Twenty- and sixty-day-old male C57BL/6 mice were used in all experiments. Mice were housed in groups of four in a temperature (25 ± 1 °C) and humidity controlled room on a 12-h light-dark cycle with food and water provided ad libitum.
All behavioral procedures were carried out during the light cycle between 7:00 and 10:00 h. All experimental procedures complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University.
Object-Location Memory Task
Object-location memory (OLM) task was performed as previ-ously described [20]. Before the training session, mice were habituated to the experimental apparatus (40 × 40 × 40 cm3) by allowing them to freely explore for 10 min for 1 day in the absence of objects. During the training session, two identical objects were presented for exploration for 10 min. During the testing session, 1 or 24 h later, object exploration was scored for 10 min. Locomotor activity and the time spent in exploring each object were recorded using a digital video camera, and scoring was performed with the behavioral tracking system
Ethovision (Noldus). Object exploration was defined as the mouse’s head being oriented toward the object within a dis-tance of 2 cm or the mouse’s nose touching the object. To analyze cognitive performance, a discrimination index was calculated as the following formula: [(time exploring the ob-ject in novel location− time exploring the object in familiar location) / (time exploring the objects in both novel and famil-iar locations)].
Contextual Fear Conditioning
The contextual fear conditioning (CFC) test was conducted using a computer-controlled context conditioning system (ENV-307A, MED Associates) as previously described [10].
Mice were placed into the conditioning chamber (15.9 × 14.0 × 12.7 cm) and allowed to explore the same context for 2 min followed by one or three aversive electrical footshocks (2 s, 0.60 mA with 30 s intershock 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 ceiling of the sound attenuating cubicle. Context-dependent freezing responses were measured 1 or 24 h after fear conditioning training.
The freezing responses were scored as the total time spent freezing in the conditioning context during the 3 min test session.
Slice Preparations and Electrophysiology
Hippocampal slices were prepared using standard procedures as previously described [20]. The mice were anesthetized with isoflurane and euthanized by decapitation. The brains were rapidly removed and placed in ice-cold sucrose artificial cere-brospinal fluid (aCSF) cutting solution [containing (in mM):
sucrose 234, KCl 2.5, CaCl20.5, MgCl2 7, NaHCO325, NaH2PO41.25, and glucose 11 at pH 7.3–7.4 and equilibrated with 95% O2–5% CO2]. Coronal hippocampal slices (250 or 400 μm) were prepared using a vibrating microtome (VT1200S; Leica) and transferred to a holding chamber of normal aCSF [containing (in mM): NaCl 117, KCl 4.7, CaCl22.5, MgCl21.2, NaHCO325, NaH2PO41.2, and glu-cose 11 at pH 7.3–7.4 and equilibrated with 95% O2–5% CO2] and maintained at room temperature for at least 1 h before use.
During each electrophysiological recording session, one slice was transferred to the recording chamber and continually perfused with oxygenated aCSF at a flow rate of 2–3 ml/min at 32.0 ± 0.5 °C. The extracellular field potential recordings were carried out using an Axoclamp-2B amplifier (Molecular Devices). Microelectrodes were pulled from microfiber-filled 1.0 mm capillary tubing on a Brown-Flaming electrode puller (Sutter Instruments). The responses were low pass filtered at 2 kHz, digitally sampled at 10 kHz, and analyzed using Mol Neurobiol
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pCLAMP 8.0 software (Molecular Devices). Postsynaptic re-sponses were evoked in CA1 stratum radiatum by extracellu-lar stimulation of Schaffer collateral/commissural afferents at 0.033 Hz with a bipolar tungsten stimulating electrode. The stimulation strength was set to elicit response for which the amplitude was 30–40% of the maximum spike-free response.
Field excitatory postsynaptic potentials (fEPSPs) were record-ed with a glass pipette fillrecord-ed with 1 M NaCl (2–3 MΩ resis-tance), and the fEPSP slope was measured from approximate-ly 20–70% of the rising phase using a least-squares regression.
Paired pulse facilitation (PPF) was assessed by using a suc-cession of paired pulses separated by intervals of 20, 40, 60, 80, 100, and 200 ms. The early-phase LTP (E-LTP) was in-duced by high-frequency stimulation (HFS), at the test pulse intensity, consisting of one or two 1 s trains of stimuli sepa-rated by an intertrain interval of 20 s at 100 Hz, and late-phase LTP (L-LTP) was induced by four 1 s trains of 100 Hz stimuli separated by an intertrain interval of 5 min. Depotentiation (DEP) was induced by application of 10 min low-frequency stimulation (LFS) at 2 Hz. The magnitudes of LTP were aver-aged the responses recorded during the last 10 min of the recording and normalized to 10 min of baseline before LTP induction. The magnitudes of DEP were calculated by com-paring the averaged responses recorded at 50–60 min after the end of LFS with the individual baseline magnitude just before each LFS application.
