Twenty- and sixty-day-old male C57BL/6 mice (lines origi-nally obtained from Charles River) were used in all experi-ments. Mice were housed in groups of four in a temperature (25 ± 1 °C)- and humidity-controlled room on a 12 h/12 h light/dark cycle (lights on at 07:00 h) with free access to food and water. All behavioral procedures were carried out during the light phase of the cycle (between 09:00 h and 15:00 h) and animals were allowed to acclimate to the behavioral testing room for at least 1 h prior to testing. To avoid variability caused by hormonal cycles in female mice, only male mice were used in this study. All procedures involving the use of animals were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the National Cheng Kung University Animal Care and Use Committee (authorization no. 106048).
Surgery and Drug Infusion
Surgery was carried out as described previously [10]. In brief, on P15 or P51, mice were bilaterally implanted under deep anesthesia by intraperitoneal injection of a mixture of 50 mg/kg zolazepam (Zoletil, Virbac, Carros, France) and 5 mg/kg xylazine hydrochloride (Rompun, Bayer, Puteaux, France) with 26-gauge guide cannulas (RWD Life Science Co., Ltd.) in the dorsal hippocampus in a stereotaxic frame
(Kopf Instruments). Coordinates for P15 mice were− 1.8 mm posterior to bregma, ± 1.5 mm bilateral to midline, and 1.3 mm ventral to brain surface, and for P51 mice were − 2.0 mm posterior to bregma, ± 1.5 mm bilateral to midline, and 1.5 mm ventral to brain surface according to the Golgi atlas of the postnatal mouse brain [25] and the stereotaxic atlas of adult mouse brain [26], respectively. The cannulas were secured to the skull with bioglue. Dummy cannulas (30 gauge) were inserted into each guide cannula following the surgery to prevent clogging. Mice were intraperitoneally ad-ministrated with ketoprofen (5 mg/kg) for post-operative an-algesia. Anisomycin (62.5μg/μl, Sigma-Aldrich), a protein-synthesis inhibitor, was microinfused bilaterally into the dor-sal hippocampus at the rate of 0.25μl/min (0.5 μl/side) 15 min before the behavioral training by using a 30-gauge needle that connected via polyethylene tubing to a Hamilton syringe.
Anisomycin was dissolved with 1 N HCl and adjusted to pH 7.4 with NaOH in the vehicle phosphate buffer solution (PBS). Drug dose was selected on the basis of published stud-ies [20,27]. The infusion cannulas were kept in place for an additional 2 min to minimize backflow of the injectant.
Histological verification of the locations of cannula tip was performed at the end of behavioral testing. Only data from animals with correct cannula implants (95% of the mice) were included in statistical analysis.
Contextual Fear Conditioning
The contextual fear conditioning (CFC) test was performed using a computer-controlled context conditioning system (ENV-307A, MED Associates) as previously described [10].
The conditioning chamber was placed inside a ventilated and sound-dampening isolation cubicle. Mice were placed into a rectangular Plexiglas conditioning chamber (15.9 × 14.0 × 12.7 cm) and allowed to explore the same context for 2 min followed by three aversive electrical footshocks (0.6 mA, 2 s duration, 30 s inter-shock interval) through a stainless steel grid floor. After the last shock, mice were allowed to explore the context for additional 2 min prior to return to their home cages. The behavior of the mice was recorded using a digital near-infrared video camera on the wall of the cubicle.
Context-dependent freezing responses were measured 1 or 24 h after fear conditioning training. For context specificity experiments, P20 mice were trained in context A (rectangular Plexiglas chamber) in a 5-min CFC session as described above. The following day, mice were tested in both condi-tioned context A and novel context B, in counterbalanced order with a 2-h inter-trial interval. Context B consisted of a rectangular box (15.9 × 14.0 × 12.7 cm), with Plexiglas white floor, 2 white sidewalls placed at 45° from the floor in order to form a triangle enclosure, a black and white check board back wall, and a transparent Plexiglas front wall. The freezing Mol Neurobiol
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responses were scored as the total time spent freezing in the conditioning or novel context during the 3-min test session.
