After a stable baseline period of a minimum of 40 min, high-frequency stimuli with parameters of two series (6 min apart) of 10 trains (250 Hz, 30 pulses) at 0.1 Hz were delivered at the test intensity to induce LTP (Gurden et al., 2000). Recordings after the second tetanus continued for 60 min. Changes of fPSP amplitudes following tetanus were calculated every 2 min as the percentage to baseline. After electrophysiological recordings, electrolytic currents (2 mA, 10 s) were delivered to produce a thermolytic lesion at the electrode tips. Animals were then given a lethal anesthetic dose and rapidly perfused with physiological saline solution followed by 10% formalin. Brains were removed, soaked in 10% formalin, and processed for histological confirmation of the recording sites.
2.7. Data analyses Because infantile age was not an effective factor indicated by two-way analysis of variance (ANOVA) [treatment (TS, TS/MS) · infantile age (PND 2–9, PND 10–17)] on any measures in this study, we pooled data from the two infantile ages for further analysis. Data derived from different litters were also pooled because there was not a significant litter effect. All data were analyzed using a one-way ANOVA. When appropriate, post hoc comparisons were followed by Duncan’s or the LSD test. All data were presented as means ± SEM.
3. Results 3.1. Body weight No significant effects of neonatal treatment (F(2, 101) = 0.21, P > .8) and the treatment · age interaction (F(4, 202) = 0.11, P > .9) were found on body weight at PND 30, 60, and 90 (data not shown), suggesting that neonatal TS and TS/MS treatments in this study did not alter their overall nutritional development. 3.2. Spontaneous activities, habituation, and the spatial discrimination task There were no significant effects of neonatal treatment on spontaneous activities in the T-maze (all P > .5), habituation training (F(2, 101) = 0.28, P > .7), and the perfor-
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mance in the spatial discrimination task (F(2, 32) = 0.06, P > .9) (data not shown). 3.3. The delayed alternation task In the first version of the delayed alternation task, there was no significant effect of neonatal treatment on the training days to reach the criterion at a delay of 0 s (F(2, 32) = 0.53, P = .59) (Fig. 2A). After 0 s training, animals were further trained for a total of 30 days, and every 5 days made up a single unit. A repeated measures one-way ANOVA indicated that treatment was not significant (F(2, 32) = 2.13, P = .14); the training time (F(5, 160) = 140.7, P < .0001) and the interaction between neonatal treatment and training time (F(10, 160) = 2.87, P < .01) exhibited significant effects, indicating that neonatal treatment-induced improvement occurred following the training process (Fig. 2B). Duncan’s test revealed that TS and TS/ MS animals achieved significantly longer delays with a choice accuracy P86.7% in unit 5 and 6 (TS: 41.6 ± 2.8 s, 49.0 ± 3.3 s; TS/MS: 39.4 ± 3.9, 47.1 ± 3.8 s, respectively) when compared with the NTS group (28.1 ± 3.1 s and 35.3 ± 4.9 s, respectively) (all P < .05). No difference was found between TS and TS/MS treatments during the 30 days training (all P > .6). In the second version of the delayed alternation task, data derived from the last 4 days whether animals were trained at 0 s or 40 s were pooled for comparison because of fluctuation of performance. No significant effect of neonatal treatment was found on 0 s training performance (F(2, 30) < 0.1) (Fig. 3A). One-way ANOVA showed a significant effect of neonatal treatment on the choice accuracy of 40 s training (F(2, 30) = 4.85, P = .015). Duncan’s test indicated that both TS (84.5 ± 1.5%) and TS/MS (84.4 ± 1.1%) animals achieved small but significantly better choice accuracy than that in the NTS group (78.3 ± 1.5%) (P < .05). No significant effects were found between TS and TS/MS animals (P > .9) (Fig. 3B). 3.4. Effects of A77636 on the SWM performance Three rats were excluded because of unexpected death or unstable baseline during testing. There were no significant effects of different doses of A77636 on the response time in all groups (data not shown), indicating that administration of these four doses of A77636 did not cause obvious effects on the locomotive abilities of the rats. A repeated measures ANOVA was performed with neonatal treatment as between-subjects and different doses of A77636 as within-subjects. No significant main effect of neonatal treatment (F(2, 34) = 0.8, P = .5) and the treatment · dose interaction (F(8, 136) = 0.8, P = .6) were shown. However, there was a significant main effect of dose (F(4, 136) = 5.66, P < .0001). The LSD post hoc test indicated that only administration of a 0.1 mg/kg dose of A77636 (81.4 ± 3.4%; P < .05) exhibited a significant facilitative effect in the NTS group when compared with saline
Fig. 2. Influence of neonatal treatment on SWM performance in the first version of the delayed alternation task. In this version of the delayed alternation task, correction trials were excluded from 16 trials per day. (A) Adult rats were trained at a delay of 0 s to reach a rigorous criterion with choice accuracy P86.7% for consecutive 2 days. Neonatal treatment did not cause significant effect on the training days to reach the criterion at a delay of 0 s. (B) After reaching the criterion at 0 s, each animal was further trained for a total of 30 days. Each unit represented 5 days. Duncan’s tests indicated that animals experiencing neonatal TS (n = 14) and TS/MS (n = 14) treatment achieved longer delays with choice accuracy P86.7% in unit 5 and 6 when compared with their NTS siblings (n = 7) (all P < .05). There was no significant difference between TS and TS/MS treatments (all P > .6). *P < .05, TS vs NTS; +P < .05, TS/MS vs NTS.
baseline. Administration of 0.01, 0.1, or 1 mg/kg doses of A77636 (78.1 ± 2.9%, 78.8 ± 2.2%, 79.4 ± 2.1%, respectively; P < .05 or .01) significantly improved the choice accuracy in TS-treated animals. In TS/MS-treated animals, both 0.1 and 1 mg/kg doses of A77636 (80.0 ± 2.1%; 80.7 ± 2.5%; both P < .01) were effective to improve the SWM performance compared with saline baseline (Fig. 4). These data suggested that neonatal TS and TS/ MS treatments led to a wider activation range than that in the NTS group.
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Fig. 4. Influence of neonatal treatment on SWM-related D1 receptor activations. This figure illustrated changes of the choice accuracy in the delayed alternation task following administration of different doses of the D1 receptor selective agonist A77636. The dashed line represents the baseline performance after saline injection. Repeated ANOVA measures indicated that there was no significant main effect of neonatal treatment (F(2, 34) = 0.8, P = .5) and the treatment · dose interaction (F(8, 136) = 0.8, P = .6). However, there was a significant main effect of dose (F(4, 136) = 5.66, P < .0001). LSD test indicated that only administration of the 0.1 mg/kg dose of A77636 exhibited significant facilitative effect in the NTS group (n = 7) when compared with saline baseline (P < .05). Administration of 0.01, 0.1, or 1 mg/kg doses of A77636 significantly improved the choice accuracy in TS-treated animals (n = 16; P < .05 or .01). In TS/MS-treated animals (n = 14), both 0.1 and 1 mg/kg doses of A77636 were effective to improve the SWM performance compared with saline baseline (P < .01). Therefore, there were more effective doses of A77636 to effectively improve the SWM performance in the TS and TS/MS animals than in their NTS siblings. *P < .05 in NTS; + P < .05, ++P < .01 in TS; ##P < .01 in TS/MS, compared with the performance following saline injection.