Whole-cell patch-clamp recordings were made from visu-alized pyramidal neurons in the CA1 region of hippocampal slices using an Axopatch 200B amplifier (Molecular Devices).
Data acquisition and analysis were performed using a digitizer (Digidata 1440A) and pCLAMP 9 software (Molecular Devices). For measurement of synaptically evoked excitatory postsynaptic currents (EPSCs), a bipolar stimulating electrode was placed in the stratum radiatum of CA1 region to stimulate Schaffer collateral/commissural afferents at 0.05 Hz and the superfusate routinely contained gabazine (10μM) to block GABAA receptor-mediated inhibitory synaptic responses.
EPSCs were recorded in voltage-clamp mode at a holding potential of− 70 mV. The composition of intracellular solution was as follows (mM): 130 cesium-methanesulfonate, 10 HEPES, 0.5 EGTA, 8 NaCl, 1 TEA, 4 Mg-ATP, 0.4 Na-GTP, 10 Na-phosphocreatine, and 1 QX-314. The NMDAR/
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid re-ceptor (AMPAR) ratio was computed by dividing the peak amplitude of the NMDAR-mediated EPSCs recorded at + 40 mV by the peak amplitude of the AMPAR-mediated EPSCs recorded at− 70 mV. The NMDAR-mediated compo-nent of EPSCs was calculated as the difference between the EPSCs measured in the absence and presence of 2-amino-5-phosphonovaleric acid (APV, 50 μM). Miniature EPSCs (mEPSCs) were recorded from CA1 pyramidal neurons held in voltage-clamp mode at a holding potential of− 70 mVin the presence of tetrodotoxin (0.5μM) and gabazine (10 μM), and
analyzed offline using a commercially available software (Mini Analysis 4.3; Synaptosoft) as previously described [21]. Detection threshold for analysis was set at twice the root-mean-square (RMS) noise levels.
Western Blotting
The microdissected hippocampal CA1 tissue samples were lysed in ice-cold Tris-HCl buffer solution (TBS; pH 7.4), con-taining a cocktail of protein phosphatase and proteinase inhib-itors, and ground with a pellet pestle (Kontes glassware) as previously described [22]. Samples were sonicated and spun down at 14,500 rpm at 4 °C for 15 min. The supernatant was then assayed for total protein concentration using Bio-Rad Bradford Protein Assay Kit (Hercules). Each sample from tissue homogenate was separated using 8 or 10% SDS-PAGE gel. Following the transfer on polyvinylidene fluoride membranes, blots were blocked in buffer solution containing 3% bovine serum albumin (BSA) and 0.4% Tween-20 in phosphate-buffered saline (PBS) (in mM: 124 NaCl, 4 KCl, 10 Na2HPO4, and 10 KH2PO4; pH 7.2) for 1 h and then blotted overnight at 4 °C with the antibodies that recognize phosphorylated calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) at Thr286 (1:1000; Cat# MA1-047; Thermo Fisher Scientific), phosphorylated GluA1 at Ser831 (1:1000;
Cat# 04-823; Millipore), GluN1 (1:1000; Cat# sc-1467; Santa Cruz Biotechnology), GluN2A (1:1000; Cat# sc-1468; Santa Cruz Biotechnology), GluN2B (1:1000; Cat# 14544S; Cell Signaling Technologies), PKMζ (1:1000; Cat# sc-216; Santa Cruz Biotechnology), protein phosphatase 1 (PP1; 1:1000;
Santa Cruz Biotechnology; mouse; sc-7482), PP2A (1:1000;
Cat# 05-421; Millipore), PP2B (1:1000; Cat# 07-069; Upstate Biotechnology), or β-actin (1:10000; Cat# MAB1501;
Millipore). It was then probed with HRP-conjugated second-ary antibody for 1 h and developed using the ECL Plus™
immunoblotting detection system (Amersham Biosciences), according to manufacturer’s instructions. The immunoblots using phosphorylation site-specific antibodies were subse-quently stripped and reprobed with the following antibodies:
anti-CaMKIIα antibody (1:1000; Cat# MA1-048; Thermo Fisher Scientific) or anti-GluA1 antibody (1:1000; Cat#
ab86141; Abcam). Immunoblots were analyzed by densitom-etry using Bio-profil BioLight PC software (Vulber Lourmat).