Novel Context Exploration
The novel context exploration (NCE) apparatus is a white acrylic square-type chamber (45 × 45 × 90 cm) with obvious markers stuck on the two walls and floor as cue. A NCE training consisted of 10 min of free exploration of the empty chamber. To familiarize the animals with the context, mice were placed in the square chamber and allowed to explore it for 6 min per day for 4 consecutive days before the onset of NCE training. The behavior of the animals was videotaped and tracked with the EthoVision XT video tracking systems (Noldus: RRID:SCR_000441). The chamber was thoroughly cleaned with 70% ethanol following each trial to remove any residual olfactory traces.
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (catFISH) was carried out using RNAscope® Multiplex Fluorescent Reagent Kit 2.0 manufacturer’s instructions (Advanced Cell Diagnostics) as
described previously [28]. Mice were deeply anesthetized with sodium Zoletil (50 mg/kg, intraperitoneally) and per-fused transcardially with sterile 4% paraformaldehyde in PBS (pH 7.4). After the perfusion, brains were quickly re-moved, frozen in liquid nitrogen, and embedded into optimal cutting temperature compound. Coronal slices were sectioned to a 20μm thickness at − 20 °C, fixed in 4% paraformalde-hyde for 15 min, and then dehydrated through graded ethanol solutions (50%, 70%, and 100%) for 5 min each. Sections were subjected to reagent Pretreat 3 at 25 °C for 30 min and then hybridized with probes at 40 °C for 2 h in a humidified oven. TheArc probe (Cat. no. 316911) was used to target Arc RNA. After hybridization, brain sections were sequentially applied with a series of probe signal amplification steps, rinsed with ACD Wash Buffer twice for 2 min between each step, and finally counterstained with DAPI (1:5000; Sigma-Aldrich) and mounted with Fluoromount-G mounting medi-um (Southern Biotech). Fluorescence images of CA1 and CA3 neurons were acquired using an Olympus FluoView FV1000 confocal microscope with sequential acquisition set-ting at a resolution of 1024 × 1024 pixels, z-stack with 15–20 optical sections. All images were imported into NIH ImageJ software (National Institutes of Health: RRID:SCR_003070)
Fig. 1 NCE promotes CFC-LTM formation in juvenile mice. a Schematic illustration of the training protocol for the CFC task. b The learning curve for three acquisition trials of CFC training for P20 and P60 mice. c, d Summary bar graphs depicting the fear memory retention test at 1 h (c) or 24 h (d) after CFC training in P20 and P60 mice. e Schematic illustration of the experimental design (upper panel). Mice received bilat-eral dorsal hippocampus injection (intra-CA1) of vehicle (VEH) or anisomycin (ANI) immediately after CFC training, and fear memory retention was evaluated 1 h after training. Summary bar graphs depicting
the fear memory retention test at 1 h was not affected by ANI treatment in both P20 and P60 mice. f Schematic illustration of the experimental designs. NCE training was done either 30–180 min before or after CFC training, and the fear memory retention was tested at 24 h after training in P20 mice. g Summary bar graphs depicting the fear memory retention 24 h after training in P20 mice without (CON) or with NCE before (Pre-) or after (Post-) CFC training. Data represent the mean ± SEM. *p < 0.05;
**p < 0.01; *** p < 0.001 compared with P60 or P20 CON mice by two-tailed unpaired Student’s t test. n.s., not significant
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for analysis, and all the parameters used were kept consistent during capturing. The cellular compartment analysis ofArc RNA follows the procedure described previously [20]. The cells containing two small intense intranuclear fluorescent foci were designated as nuclear-positive (Nuc) neurons. The cells containing perinuclear and cytoplasmic labeling in multiple optical sections were designated as cytoplasm-positive (Cyto) neurons. The cells containing both intranuclear and cytoplasmic Arc-positive signals were designated as Nuc and Cyto doubleArc+neurons. Three sections corresponding to each region of interest were chosen from each mouse.
Immunohistochemistry and Quantification
Immunohistochemistry procedures were carried out as previously described [10]. Immediately after fear condi-tioning, mice were deeply anesthetized with 5%
isoflurane and perfused transcardially with PBS and 4%
paraformaldehyde. After the perfusion, brains were re-moved and continue to fix in 4% paraformaldehyde for
24 h at 4 °C and then cryoprotected by immersion in 30% sucrose at 4 °C for 48 h. Coronal slices were sec-tioned to a 40-μm thickness, washed with 0.3% Triton X-100, and then incubated for blocking with solution containing 3% bovine serum albumin in PBS. After blocking, the sections were incubated in the primary an-tibodies against phosphorylated cAMP response element-binding protein at Ser133 (pCREB, 1:3000; Cat. no. 06-519; Millipore) overnight at 4 °C in PBS with 0.3%
Triton X-100. Finally, sections were washed with TBS containing 0.1% Tween-20 and then incubated with the secondary Alexa Fluor 568 antibodies (1:2000; Cat. no.