3.5. vHIP–mPFC LTP
Fig. 3. Influence of neonatal treatment on SWM performance in the second version of the delayed alternation task. In this version of the delayed alternation task, correction trials were included in the 16 trials per day. (A) This figure showed the averaged choice accuracy of the last 4 days when rats were trained at a delay of 0 s for a total of 10 days. No significant effect of neonatal treatment was found on 0 s training performance (F(2, 30) < 0.1). (B) This figure showed the averaged choice accuracy of the last 4 days when rats were trained at delay 40 s for a total of 14 days. One-way ANOVA revealed a significant effect of neonatal treatment on the choice accuracy of 40 s training (F(2, 30) = 4.85, P = .015). Duncan’s test indicated that TS (n = 13) and TS/MS (n = 13) animals achieved mild but significantly better choice accuracy than that in the NTS group (n = 7) (both P < .05). No significant effects were found between TS and TS/MS animals (P > .9). *P < .05, TS vs NTS; +P < .05, TS/MS vs NTS.
As shown in Fig. 5A, application of two series of high-frequency stimuli to the CA1/subicular region induced long-lasting enhancement of the mPFC field potentials compared with baseline in all three groups (repeated ANOVA, all P < .0001). During the 6 min interval after 1st tetanus, one-way ANOVA revealed no significant main effect of neonatal treatment (F(2, 22) = 1.20, P = .3). After the 2nd tetanus, there was a significant effect of neonatal treatment on the amplitude of fPSP during 0–30 min (F(2, 22) = 3.54, P < .05) and 30–60 min (F(2, 22) = 4.33, P < .05). Duncan’s tests indicated that both TS and TS/MS groups achieved significantly higher fPSP amplitudes during 0– 30 min and 30–60 min than those in NTS animals (all P < .05) (Fig. 5B). There was no significant difference between the TS and TS/MS groups (all P > .7). These data showed that early life TS and TS/MS experience led to an enhanced effect on the induction and maintenance of vHIP–mPFC LTP in adulthood.
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Fig. 5. Influence of neonatal treatment on vHIP–mPFC LTP. (A) Y coordination indicated the amplitude changes relative to the fPSP baseline amplitude before tetanus. Following application of two series of tetanus (arrows) to the CA1/subicular region, long-lasting enhancement of the fPSP in the mPFC region was recorded in all three groups (All P < .0001). The inserted figure shows the typical waves before (inner) and after (outer) tetanic stimulation. Calibration: 10 ms, 0.2 mV. (B) Illustrated fPSP amplitude every 30 min after the second tetanus. One-way ANOVA showed that neonatal treatment significantly altered the fPSP amplitude during 0–30 min (F(2, 22) = 3.54, P < .05) and 30–60 min (F(2, 22) = 4.33, P < .05) after the second tetanus. Duncan’s test indicated that both TS (n = 9) and TS/MS (n = 8) animals achieved significantly higher fPSP amplitude than NTS animals (n = 8) during 0– 30 min, and 30–60 min (all P < .05). There was no difference between TS and TS/MS groups (all P > .7). *P < .05, TS vs NTS; +P < .05, TS/MS vs NTS.
4. Discussion 4.1. Effects of neonatal treatment on SWM performance in adulthood In the present study, a split-litter design was used to eliminate other confounding factors such as heredity and maternal disturbance (Smotherman, Wiener, Mendoza, & Levine, 1977), and alteration of maternal care behaviors (Liu et al., 1997). We also paid attention to reduce transient novelty exposure that has been shown to affect the development of cognitive functions (Tang, 2001) by grab-
bing some nest sawdust with a gloved hand before pups were manipulated. In this way, specific long-term effects of neonatal TS on SWM function were explored. As a result, we found that neonatal tactile stimulation treatment led to delay-dependently improved performance in two versions of the delayed alternation task, but did not cause significant alterations in the habituation training and the spatial discrimination task. The habituation may be regarded as operational conditioned learning, that is, once the T-maze gate was lifted (conditioned signal), rats must run into the goal arms (operation, spatial locations) to get a food reward (unconditioned signal). The spatial dis-
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crimination task is to verify the spatial reference memory, which requires locomotion and motivation, just as the delayed alternation task does. Therefore, these data indicated that neonatal TS and TS/MS treatments task- and delay-dependently improved SWM function in adulthood when compared with their NTS siblings. It was reported previously that aged, but not adult, rats experiencing early handling showed better performance in the Morris maze task than nonhandling controls (Meaney et al., 1988). Whether neonatal tactile stimulation could influence spatial long-term memory during the aging process or in tasks involving more emotional factors needs further exploration. 4.2. Effects of neonatal treatment on vHIP–mPFC LTP The vHIP–mPFC glutamatergic neural pathway has been demonstrated to play an important role in SWM function. For example, blockade of unilateral vHIP and contralateral mPFC resulted in impaired SWM in the delayed spatial win-shift task and the delayed alternation task (Floresco et al., 1997; Seamans et al., 1998; Wang & Cai, 2006), suggesting that this neural pathway could be critical for online information encoding and refreshing. In the present study, neonatal tactile stimulation significantly enhanced the amplitude of the vHIP–mPFC LTP, an important form of synaptic plasticity widely thought to be a basic cellular mechanism of learning and memory (Bliss & Collingridge, 1993; Bliss & Lomo, 1973; Malenka & Nicoll, 1993, 1999), when compared with their NTS siblings in adulthood. External novel stimuli (e.g., early handling, novelty exposure) have been reported to enhance hippocampal LTP and spatial learning and memory (Akers et al., 2006; Tang, Akers, Reeb, Romeo, & McEwen, 2006; Tang & Zou, 2002; Wilson, Wilner, Kurz, & Nadel, 1986). Our data suggest that neonatal TS is an effective way to improve development of synaptic plasticity and the enhancement of vHIP–mPFC LTP could be one mechanism underlying the facilitative effects of TS on SWM function. 4.3. Effects of neonatal treatment on D1 receptor activation Behavioral and electrophysiological studies have shown that postsynaptic D1 receptors of the mPFC play an important role for SWM function in monkeys (Didriksen, 1995; Sawaguchi & Goldman-Rakic, 1991, 1994; Sawaguchi, Matsumura, & Kubota, 1988) and rodents (Seamans et al., 1998. Murphy et al., 1996; Murphy et al., 1996; Zahrt et al., 1997). Based on these studies, activation of prefrontal D1 receptors effectively modulates information maintenance during delay duration and inhibits irrelevant information in SWM performance (Durstewitz, Seamans, & Sejnowski, 2000). An ‘inverted U-shape’ relationship between D1 receptor activation and SWM has demonstrated that there is a narrow intermediate range for optimal D1 receptor stimulation while hypo- and hyper-