Only film exposures that were not saturated were used for quantification analysis.
Immunofluorescence
Immunofluorescence was performed as previously described [20]. Immediately after fear conditioning, mice were deeply anesthetized through intraperitoneal injection of sodium pen-tobarbital (100 mg/kg) and perfused transcardially with PBS and 4% paraformaldehyde. After the perfusion, the brains Mol Neurobiol
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were removed and fixed in 4% paraformaldehyde for 24 h at 4 °C and then transferred to the solution containing 30% su-crose that immersed in 4 °C for at least 48 h before slicing.
Coronal slices were sectioned to a 40-μm thickness, washed with 0.3% Triton X-100, and then incubated for blocking with solution containing 3% goat serum in PBS. After blocking, the sections were incubated in the primary antibodies against phosphorylated cAMP response element-binding protein at Ser133 (pCREB, 1:1000; Cat# 06-519; Millipore) or neuronal nuclei (NeuN, 1:2000; Cat# MAB377; Millipore) overnight at 4 °C in PBS with 0.1% Triton X-100. Finally, sections were washed with TBS containing 0.1% Tween-20 and then incu-bated with the secondary Alexa Fluor 488 or Alexa Fluor 568 antibodies (Invitrogen) for 1 h at room temperature. The im-munostained sections were collected on separate gelatin-subbed glass slides, rinsed extensively in PBS, and mounted with ProLong Gold Antifade Reagent (Invitrogen).
Fluorescence images of neurons were obtained using an Olympus FluoView FV1000 confocal microscope with se-quential acquisition setting at a resolution of 1024 × 1024 pixels and a sampling of six consecutive optical sections in the Z-stack. The high magnification images were recorded with an Olympus Plan Apochromat × 60 oil-immersion objec-tive (1.42 numerical aperture and 0.15 working distance). All images were imported into NIH ImageJ software (National Institutes of Health) for analysis, and all the parameters used were kept consistent during capturing.
Hippocampal Cannula Implantation and Drug Injection
On P11 and P51, mice were bilaterally implanted under deep pentobarbital (50 mg/kg, i.p. supplemented as required) anes-thesia with 28-gauge guide cannulas (Plastics One Inc., Roanoke, VA) in the dorsal hippocampus. Coordinates for P11 mice were− 1.8 mm posterior to bregma, ± 1.5 mm bilat-eral 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 [23] and the stereo-taxic atlas of adult mouse brain [24]. The cannulas were fixed to the skull with dental cement. Dummy cannulas (33 gauge) were inserted into each guide cannula following the surgery to prevent clogging. Mice were allowed to recover from surgery for 1 week prior to cannula infusion and behavioral training.
Cyclosporin A (0.2μg/μl) or ifenprodil (5 μg/μl) was bilater-ally administered into the hippocampus at the rate of 0.25μl/
min (0.5 μl/side) 15 min before the behavioral training by using a 33-gauge needle that connected via polyethylene tub-ing to a Hamilton syrtub-inge. Cyclosporin A was dissolved in 10% dimethyl sulfoxide (DMSO) in PBS. Ifenprodil was dis-solved in PBS. Drug dose was selected on the basis of pub-lished studies [25,26]. The infusion cannulas were kept in
place for an additional 2 min to minimize backflow of the injectant. Histological verification of the cannula locations was performed at the end of behavioral testing. Mice with misplaced cannulas were excluded from behavioral analysis.
Statistical Analysis
The results are presented as mean ± SEM. Statistical analyses were performed using the Prism 6 software package (GraphPad Software). To compare the difference between the means of two distributions, we first determined whether the distributions of values were Gaussian using the Shapiro-Wilk test. For Gaussian distributions, we calculatedp values using unpaired Student’s t test, while for non-Gaussian distri-butions, we used Mann-WhitneyU test. Because the distribu-tions of LTP and DEP magnitudes were not Gaussian, the Mann-Whitney U test was used to compare differences be-tween two independent groups. The significance of the differ-ence between multiple groups was calculated by two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analyses. The number of animals used is indicated byn.
Electrophysiological values across multiple slices or neurons from the same animal were averaged to yield a single value for each animal. Probability values ofp ≤ 0.05 were considered to represent significant differences.