A-11036; Invitrogen) for 1 h at room temperature.
N u c l e i w e r e c o u n t e r s t a i n e d w i t h d i a m i d i n o 2 -phenylindole (DAPI, 1:5000; Cat. no. D9542; Sigma-Aldrich). Fluorescence microscopic images of neurons were obtained using an Olympus FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan). For quantification of pCREB immunopositivity, pCREB+ neurons were determined only when cells were co-Fig. 2 Inhibition of protein synthesis prevents NCE-induced CFC-LTM
formation in P20 mice. a Schematic illustration of the experimental de-signs. NCE was done 60 min before CFC training. Mice received bilateral dorsal hippocampus injection (intra-CA1) of vehicle (VEH) or anisomycin (ANI) immediately after NCE or CFC training. Fear memory retention was evaluated 24 h after CFC training. b Summary bar graphs depicting the fear memory retention test at 24 h in mice receiving VEH or ANI treatment. NCE-induced CFC-LTM is impaired by bilateral intra-CA1 infusions of ANI immediately after NCE, but not immediately after
CFC training. c Schematic illustration of the experimental designs. Mice were familiarized with the square chamber for 6 min per day for 4 con-secutive days before the beginning of behavioral training and testing.
NCE was done 60 min before CFC training, and the fear memory reten-tion was tested at 24 h after CFC training. d Summary bar graphs depicting the fear memory retention test at 24 h in mice receiving famil-iarized or novelty treatment. Data represent the mean ± SEM. *p < 0.05;
**p < 0.01 compared with VEH or familiarized group by two-tailed un-paired Student’s t test. n.s., not significant
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localized with DAPI staining and used visual-based es-timation every sixth coronal section of the hippocampal region. For each animal, pCREB+ neurons were counted from both hemispheres and averaged over six sections that extend from approximately 1.70 to 1.90 mm poste-rior to Bregma according to the developing mouse brain atlas [25]. The entire hippocampal region was sampled in each section and was defined individually per section by hand. The fluorescence intensity of pCREB+ cells was quantitated as nuclear pixel density above the back-ground and classified into three subclasses as follows:
low-pCREB, less than 10-fold above the background;
medium-pCREB, 10–20-fold above the background;
high-pCREB, more than 20-fold above the background.
All images were imported into NIH ImageJ software for analysis, and all the parameters used were kept consis-tent during capturing.
Drug Treatment
(R)-(+)-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390, 0.2 mg/kg,
Sigma-Aldrich) was dissolved in 0.9% NaCl and administered intra-peritoneally. Drug dose was selected on the basis of the pub-lished study [20].
Statistical Analysis
No statistical methods were used to predetermine sample size, but our sample sizes were based on previous work of a similar nature by our laboratory [10]. No randomization was used to collect data. The results are presented as mean ± SEM. All sta-tistical analyses were performed using the GraphPad Prism 6 software (RRID:SCR_002798). To compare the difference be-tween the two population means, we first determined whether the data were normally distributed using the Shapiro-Wilk test. For normal distributions, we calculated p values using two-tailed unpaired Student’s t test, while for non-normal distributions, we used the Mann-WhitneyU test. The significance of the dif-ference between multiple groups was calculated by one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analyses. Unless otherwise specified, n represents the number of animals used. Differences were consid-ered as significant atp ≤ 0.05.
Fig. 3 Confirmation of bilateral cannula placement in the dorsal hippocampus. a Representative Nissl-stained coronal brain sec-tions showing bilateral cannula tracks targeting the dorsal hippo-campus of P20 mice. b, c Schematic illustrations showing distribution of cannula tips in the experiments shown in Fig.1e.