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stimulation leads to adverse SWM performance (for a review, see Arnsten, 1998). Therefore, it is reasonable that systemic administration of A77636 could act mainly at postsynaptic D1 receptors in the mPFC to affect SWM performance. As a result, we found that there was only one dose (0.1 mg/kg) of A77636 to effectively improve SWM performance in the NTS group, while there were three effective doses (0.01, 0.1, and 1 mg/kg) in the TS group and two effective doses (0.1 and 1 mg/kg) in the TS/MS group, indicating that the optimal activation range of D1 receptor was wider in TS and TS/MS animals than in NTS animals. Previously, it was shown in monkeys (Ljungberg, Apicella, & Schultz, 1991; Watanabe, Kodama, & Hikosaka, 1997) and rats (Rossetti & Sonia, 2005) that there is significant elevation of DA release in the PFC when the animals perform a SWM task. The finding that administration of a 1 mg/kg dose of A77636 significantly improved SWM performance in TS and TS/MS animals, but not in NTS animals, implies that the activation of prefrontal D1 receptors in TS and TS/MS animals is easier for maintaining an optimal status following elevation of DA release in the SWM task than in NTS animals, and that this then leads to improved SWM performance behaviorally. The lack of significant between-subject effects of neonatal treatment on SWM performance after administration of A77636 could be because of a ‘ceiling’ effect where all three groups achieved similar maximum response after A77636 administration (NTS: 81.4 ± 3.4%; TS: 79.4 ± 2.1%; TS/ MS: 80.7 ± 2.5%). This ‘ceiling’ effect could be related to indiscernible differences of mesocortical D1 receptor densities among groups as demonstrated by previous studies where early handling and maternal care did not affect D1 receptor densities in the nucleus accumbens and PFC (Brake et al., 2004; Meaney et al., 2002; Zhang et al., 2005). It should be mentioned that there was also an ‘inverted U-shape’ relationship between the mPFC D1 receptor activation and the induction of vHIP–mPFC LTP. For example, local injection of the D1 receptor antagonist SCH23390 into mPFC impaired vHIP–mPFC LTP. Conversely, directly infusion of 0.1 and 1 mM doses of the D1 receptor agonist SKF81297 into mPFC facilitated vHIP–mPFC LTP. But infusion of a higher dose of 5 mM did not result in facilitative effect (Gurden et al., 2000). Furthermore, prefrontal DA release increased following high-frequency stimuli at vHIP, and enhanced vHIP–mPFC LTP (Gurden et al., 1999). Therefore, the wider activation range of mPFC D1 receptors is also beneficial for the induction of vHIP–mPFC LTP. The mechanism underlying the beneficial effects of neonatal TS on prefrontal D1 receptor activation is not yet known. A possible explanation may be related to the enhanced efficiency of D1 receptor-coupled Gs signal transduction pathway (for a review, see Jay, 2003). In this case, adenylyl cyclase is more easily activated by low doses (e.g., 0.01 mg/kg) of A77636 to increase intracellular cAMP in TS animals than in NTS animals. After administration of
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high doses (e.g., 1 mg/kg) of A77636, activities of negative feedback signals such as protein phosphatase 1 and calcineurin may also be enhanced to maintain D1 receptor stimulation under an optimal state in TS animals. Only by continuing to increase A77636 doses and thus potentially overloading the negative modulation might D1 receptors exhibit hyperactivation. This explanation is supported to some extent by findings that the D1 receptor-related cAMP-PKA pathway contributed to SWM function (Aujla & Beninger, 2001; Runyan & Dash, 2005) and vHIP– mPFC LTP (Gurden et al., 2000). 4.4. Long-term effects of neonatal MS experience In this study, we used a relatively short period (1 h daily) of MS to reduce physiological stressful factors, especially hunger and thirst; therefore, this kind of short-period MS paradigm could be considered as a psychological stressful factor. Actually, pups have been shown to exhibit a more obvious stress response following a progressively increased separation time course (Levine, Huchton, Wiener, & Rosenfeld, 1992). Furthermore, we made an effort to reduce the influence of novelty exposure by placing pups in a cup bedded with sawdust taken from their own nest, allowing these pups to be in contact with familiar olfactory cues and hear ultrasonic vocalization from other separated pups in the same incubator. As a result, TS/MS animals exhibited significantly enhanced SWM performance and vHIP–mPFC LTP when compared with the NTS animals, and there was no significant difference when comparing TS/MS and TS animals, suggesting that short periods of neonatal MS as a psychological factor was not stressful enough to counteract the TS-induced beneficial effect on mPFC-related cognitive function and neural plasticity. The lack of an MS-induced effect could be related with to the stress-hyporesponsive period, in which adrenal cortical response of pups is resistant to stressors in rodents, thus preventing hypersecretion of corticoids (Sapolsky & Meaney, 1986). Differently from those studies that found an adverse effect of MS (mentioned in the Introduction), several studies did not find a significant effect of long-time (3–6 h) MS on fear behavior and cognitive tasks (Lehmann et al., 2002; Shalev & Kafkafi, 2002; Stanton, Crofton, & Lau, 1992), and even MS led to reduced stressful response and improved learning and memory (Lehmann, Stohr, & Feldon, 2000; Ogawa et al., 1994; Pryce et al., 2001). These discrepancies might be related to inconsistency of MS manipulations, including the time course during which pups are separated from their mother, repeated times, temperature, familiar or novel environment, and so on. In our study, we actually investigated TS/MS-induced effects on SWM function. Whether this kind of MS paradigm alone could influence neural development and cause behavioral alterations needs further exploration. In conclusion, our findings that neonatal tactile stimulation effectively improved the SWM performance, the syn-
aptic plasticity of vHIP–mPFC, and mesocortical dopaminergic D1 receptor activation suggest that neonatal stimulation during infancy would be a meaningful way of improving later cognitive development and possibly provide a protective mechanism against the development of neuropsychological diseases such as attention-deficit hyperactivity disorder (ADHD), which involves working memory deficit (Karatekin & Asarnow, 1998). Acknowledgments We are grateful to Mrs. Xi-Lan Chen, Hua-Xian Zhang, and Ruo-Yun Yang for their excellent technical assistance. This work was supported by the National Natural Science Foundation (30070251) and the National Basic Research Program (G1999054000) of China to Prof. Jing-Xia Cai. References Akers, K. G., Nakazawa, M., Romeo, R. D., Connor, J. A., McEwen, B. S., & Tang, A. C. (2006). Early life modulators and predictors of adult synaptic plasticity. The European Journal of Neuroscience, 24, 547–554. Arnsten, A. F. T. (1998). Catecholamine modulation of prefrontal cortical cognitive function. Trends in Cognitive Sciences, 11, 436–447. Aujla, H., & Beninger, R. J. (2001). Hippocampal–prefrontocortical circuits: PKA inhibition in the prefrontal cortex impairs delayed nonmatching in the radial maze in rats. Behavioral Neuroscience, 115, 1204–1211. Becker, J. T., Walker, J. A., & Olton, D. S. (1980). Neuroanatomical bases of spatial memory. Brain Research, 200, 307–320. Biagini, G., Pich, E. M., Carani, C., Marrama, P., & Agnati, L. F. (1998). Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field. International Journal of Developmental Neuroscience, 16, 187–197. Bliss, T. V., & Collingridge, G. L. (1993). A synaptic model of memory: Long-term potentiation in the hippocampus. Nature, 361, 31–39. Bliss, T. V., & Lomo, T. (1973). Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. The Journal of Physiology, 232, 331–356. Brake, W. G., Zhang, T. Y., Diorio, J., Meaney, M. J., & Gratton, A. (2004). Influence of early postnatal rearing conditions on mesocorticolimbic dopamine and behavioural responses to psychostimulants and stressors in adult rats. The European Journal of Neuroscience, 19, 1863–1874. Brito, G. N., & Brito, L. S. (1990). Septohippocampal system and the prelimbic sector of frontal cortex: A neuropsychological battery analysis in the rat. Behavioral Brain Research, 36, 127–146. Brito, G. N., Thomas, G. J., Davis, B. J., & Gingold, S. I. (1982). Prelimbic cortex, mediodorsal thalamus, septum, and delayed alternation in rats. Experimental Brain Research., 46, 52–58. Didriksen, M. (1995). Effects of antipsychotics on cognitive behaviour in rats using the delayed non-match to position paradigm. European Journal of Pharmacology, 281, 241–250. Durstewitz, D., Seamans, J. K., & Sejnowski, T. J. (2000). Dopaminemediated stabilization of delay-period activity in a network model of prefrontal cortex. Journal of Neurophysiology, 83, 1733–1750. Floresco, S. B., Seamans, J. K., & Phillips, A. G. (1997). Selective roles for hippocampal, prefrontal, cortical, and ventral striatal circuits in radialarm maze tasks with or without a delay. The Journal of Neuroscience, 17, 1880–1890. Gurden, H., Takita, M., & Jay, T. M. (2000). Essential role of D1 but not D2 receptors in the NMDA receptor-dependent long-term potentia-