Results
Infant and Adult Mice Show Different Memory Retentions
We first compared the ability of infant (P20) and adult (P60) mice to form enduring hippocampus-dependent memories using the OLM and CFC tasks. As shown in Fig.1a, when mice were tested short-term OLM 1 h after training, both P20 and P60 mice had intact memory retention. They both spent more time exploring the object in the novel location than the one in the original location (t(14)= 0.20,p = 0.85; unpaired Student’s t test). However, when tested long-term OLM 24 h after training, P20 mice exhibited significantly reduced dis-crimination index compared to P60 mice (t(35)= 3.61, p <
0.001; unpaired Student’s t test; Fig.1b), whereas no differ-ences between the groups in overall exploration of objects were observed. In CFC tests, both short-term memory and long- term memory were assessed. There was no difference between P20 and P60 mice in memory retention test 1 h (short-term memory) after a single context-shock pairing fear conditioning training (t(8)= 0.85,p = 0.42; unpaired Student’s t test; Fig.1c). However, P20 mice froze significantly less than P60 mice when tested 24 h postconditioning (long-term memory) (t(19)= 3.80, p < 0.01; unpaired Student’s t test;
Fig.1d). When mice received fear conditioning to contextual Mol Neurobiol
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stimuli paired with three shocks, P20 and P60 mice exhibited comparable levels of freezing in the conditioning context when tested 24 h postconditioning (t(22)= 0.30,p = 0.77; un-paired Student’s t test; Fig.1e). P20 and P60 mice have no differences in foot-shock pain sensitivity.
Because CREB phosphorylation (pCREB) at Ser133 cor-relates with memory formation [27, 28], we compared the percentages of CA1 neurons expressing pCREB immediately after a single context-shock pairing fear conditioning. We ob-served significantly lower percentages of CA1 neurons expressed pCREB after a single context-shock pairing fear conditioning training in both dorsal (DH) and ventral hippo-campus (VH) of P20 mice compared to those in P60 mice (DH:t(10)= 2.61,p < 0.05; VH: t(10)= 2.30,p < 0.05; unpaired Student’s t test; Fig.2a, b). However, there was no difference between P20 and P60 mice in percentages of CA1 neurons expressed pCREB immediately after three context-shock pairing fear conditioning training in both DH (t(6)= 0.85, p = 0.43; unpaired Student’s t test) and VH (t(6)= 1.31,p = 0.24; unpaired Student’s t test; Fig.2c, d).
Infant and Adult Mice Show Different Synaptic Transmissions and Plasticities
To investigate the potential cellular basis of IA, we compared the basal synaptic transmission and the induction of long-term syn-aptic plasticity at Schaffer collateral-CA1 synapses in hippocam-pal slices from P20 and P60 mice. Consistent with previous findings [29], we found that P20 mice exhibited smaller fEPSP slopes at different stimulus intensities compared to P60 mice.
Two-way ANOVA revealed a significant age × stimulus inten-sity interaction (F(11120)= 2.72,p < 0.01), a significant effect of age (F(1,120)= 33.09,p < 0.001), and a significant effect of stim-ulus intensity (F(11120)= 101.7,p < 0.001; Fig.3a). To determine whether P20 and P60 mice have different presynaptic function, we examined the PPF, a transient form of presynaptic plasticity.
As shown in Fig.3b, pairs of presynaptic fiber stimulation pulses delivered over an interpulse interval range of 20–200 ms evoked higher amounts of PPF ratio in slices from P20 mice than those from P60 mice. Two-way repeated measure ANOVA revealed a significant effect of age (F(1,60)= 25.70, p < 0.01) and a Fig. 1 Infant and adult mice show differential retention of OLM and CFC
memories. a, b Schematic representations of the OLM task (upper panel).
Mice were habituated to the experimental apparatus for 10 min and 24 h before OLM training session. During the training session, mice were given 10 min to explore two identical objects. For retention test, one object was placed in the familiar location and one was moved to a different location.
The discrimination index was calculated as the difference in time spent exploring the novel location minus the time spent exploring the familiar location over the total exploration time during the test session. Summary bar graphs depicting the memory retention testing at 1 h (a) (n = 8 in each
group) or 24 h (b) after training (n = 18 in each group). c, d Schematic representations of the training protocol for the CFC test (upper panel).
Summary bar graphs depicting the fear memory retention test at 1 h (c) (n = 5 in each group) or 24 h (d) after receiving one conditioned stimulus (CS) × unconditioned stimulus (US) paired training (P20,n = 11 and P60, n = 10) in P20 and P60 mice. e Schematic representations of the training protocol for the CFC test (upper panel). Summary bar graphs depicting the fear memory retention test at 24 h after receiving three CS × US paired training (n = 14 in each group) in P20 and P60 mice. ***p < 0.001 com-pared with P60 group. Error bars indicate SEM
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significant effect of interpulse interval (F(5,60)= 4.88,p < 0.001), but a non-significant effect for the interaction between age and interpulse interval (F(5,60)= 0.05,p = 0.99). However, we found no significant difference in the NMDAR/AMPAR ratio of evoked EPSCs in slices from P20 and P60 mice (t(9)= 0.33, p = 0.75; unpaired Student’s t test; Fig.3c). Furthermore, the frequency (t(10)= 4.51, p < 0.01; unpaired Student’s t test) but not the amplitude (t(10)= 0.34,p = 0.74; unpaired Student’s t test) of mEPSCs was significantly lower in slices from P20 mice compared to those from P60 mice (Fig.3d).