Blue and red dots represent mice received bilateral anisomycin (ANI) or vehicle (VEH) injections into the dorsal hippocampus, re-spectively, at P20 (b) and P60 (c).
d, e Schematic illustrations showing distribution of cannula tips in the experiments shown in Fig.2b. Blue and red dots repre-sent P20 mice received bilateral VEH or ANI injections into the dorsal hippocampus, respectively, immediately after CFC training (d) or NCE (e)
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Results
The Promotion of CFC-LTM Formation by NCE Requires Hippocampal Protein Synthesis
We first compared the ability of juvenile (P20) and adult (P60) mice to form enduring contextual fear mem-ory. Mice were allowed to acclimate to the conditioning chamber for 10 min 1 day before training. Context-dependent freezing responses were measured 1 or 24 h after CFC training (Fig. 1a). In CFC training session, there was no significant difference between P20 and P60 mice in fear acquisition (F(3,80)= 0.04, p = 0.98;
two-way ANOVA; Fig. 1b). In CFC tests, both STM and LTM were assessed. There was no significant dif-ference between P20 and P60 mice in memory retention test 1 h (STM) after CFC training (t(20)= 0.50, p = 0.63;
two-tailed unpaired Student’s t test; Fig. 1c). However, P20 mice froze significantly less than P60 mice when tested 24 h after CFC training (LTM) (t(16)= 2.35, p = 0.03; two-tailed unpaired Student’s t test; Fig. 1d), confirming that juvenile mice have an impaired ability
to form LTM. To investigate whether STM formation required new protein synthesis, a protesynthesis hibitor anisomycin or vehicle was bilaterally infused in-to the CA1 region of the dorsal hippocampus immedi-ately after CFC training. Figure 1 e depicts no differ-e n c differ-e s i n S T M r differ-e t differ-e n t i o n b differ-e t w differ-e differ-e n v differ-e h i c l differ-e - a n d anisomycin-treated groups in both P20 (t(12)= 0.46, p = 0.65; two-tailed unpaired Student’s t test) and P60 mice (t(12)= 0.08, p = 0.94; two-tailed unpaired Student’s t test).
In an attempt to explore whether behavioral tagging effect occurs in juvenile mice, the effect of spatial novelty on the formation of CFC-LTM in P20 mice was examined. We sub-mitted mice to a 10-min NCE at different time points before or after CFC training (Fig.1f). We found that STM was trans-formed into LTM when NCE was done 30, 60, or 180 min before or 30 or 60 min after CFC training (F(6,46)= 5.40, p < 0.001; one-way ANOVA; Fig.1g). However, this trans-formation was not observed when NCE was done 180 min after CFC training. These results suggest the existence of a time-dependent behavioral tagging process in juvenile mice.
It was proposed that the formation of a novelty-promoted LTM is dependent on protein synthesis [16–18,20]. Therefore, we sought to investigate whether the formation of CFC-LTM by a prior NCE was prevented by infusion of anisomycin into the CA1 region of the dorsal hippocampus (Fig.2a). We found that the administration of anisomycin, immediately after NCE but not CFC training, impaired LTM retention for the CFC task (F(1,26)= 4.19,p = 0.05; two-way ANOVA; Fig.2b). Post hoc analysis showed a significant vehicle vs. anisomycin group dif-ference after NCE (p = 0.004) but not after CFC (p = 0.60). The location of cannula tips for mice receiving bilateral anisomycin or vehicle injections into the CA1 region of the dorsal hippocampus immediately after CFC training or NEC included in the behavioral analyses are shown in Fig.3. These results suggest that new protein synthesis elicited by NCE is necessary to promote CFC-LTM formation.
It has been recently demonstrated that pre-familiarized to the context before subsequent exposure does not af-fect the behavioral tagging efaf-fect in P17 infant rats [29].
However, familiarization has been to shown to abolish behavioral tagging effect in adult animals [16–18, 20].
To examine whether the promotion of CFC-LTM was due to the novel nature of NCE, a group P20 mice was familiarized with the context for 6 min per day for 4 consecutive days before the onset of NCE (Fig. 2c).