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Neurobiology of Learning and Memory 89 (2008) 397–406 www.elsevier.com/locate/ynlme
Neonatal tactile stimulation enhances spatial working memory, prefrontal long-term potentiation, and D1 receptor activation in adult rats Ming Zhang, Jing-Xia Cai
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Division of Brain and Behavior, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang East Road, Kunming, Yunnan 650223, China Received 20 March 2007; revised 8 October 2007; accepted 31 October 2007 Available online 20 February 2008
Abstract Environmental stimuli during neonatal periods play an important role in the development of cognitive function. In this study, we examined the long-term effects of neonatal tactile stimulation (TS) on spatial working memory (SWM) and related mechanisms. We also investigated whether TS-induced effects could be counteracted by repeated short periods of maternal separation (MS). Wistar rat pups submitted to TS were handled and marked transiently per day during postnatal days 2–9 or 10–17. TS/MS pups were stimulated in the same way as TS pups and then individually separated from their mother for 1 h/day. Their nontactile stimulated (NTS) siblings served as controls. In adulthood, TS and TS/MS rats showed better performance in two versions of the delayed alternation task and superior in vivo long-term potentiation of the hippocampo–prefrontal cortical pathway when compared with controls. Furthermore, there were more doses of A77636 (a selective dopamine D1 agonist) to significantly improve SWM performance in TS and TS/MS rats than in NTS rats, suggesting that activation of prefrontal D1 receptors in TS and TS/MS rats is more optimal for SWM function than in NTS rats. MS did not counteract TS-induced effects because no significant difference was found between TS/MS and TS animals. These data indicate that in early life, external tactile stimulation leads to long-term facilitative effects in SWM-related neural function. 2007 Elsevier Inc. All rights reserved. Keywords: Tactile stimulation; Maternal separation; Working memory; Synaptic plasticity; Dopamine
1. Introduction During the neonatal period, the immature nervous system can be highly susceptible to even mild environmental interventions. For instance, it has been widely shown that repetitive brief early handling leads to improved development of the neuroendocrine system and enhanced capacities in dealing with stressful events in later stages of development (Levine, 1957; Levine, Alpert, & Levis, 1957; Meaney, Aiken, Bhatnager, Vanberkel, & Sapolsky, 1988). Previously, some studies reported that postnatal tactile stimulation (TS) as one kind of external sensory stimuli effectively accelerated the maturation of cortical pyramidal
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Corresponding author. Fax: +86 871 5191823. E-mail address: [email protected] (J.-X. Cai).
1074-7427/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2007.10.010
neurons (Schapiro & Vukovich, 1970), prevented maternal deprivation-induced alteration of neural stress markers (e.g., adrenocorticotropin hormone (ACTH), and corticotropin-releasing hormone (CRH) mRNA) (van Oers, de Kloet, Whelan, & Levine, 1998), pain sensitivity (Stephan, Helfritz, Pabst, & von Ho¨rsten, 2002), and improved passive avoidance response in adulthood (Zhang & Cai, 2006), suggesting that postnatal TS is also an effective way to influence functions of the developing brain. In the present study, we investigated whether postnatal TS experience could produce long-term beneficial effects on spatial working memory (SWM) in adulthood. SWM is an important cognitive function depending on the integrity of the prefrontal cortex (PFC) and hippocampus (Becker, Walker, & Olton, 1980; Brito & Brito, 1990; Brito, Thomas, Davis, & Gingold, 1982; Meck, Church, & Olton, 1984).
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Several lines of evidence have indicated that the prelimbic subarea of the medial PFC (mPFC) receives glutamatergic projection from the CA1/subicular region of the ventral hippocampus (vHIP) in rodents (Jay, Thierry, Wiklund, & Glowinski, 1992; Jay & Witter, 1991; Swanson, 1981). This vHIP–mPFC neural pathway has been shown to be involved in information encoding processes, behavioral inhibition, and SWM function (Floresco, Seamans, & Phillips, 1997; Izaki, Hori, & Nomura, 2000; Izaki, Maruki, Hori, & Nomura, 2001; Wang & Cai, 2006). Interestingly, both SWM performance (Murphy, Arnsten, Goldman-Rakic, & Roth, 1996; Murphy, Arnsten, Jentsch, & Roth, 1996; Seamans, Floresco, & Phillips, 1998; Zahrt, Taylor, Mathew, & Arnsten, 1997) and vHIP– mPFC long-term potentiation (LTP) (Gurden, Takita, & Jay, 2000; Gurden, Tassin, & Jay, 1999) can be modulated by the mPFC dopaminergic D1 receptor with a narrow range of optimal activation. The development of the mesocorticolimbic dopamine system is susceptible to early life environmental variations, such as early handling and maternal care (Brake, Zhang, Diorio, Meaney, & Gratton, 2004; Meaney, Brake, & Gratton, 2002; Zhang, Chretien, Meaney, & Gratton, 2005). Therefore, we also explored TS-induced effects on vHIP–mPFC LTP and prefrontal D1 receptor activation. Another type of postnatal manipulation, maternal separation (MS), may serve as an adverse stressful event for CNS development and yield behavioral disorders opposite to those caused by early handling and TS, such as increased anxiety behavior (Biagini, Pich, Carani, Marrama, & Agnati, 1998; Huot, Thrivikraman, Meaney, & Plotsky, 2001; Kalinichev, Easterling, & Holtzman, 2002; Kalinichev, Easterling, Plotsky, & Holtzman, 2002) and impaired spatial learning and memory (Huot, Plotsky, Lenox, & McNamara, 2002; Huot et al., 2001; Oitzl, Workel, Fluttert, Frosch, & De Kloet, 2000). Reports of several previous studies indicated that MS-induced stress response in rat pups was effectively reversed by stoking treatment (Suchecki, Rosenfeld, & Levine, 1993; van Oers et al., 1998). On the other hand, whether TS-induced effects could be counteracted by additional MS treatment remains unknown. Considering both psychological (MS per se) and physiological (e.g., hunger and thirst) stressful factors could be involved in a long-term (e.g., 3–24 h) MS procedure, we employed a repeated 1 h/d MS paradigm involving less physiological stress to test whether MS as a psychological factor could be stressful enough to cause adverse response against TS-induced neurobehavioral effects. 2. Materials and methods 2.1. Animals Juvenile Wistar rats were obtained from Kunming General Hospital and housed in a laboratory colony. During 3–4 months of age, female rats were mated with breeder males (3:1 or 2:1) and housed individually after pregnancy. Both sexes of offspring were housed with their mothers until
weaning, and only male offspring were used in this study. Food and water were available ad libitum. The colony room was maintained at 22 ± 1 C on a 12 h light/dark cycle (light on: 7:00–19:00). All experiments were conducted during the light phase. The animal experimental protocols were compatible with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health in 1996.