We next compared the magnitude of hippocampal CA1 LTP in slices from P20 and P60 mice. In slices from P60 mice, a single train of HFS (100 Hz, 1 s) induced a robust E-LTP (41.7 ± 7.0%), whereas a significantly faster decay of E-LTP was observed in slices from P20 mice (10.2 ± 5.1%,p < 0.01;
Mann-WhitneyU test; Fig.3e, h). In contrast, we observed no significant difference between P20 and P60 mice in the induc-tion of E-LTP by two trains of HFS (p = 0.48; Mann-Whitney U test; Fig.3f, h) or L-LTP by four trains of HFS (p = 0.68;
Mann-WhitneyU test; Fig.3g, h). To compare the ability of LFS-induced DEP in slices from P20 and P60 mice, LFS was applied 10 or 30 min after two trains of HFS. The magnitude of DEP was similar between P20 and P60 mice when LFS was applied 10 min after HFS (p = 0.18; Mann-Whitney U test;
Fig.3i, k). In contrast, when LFS was applied 30 min after HFS, the higher magnitude of DEP was observed in slices from P20 mice compared to those from P60 mice (p = 0.03;
Mann-WhitneyU test; Fig.3j, k).
Infant and Adult Mice Show Different Expression Profiles of Protein Kinases and Phosphatases
We then compared the hippocampal CA1 expression profiles of molecules known to play critical roles in LTP and memory formation, either in basal condition or following LTP induction, using Western blot analyses in DH CA1 tissue extracts from P20 and P60 mice. P20 mice had a significant higher level of GluN2B protein compared to P60 mice (t(10)= 3.24, p < 0.01;
DH
Fig. 2 The expression of pCREB in CA1 pyramidal neurons immediately after context-shock pairing fear conditioning. a Representative immuno-fluorescence images showing pCREB-expressing neurons in the dorsal (DH) or ventral (VH) hippocampal CA1 region of P20 and P60 mice immediately after fear conditioning. Augmented figures (lower panel) showing pCREB-expressing CA1 neurons in rectangle area. b Quantitative analysis of percentage of CA1 neurons expressing pCREB (n = 6 in each group). c Representative immunofluorescence images
showing pCREB-expressing neurons in the dorsal (DH) or ventral (VH) hippocampal CA1 region of P20 and P60 mice immediately after three context-shock pairing fear conditioning training. Augmented figures (lower panel) showing pCREB-expressing CA1 neurons in rectangle ar-ea. d Quantitative analysis of percentage of CA1 neurons expressing pCREB (n = 4 in each group). *p < 0.05 compared with P60 group.
Error bars indicate SEM
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unpaired Student’s t test), whereas the levels of GluN1 and GluN2A proteins were not different between groups (Fig.4a).
In addition, there was a significant higher level of PKMζ (t(12)= 4.08,p < 0.01; unpaired Student’s t test; Fig.4b) and PP2B (t(12)= 3.64,p < 0.01; unpaired Student’s t test; Fig.4c) in P20 mice compared to P60 mice. No significant differences were found in the levels of CaMKIIα, GluA1, PP1, and PP2A be-tween P20 and P60 mice in basal condition (Fig.4b, c). To determine LTP-associated activation of CaMKII and GluA1 phosphorylation, slices were harvested at different time points after a single train of HFS and measured for their pCaMKIIα
(Thr286) and pGluA1 (Ser831) and total CaMKIIα and GluA1 levels. In slices from P60 mice, HFS led to pronounced and significant increase in pCaMKIIα and pGluA1 levels, which continued up to 60 min after HFS. In contrast, HFS in slices from P20 mice did not significantly alter pCaMKIIα and pGluA1 levels. Two-way ANOVA followed by Bonferroni’s post hoc test revealed that P20 mice had significant lower levels of pCaMKIIα (F(4,70)= 3.69, p < 0.01) and pGluA1 (F(4,70)= 4.43, p < 0.01) compared to P60 mice (p < 0.01; Fig. 4d–f).
There were no differences between the groups in total
There were no differences between the groups in total