We observed a significant reduction in LTM retention by a familiarization experience in P20 mice (t(14)= 2.26,p = 0.04; two-tailed unpaired Student’s t test; Fig. 2d). We next asked whether this NCE-induced CFC-LTM could have effect on the quality of the CFC memory represen-tation by examining context discrimination. Toward this end, P20 mice were subjected to NCE training 60 min Fig. 4 NCE-induced CFC-LTM shows context specificity in P20 mice. a
Schematic illustration of the experimental design to examine context discrimination. NCE was done 1 h before CFC training. Twenty-four hours after CFC training, mice were tested for fear memory retention in either the original training context A or a novel context B, followed by exposure to the opposite context 2 h later in a counterbalanced design. b Summary bar graphs depicting the freezing levels of mice in original training context A or a novel context B after CFC training. Mice can discriminate between the conditioned context A and the new context B.
Data represent the mean ± SEM. ***p < 0.001 compared with context A by two-tailed unpaired Student’s t test
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before CFC training in context A. Twenty-four hours later, mice received memory retention tests in both con-d i t i o n e con-d c o n t e x t A a n con-d n o v e l c o n t e x t B , i n counterbalanced order with a 2-h inter-trial interval (Fig. 4a). We found that mice froze significantly more in their training context A than in the novel context B (t(14)= 12.96, p < 0.001; two-tailed paired Student’s t test; Fig.4b), regardless of the order of the test.
An Increase in the Overlapping Neurons Following NCE
Previous work demonstrated that a shared neuronal en-semble is important for behavioral tagging [20]. We therefore hypothesized that inputs from two different sources of information, CFC and NCE, may converge at the same hippocampal neuronal ensembles when be-havioral tagging is successfully achieved. To visualize
neuronal ensembles that were activated during behavior-al tagging process, we performed cell compartment anbehavior-al- anal-ysis of temporal activity using catFISH to detect the i m m e d i a t e e a r l y g e n e , Arc (activity-regulated cytoskeleton-associated protein) (Fig. 5a). We took ad-vantage of the dynamic subcellular localization of arc RNA in which it localizes to the nucleus and cytoplasm 5 min and 25–35 min, respectively, after its transcrip-tional induction [30]. The detection of cytoplasmic and nuclear Arc RNA was used to distinguish neuronal en-sembles engaged by CFC and NCE, respectively (Fig.
5b). Five minutes after NCE, we harvested the brain and performed catFISH to analyze expression of Arc in both CA1 and CA3 pyramidal neurons of the dorsal hippocampus. In the CA1 region, the expression of cy-toplasmic or nuclear Arc+ cells was comparable between the novelty and familiarized groups. However, a signif-icant increase in the proportion of double-positive cells Fig. 5 Increasing overlapping hippocampal CA1 cell ensemble when
behavioral tagging is successfully achieved. a Schematic illustration of the experimental design for the catFISH experiments. Mice were familiarized with the square chamber for 6 min per day for 4 consecutive days before the beginning of behavioral training and subsequent catFISH analysis. CFC training was done 30 min before NCE and mice were sacrificed 5 min after the behavioral session. b Scheme of intracellular localization ofArc RNA signal used to detect and compare cell ensemble activated during NCE and CFC training. c, d Representative z-stack images ofArc catFISH signals captured in slices
from the CA1 (c) and CA3 (d) region of home cage, familiarized, and novelty groups. TheArc RNA signal and DAPI nuclear staining are shown in green and blue, respectively. Scale bar, 25μm. Bottom left panel: summary bar graphs depicting the percentages of cell containing cytoplasmic or nuclearArc RNA in DAPI-positive cells in the CA1 (c) and CA3 (d) region. Bottom right panel: summary bar graphs depicting the percentages of cytoplasmic and nuclearArc RNA double-positive cells (yellow arrowheads) in the CA1 and CA3 region. Data represent the mean ± SEM. *p < 0.05 compared with home cage or familiarized group by two-tailed unpaired Student’s t test. n.s., not significant Mol Neurobiol
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(i.e., Arc signals detected in both the cytoplasm and the nucleus) was observed in the novelty group compared to the familiarized group (F(2,18)= 81.05, p < 0.0001; one-way ANOVA; Fig. 5c). Post hoc analysis showed a significant difference (p = 0.002) between the novelty
(i.e., Arc signals detected in both the cytoplasm and the nucleus) was observed in the novelty group compared to the familiarized group (F(2,18)= 81.05, p < 0.0001; one-way ANOVA; Fig. 5c). Post hoc analysis showed a significant difference (p = 0.002) between the novelty