2.2. TS and MS procedure The pup birthday was defined as postnatal day 0 (PND 0). Each litter was culled to 9–12 pups with an effort to balance the ratio of sexes. A splitlitter design was introduced (Fig. 1), so there was generally only one male pup per litter for each group. In the same litter, male pups were randomly located to one of the following groups: the nontactile stimulated group (NTS), PND 2–9 TS, PND 10–17 TS, PND 2–9 TS/MS, and PND 10– 17 TS/MS. The two neonatal periods (PND 2–9, 10–17) were chosen for consideration to compare which period was more critical for external stimulus during the stress-hyporesponsive period (Sapolsky & Meaney, 1986). The TS and TS/MS manipulations were conducted between 09:00 and 13:00 once daily from PND 2 through PND 9, or from PND 10 through PND 17. The dam was removed for 3–4 min, during which TS and TS/ MS pups were handled and marked. The experimenter would grab some sawdust from the nest with a gloved hand to reduce the possibility of transient novelty exposure that has been shown to affect the development of cognitive functions (Tang, 2001). The TS pups were picked up individually and the pup’s back or rear area was stained regularly with a brush dipped in liquid dye (0.1% picric acid) 10 times for approximately 30 s. The dye remnant was absorbed with soft paper and then the pup was placed back in the home cage. Pups allotted to TS/MS were also handled and marked in the same way as the TS pups, but on the contralateral skin to distinguish them from siblings with different treatment in the same nest, and then individually placed into a circular cup (11 cm diameter) bedded with sawdust taken from its own nest. The cups were immediately put into an incubator (30 ± 0.5 C) for 1 h daily. Pups assigned to NTS were left undisturbed and served as controls. Cage cleaning was performed only once a week in an effort not to touch pups any more. At PND 22, dams were taken away and 3–5 male siblings of different groups were housed in one Plexiglas cage until testing in adulthood. As body weight is an indication of general nutritional development, all subjects were weighed at PND 30, 60, and 90.
2.3. Working memory protocol The apparatus was a wooden and brown T-maze with a start arm (90 cm · 13 cm · 25 cm) and two choice arms (65 cm · 13 cm · 25 cm). A start box (25 cm · 13 cm · 25 cm) was separated by a removable door
Fig. 1. Illustration of the split-litter design used in the present study. This figure shows an example of a litter including a dam and five male pups (female pups are not shown). T1, T2: pups received tactile stimulation (TS) during PND 2–9 or 10–17, respectively. M1, M2: pups received TS and then were separated from their mother (i.e., tactile stimulation and maternal separation, TS/MS) for 1 h per day during PND 2–9 or 10–17, respectively. N: pups received nontactile stimulation (NTS) and served as controls.
M. Zhang, J.-X. Cai / Neurobiology of Learning and Memory 89 (2008) 397–406 from the start arm. There was a food well (3 cm diameter and 1 cm deep) located at the end of each choice arm. The T-maze was 70 cm above the ground in a behavioral testing room that was adjacent to the colony room. Beginning at PND 85, each rat was handled for 5 min per day for 5 days. During PND 90–92, each rat was allowed to freely explore the T-maze for 15 min daily. The floor of T-maze was divided into grids every 13 cm. The spontaneous activities (i.e., locomotion, erection, and grooming) were recorded. No food except water was provided following the last session of the spontaneous activity. After 2 days of food restriction, each rat was trained to run a T-maze from the start box to the food wells in which a sunflower seed was put to minimize the need for dietary restriction. They habituated to the T-maze, including the movement of the door. Each rat was immediately fed 12–15 g of chow after training. All rats reached the criterion of eating 16 seeds from food wells within 5 min for two consecutive days after 4–7 days training. This habituation procedure aimed at minimizing affective factors and ensuring the veracity of delays used in the delayed alternation task as following. Two versions of the delayed alternation task were used in the present study. In both versions, 16 trials made up a session per day. A rat was reinforced for entering either arm on the first trial, which was excluded from the session. In the subsequent 15 trials, it was reinforced only if it entered the maze arm that was not chosen on the immediately preceding trial. At first, a ‘‘correction’’ version was used to promote training and evaluate working memory capacity as much as possible (Izaki et al., 2001; Zhang & Cai, 2005). That is, if a rat made an error choice, the following correction trials were excluded from the error choice. The trained rat was placed in the start box during the intertrial delay, which was initially ‘‘0 s’’ (i.e., approximately 1–2 s, as true 0 s is not possible). When a rat’s choice accuracy at a delay of 0 s reached P86.7% for consecutive 2 days, it was further trained for 30 days. The delay was gradually increased or decreased at 5 s intervals depending on whether the choice accuracy reached P86.7%. If it was true, the delay was increased on the next day. Otherwise, the delay was decreased and 0 s served as the ‘‘floor’’. During intertrial delay, the start box was covered with transparent Plexiglas and the choice arms were cleaned with water. Another batch of naı¨ve animals were used in the second version of the delayed alternation task. That is, the correction trials were included in the total choice. Rats were trained at the delay 0 s for 10 days, and then were further trained at a delay of 40 s for 14 days. The 40 s delay was selected because we considered that 40 s would be suitable to distinguish the SWM performance between different groups depending on the training data of the first version of the delayed alternation task.
2.4. Spatial discrimination task Naı¨ve animals were trained in a spatial discrimination task as a control task with similar locomotive and motivational demands as those in the delayed alternation task. Animals of each group were trained to run the left or right choice arm of the T-maze. The intertrial interval was 40 s, and 16 trials made up one session daily. The criterion was to reach a choice accuracy P93.3% for two consecutive days.
2.5. Drug administration After the delayed alternation task, 8 litters were used in the behavioral pharmacological experiment. Investigators were blind to animal treatment and drug doses. Herein, all rats were tested in the second version of the delayed alternation task in which 11 trials made up one session daily and the first trial was excluded. The rats were initially adapted to intraperitoneal (i.p.) injections of saline. The sterile saline (1 ml/kg) was injected 30 min prior to cognitive testing. Once the total choice accuracy of saline treatments scored 70% for at least three consecutive training days, 0.001, 0.01, 0.1, or 1 mg/kg doses of A77636 (( )-(1R,3S)-3-adamantyl-1-(aminomethyl)-3,4-dihydro-5,6-dihydro-1H-2-benzopyran hydrochloride; Sigma, St. Louis, MO), a selective dopamine D1 receptor full agonist, was administered in a counterbalanced order. After injection of each dose
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of A77636, the saline injections were continued to ensure the choice accuracy baseline at 70%. A minimum of seven washing-out days were inserted between drug administrations.
2.6. Electrophysiological recording Since there was no obvious effect of infantile age on behavioral testing, pups were only manipulated during PND 2–9 in the electrophysiological studies. When at 3 months, naı¨ve male rats were anesthetized with sodium pentobarbital (50–55 mg/kg, i.p.) and placed in a stereotaxic frame (Model I; Jiangwan, China). An extracellular recording electrode and a stimulating electrode consisting of a pair of Teflon-coated wires (90% platinum and 10% iridium, 75 lm outer diameter), were respectively implanted into the PL subarea (coordinates from bregma: AP = 3.3–3.4 mm, ML = 0.7–0.9 mm, DV = 3.5–4.2 mm) and the ipsilateral CA1/subicular region (AP = 5.6–6.3 mm, ML = 5.2–5.5 mm, DV = 4.0–6.0 mm), according to the atlas of Paxinos and Watson (1986). Placements of the electrodes were adjusted to obtain the maximum amplitude of the field postsynaptic potential (fPSP). The test fPSPs were evoked with constant current pulses (0.1 ms duration) delivered every 30 s. The stimulation intensity (0.20–0.40 mA) was used to evoke fPSP amplitude at 70% of the maximum. After a stable baseline period of a minimum of 40 min, high-frequency stimuli with parameters of two series (6 min apart) of 10 trains (250 Hz, 30 pulses) at 0.1 Hz were delivered at the test intensity to induce LTP (Gurden et al., 2000). Recordings after the second tetanus continued for 60 min. Changes of fPSP amplitudes following tetanus were calculated every 2 min as the percentage to baseline. After electrophysiological recordings, electrolytic currents (2 mA, 10 s) were delivered to produce a thermolytic lesion at the electrode tips. Animals were then given a lethal anesthetic dose and rapidly perfused with physiological saline solution followed by 10% formalin. Brains were removed, soaked in 10% formalin, and processed for histological confirmation of the recording sites.
2.7. Data analyses Because infantile age was not an effective factor indicated by two-way analysis of variance (ANOVA) [treatment (TS, TS/MS) · infantile age (PND 2–9, PND 10–17)] on any measures in this study, we pooled data from the two infantile ages for further analysis. Data derived from different litters were also pooled because there was not a significant litter effect. All data were analyzed using a one-way ANOVA. When appropriate, post hoc comparisons were followed by Duncan’s or the LSD test. All data were presented as means ± SEM.
3. Results 3.1. Body weight No significant effects of neonatal treatment (F(2, 101) = 0.21, P > .8) and the treatment · age interaction (F(4, 202) = 0.11, P > .9) were found on body weight at PND 30, 60, and 90 (data not shown), suggesting that neonatal TS and TS/MS treatments in this study did not alter their overall nutritional development. 3.2. Spontaneous activities, habituation, and the spatial discrimination task There were no significant effects of neonatal treatment on spontaneous activities in the T-maze (all P > .5), habituation training (F(2, 101) = 0.28, P > .7), and the perfor-
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mance in the spatial discrimination task (F(2, 32) = 0.06, P > .9) (data not shown). 3.3. The delayed alternation task In the first version of the delayed alternation task, there was no significant effect of neonatal treatment on the training days to reach the criterion at a delay of 0 s (F(2, 32) = 0.53, P = .59) (Fig. 2A). After 0 s training, animals were further trained for a total of 30 days, and every 5 days made up a single unit. A repeated measures one-way ANOVA indicated that treatment was not significant (F(2, 32) = 2.13, P = .14); the training time (F(5, 160) = 140.7, P < .0001) and the interaction between neonatal treatment and training time (F(10, 160) = 2.87, P < .01) exhibited significant effects, indicating that neonatal treatment-induced improvement occurred following the training process (Fig. 2B). Duncan’s test revealed that TS and TS/ MS animals achieved significantly longer delays with a choice accuracy P86.7% in unit 5 and 6 (TS: 41.6 ± 2.8 s, 49.0 ± 3.3 s; TS/MS: 39.4 ± 3.9, 47.1 ± 3.8 s, respectively) when compared with the NTS group (28.1 ± 3.1 s and 35.3 ± 4.9 s, respectively) (all P < .05). No difference was found between TS and TS/MS treatments during the 30 days training (all P > .6). In the second version of the delayed alternation task, data derived from the last 4 days whether animals were trained at 0 s or 40 s were pooled for comparison because of fluctuation of performance. No significant effect of neonatal treatment was found on 0 s training performance (F(2, 30) < 0.1) (Fig. 3A). One-way ANOVA showed a significant effect of neonatal treatment on the choice accuracy of 40 s training (F(2, 30) = 4.85, P = .015). Duncan’s test indicated that both TS (84.5 ± 1.5%) and TS/MS (84.4 ± 1.1%) animals achieved small but significantly better choice accuracy than that in the NTS group (78.3 ± 1.5%) (P < .05). No significant effects were found between TS and TS/MS animals (P > .9) (Fig. 3B). 3.4. Effects of A77636 on the SWM performance Three rats were excluded because of unexpected death or unstable baseline during testing. There were no significant effects of different doses of A77636 on the response time in all groups (data not shown), indicating that administration of these four doses of A77636 did not cause obvious effects on the locomotive abilities of the rats. A repeated measures ANOVA was performed with neonatal treatment as between-subjects and different doses of A77636 as within-subjects. No significant main effect of neonatal treatment (F(2, 34) = 0.8, P = .5) and the treatment · dose interaction (F(8, 136) = 0.8, P = .6) were shown. However, there was a significant main effect of dose (F(4, 136) = 5.66, P < .0001). The LSD post hoc test indicated that only administration of a 0.1 mg/kg dose of A77636 (81.4 ± 3.4%; P < .05) exhibited a significant facilitative effect in the NTS group when compared with saline
Fig. 2. Influence of neonatal treatment on SWM performance in the first version of the delayed alternation task. In this version of the delayed alternation task, correction trials were excluded from 16 trials per day. (A) Adult rats were trained at a delay of 0 s to reach a rigorous criterion with choice accuracy P86.7% for consecutive 2 days. Neonatal treatment did not cause significant effect on the training days to reach the criterion at a delay of 0 s. (B) After reaching the criterion at 0 s, each animal was further trained for a total of 30 days. Each unit represented 5 days. Duncan’s tests indicated that animals experiencing neonatal TS (n = 14) and TS/MS (n = 14) treatment achieved longer delays with choice accuracy P86.7% in unit 5 and 6 when compared with their NTS siblings (n = 7) (all P < .05). There was no significant difference between TS and TS/MS treatments (all P > .6). *P < .05, TS vs NTS; +P < .05, TS/MS vs NTS.
baseline. Administration of 0.01, 0.1, or 1 mg/kg doses of A77636 (78.1 ± 2.9%, 78.8 ± 2.2%, 79.4 ± 2.1%, respectively; P < .05 or .01) significantly improved the choice accuracy in TS-treated animals. In TS/MS-treated animals, both 0.1 and 1 mg/kg doses of A77636 (80.0 ± 2.1%; 80.7 ± 2.5%; both P < .01) were effective to improve the SWM performance compared with saline baseline (Fig. 4). These data suggested that neonatal TS and TS/ MS treatments led to a wider activation range than that in the NTS group.
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Fig. 4. Influence of neonatal treatment on SWM-related D1 receptor activations. This figure illustrated changes of the choice accuracy in the delayed alternation task following administration of different doses of the D1 receptor selective agonist A77636. The dashed line represents the baseline performance after saline injection. Repeated ANOVA measures indicated that there was no significant main effect of neonatal treatment (F(2, 34) = 0.8, P = .5) and the treatment · dose interaction (F(8, 136) = 0.8, P = .6). However, there was a significant main effect of dose (F(4, 136) = 5.66, P < .0001). LSD test indicated that only administration of the 0.1 mg/kg dose of A77636 exhibited significant facilitative effect in the NTS group (n = 7) when compared with saline baseline (P < .05). Administration of 0.01, 0.1, or 1 mg/kg doses of A77636 significantly improved the choice accuracy in TS-treated animals (n = 16; P < .05 or .01). In TS/MS-treated animals (n = 14), both 0.1 and 1 mg/kg doses of A77636 were effective to improve the SWM performance compared with saline baseline (P < .01). Therefore, there were more effective doses of A77636 to effectively improve the SWM performance in the TS and TS/MS animals than in their NTS siblings. *P < .05 in NTS; + P < .05, ++P < .01 in TS; ##P < .01 in TS/MS, compared with the performance following saline injection.
3.5. vHIP–mPFC LTP
Fig. 3. Influence of neonatal treatment on SWM performance in the second version of the delayed alternation task. In this version of the delayed alternation task, correction trials were included in the 16 trials per day. (A) This figure showed the averaged choice accuracy of the last 4 days when rats were trained at a delay of 0 s for a total of 10 days. No significant effect of neonatal treatment was found on 0 s training performance (F(2, 30) < 0.1). (B) This figure showed the averaged choice accuracy of the last 4 days when rats were trained at delay 40 s for a total of 14 days. One-way ANOVA revealed a significant effect of neonatal treatment on the choice accuracy of 40 s training (F(2, 30) = 4.85, P = .015). Duncan’s test indicated that TS (n = 13) and TS/MS (n = 13) animals achieved mild but significantly better choice accuracy than that in the NTS group (n = 7) (both P < .05). No significant effects were found between TS and TS/MS animals (P > .9). *P < .05, TS vs NTS; +P < .05, TS/MS vs NTS.
As shown in Fig. 5A, application of two series of high-frequency stimuli to the CA1/subicular region induced long-lasting enhancement of the mPFC field potentials compared with baseline in all three groups (repeated ANOVA, all P < .0001). During the 6 min interval after 1st tetanus, one-way ANOVA revealed no significant main effect of neonatal treatment (F(2, 22) = 1.20, P = .3). After the 2nd tetanus, there was a significant effect of neonatal treatment on the amplitude of fPSP during 0–30 min (F(2, 22) = 3.54, P < .05) and 30–60 min (F(2, 22) = 4.33, P < .05). Duncan’s tests indicated that both TS and TS/MS groups achieved significantly higher fPSP amplitudes during 0– 30 min and 30–60 min than those in NTS animals (all P < .05) (Fig. 5B). There was no significant difference between the TS and TS/MS groups (all P > .7). These data showed that early life TS and TS/MS experience led to an enhanced effect on the induction and maintenance of vHIP–mPFC LTP in adulthood.
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Fig. 5. Influence of neonatal treatment on vHIP–mPFC LTP. (A) Y coordination indicated the amplitude changes relative to the fPSP baseline amplitude before tetanus. Following application of two series of tetanus (arrows) to the CA1/subicular region, long-lasting enhancement of the fPSP in the mPFC region was recorded in all three groups (All P < .0001). The inserted figure shows the typical waves before (inner) and after (outer) tetanic stimulation. Calibration: 10 ms, 0.2 mV. (B) Illustrated fPSP amplitude every 30 min after the second tetanus. One-way ANOVA showed that neonatal treatment significantly altered the fPSP amplitude during 0–30 min (F(2, 22) = 3.54, P < .05) and 30–60 min (F(2, 22) = 4.33, P < .05) after the second tetanus. Duncan’s test indicated that both TS (n = 9) and TS/MS (n = 8) animals achieved significantly higher fPSP amplitude than NTS animals (n = 8) during 0– 30 min, and 30–60 min (all P < .05). There was no difference between TS and TS/MS groups (all P > .7). *P < .05, TS vs NTS; +P < .05, TS/MS vs NTS.
4. Discussion 4.1. Effects of neonatal treatment on SWM performance in adulthood In the present study, a split-litter design was used to eliminate other confounding factors such as heredity and maternal disturbance (Smotherman, Wiener, Mendoza, & Levine, 1977), and alteration of maternal care behaviors (Liu et al., 1997). We also paid attention to reduce transient novelty exposure that has been shown to affect the development of cognitive functions (Tang, 2001) by grab-
bing some nest sawdust with a gloved hand before pups were manipulated. In this way, specific long-term effects of neonatal TS on SWM function were explored. As a result, we found that neonatal tactile stimulation treatment led to delay-dependently improved performance in two versions of the delayed alternation task, but did not cause significant alterations in the habituation training and the spatial discrimination task. The habituation may be regarded as operational conditioned learning, that is, once the T-maze gate was lifted (conditioned signal), rats must run into the goal arms (operation, spatial locations) to get a food reward (unconditioned signal). The spatial dis-
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crimination task is to verify the spatial reference memory, which requires locomotion and motivation, just as the delayed alternation task does. Therefore, these data indicated that neonatal TS and TS/MS treatments task- and delay-dependently improved SWM function in adulthood when compared with their NTS siblings. It was reported previously that aged, but not adult, rats experiencing early handling showed better performance in the Morris maze task than nonhandling controls (Meaney et al., 1988). Whether neonatal tactile stimulation could influence spatial long-term memory during the aging process or in tasks involving more emotional factors needs further exploration. 4.2. Effects of neonatal treatment on vHIP–mPFC LTP The vHIP–mPFC glutamatergic neural pathway has been demonstrated to play an important role in SWM function. For example, blockade of unilateral vHIP and contralateral mPFC resulted in impaired SWM in the delayed spatial win-shift task and the delayed alternation task (Floresco et al., 1997; Seamans et al., 1998; Wang & Cai, 2006), suggesting that this neural pathway could be critical for online information encoding and refreshing. In the present study, neonatal tactile stimulation significantly enhanced the amplitude of the vHIP–mPFC LTP, an important form of synaptic plasticity widely thought to be a basic cellular mechanism of learning and memory (Bliss & Collingridge, 1993; Bliss & Lomo, 1973; Malenka & Nicoll, 1993, 1999), when compared with their NTS siblings in adulthood. External novel stimuli (e.g., early handling, novelty exposure) have been reported to enhance hippocampal LTP and spatial learning and memory (Akers et al., 2006; Tang, Akers, Reeb, Romeo, & McEwen, 2006; Tang & Zou, 2002; Wilson, Wilner, Kurz, & Nadel, 1986). Our data suggest that neonatal TS is an effective way to improve development of synaptic plasticity and the enhancement of vHIP–mPFC LTP could be one mechanism underlying the facilitative effects of TS on SWM function. 4.3. Effects of neonatal treatment on D1 receptor activation Behavioral and electrophysiological studies have shown that postsynaptic D1 receptors of the mPFC play an important role for SWM function in monkeys (Didriksen, 1995; Sawaguchi & Goldman-Rakic, 1991, 1994; Sawaguchi, Matsumura, & Kubota, 1988) and rodents (Seamans et al., 1998. Murphy et al., 1996; Murphy et al., 1996; Zahrt et al., 1997). Based on these studies, activation of prefrontal D1 receptors effectively modulates information maintenance during delay duration and inhibits irrelevant information in SWM performance (Durstewitz, Seamans, & Sejnowski, 2000). An ‘inverted U-shape’ relationship between D1 receptor activation and SWM has demonstrated that there is a narrow intermediate range for optimal D1 receptor stimulation while hypo- and hyper-
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stimulation leads to adverse SWM performance (for a review, see Arnsten, 1998). Therefore, it is reasonable that systemic administration of A77636 could act mainly at postsynaptic D1 receptors in the mPFC to affect SWM performance. As a result, we found that there was only one dose (0.1 mg/kg) of A77636 to effectively improve SWM performance in the NTS group, while there were three effective doses (0.01, 0.1, and 1 mg/kg) in the TS group and two effective doses (0.1 and 1 mg/kg) in the TS/MS group, indicating that the optimal activation range of D1 receptor was wider in TS and TS/MS animals than in NTS animals. Previously, it was shown in monkeys (Ljungberg, Apicella, & Schultz, 1991; Watanabe, Kodama, & Hikosaka, 1997) and rats (Rossetti & Sonia, 2005) that there is significant elevation of DA release in the PFC when the animals perform a SWM task. The finding that administration of a 1 mg/kg dose of A77636 significantly improved SWM performance in TS and TS/MS animals, but not in NTS animals, implies that the activation of prefrontal D1 receptors in TS and TS/MS animals is easier for maintaining an optimal status following elevation of DA release in the SWM task than in NTS animals, and that this then leads to improved SWM performance behaviorally. The lack of significant between-subject effects of neonatal treatment on SWM performance after administration of A77636 could be because of a ‘ceiling’ effect where all three groups achieved similar maximum response after A77636 administration (NTS: 81.4 ± 3.4%; TS: 79.4 ± 2.1%; TS/ MS: 80.7 ± 2.5%). This ‘ceiling’ effect could be related to indiscernible differences of mesocortical D1 receptor densities among groups as demonstrated by previous studies where early handling and maternal care did not affect D1 receptor densities in the nucleus accumbens and PFC (Brake et al., 2004; Meaney et al., 2002; Zhang et al., 2005). It should be mentioned that there was also an ‘inverted U-shape’ relationship between the mPFC D1 receptor activation and the induction of vHIP–mPFC LTP. For example, local injection of the D1 receptor antagonist SCH23390 into mPFC impaired vHIP–mPFC LTP. Conversely, directly infusion of 0.1 and 1 mM doses of the D1 receptor agonist SKF81297 into mPFC facilitated vHIP–mPFC LTP. But infusion of a higher dose of 5 mM did not result in facilitative effect (Gurden et al., 2000). Furthermore, prefrontal DA release increased following high-frequency stimuli at vHIP, and enhanced vHIP–mPFC LTP (Gurden et al., 1999). Therefore, the wider activation range of mPFC D1 receptors is also beneficial for the induction of vHIP–mPFC LTP. The mechanism underlying the beneficial effects of neonatal TS on prefrontal D1 receptor activation is not yet known. A possible explanation may be related to the enhanced efficiency of D1 receptor-coupled Gs signal transduction pathway (for a review, see Jay, 2003). In this case, adenylyl cyclase is more easily activated by low doses (e.g., 0.01 mg/kg) of A77636 to increase intracellular cAMP in TS animals than in NTS animals. After administration of
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high doses (e.g., 1 mg/kg) of A77636, activities of negative feedback signals such as protein phosphatase 1 and calcineurin may also be enhanced to maintain D1 receptor stimulation under an optimal state in TS animals. Only by continuing to increase A77636 doses and thus potentially overloading the negative modulation might D1 receptors exhibit hyperactivation. This explanation is supported to some extent by findings that the D1 receptor-related cAMP-PKA pathway contributed to SWM function (Aujla & Beninger, 2001; Runyan & Dash, 2005) and vHIP– mPFC LTP (Gurden et al., 2000). 4.4. Long-term effects of neonatal MS experience In this study, we used a relatively short period (1 h daily) of MS to reduce physiological stressful factors, especially hunger and thirst; therefore, this kind of short-period MS paradigm could be considered as a psychological stressful factor. Actually, pups have been shown to exhibit a more obvious stress response following a progressively increased separation time course (Levine, Huchton, Wiener, & Rosenfeld, 1992). Furthermore, we made an effort to reduce the influence of novelty exposure by placing pups in a cup bedded with sawdust taken from their own nest, allowing these pups to be in contact with familiar olfactory cues and hear ultrasonic vocalization from other separated pups in the same incubator. As a result, TS/MS animals exhibited significantly enhanced SWM performance and vHIP–mPFC LTP when compared with the NTS animals, and there was no significant difference when comparing TS/MS and TS animals, suggesting that short periods of neonatal MS as a psychological factor was not stressful enough to counteract the TS-induced beneficial effect on mPFC-related cognitive function and neural plasticity. The lack of an MS-induced effect could be related with to the stress-hyporesponsive period, in which adrenal cortical response of pups is resistant to stressors in rodents, thus preventing hypersecretion of corticoids (Sapolsky & Meaney, 1986). Differently from those studies that found an adverse effect of MS (mentioned in the Introduction), several studies did not find a significant effect of long-time (3–6 h) MS on fear behavior and cognitive tasks (Lehmann et al., 2002; Shalev & Kafkafi, 2002; Stanton, Crofton, & Lau, 1992), and even MS led to reduced stressful response and improved learning and memory (Lehmann, Stohr, & Feldon, 2000; Ogawa et al., 1994; Pryce et al., 2001). These discrepancies might be related to inconsistency of MS manipulations, including the time course during which pups are separated from their mother, repeated times, temperature, familiar or novel environment, and so on. In our study, we actually investigated TS/MS-induced effects on SWM function. Whether this kind of MS paradigm alone could influence neural development and cause behavioral alterations needs further exploration. In conclusion, our findings that neonatal tactile stimulation effectively improved the SWM performance, the syn-
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