Male Sprague Dawley rats were used in this study (Table 1). After weaning, same-sex groups of 2–3 rats were housed in plastic cages (length: 25 cm; width: 40 cm; height: 25 cm) at a constant temperature of 23°C ± 1°C under a constant cycle of light and dark (lights on: 8:00 am to 8:00 pm). We counted the parturition day as postnatal day 1, and litters were reduced to 10 pups per cage on day 2. The pups were weaned on postnatal day 20 or 21 and housed in group cages with free access to food and water. The rats were individually housed at least 24 h prior to the experiments, to avoid any episodic experience. Food (MF, Oriental Yeast Co. Ltd, Tokyo Japan) and tap water were available ad libitum in all experimental periods. All animal housing and surgical procedures followed the guidelines of the Institutional Animal Care and Use Committee of Yamaguchi University. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Yamaguchi University (Approval No. 04-S02). the These guidelines comply with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The study reported in accordance with ARRIVE guidelines.
Table 1 Postnatal groups and body weight
Postnatal weeks Postnatal days Body weight (g) Number of rats
2 16.0 ± 0.1 34.7 ± 0.7 73
3 21.5 ± 0.1 53.2 ± 1.0 49
4 28.6 ± 0.2 103.4 ± 2.2 59
8 56.1 ± 0.2 307.2 ± 6.3 49
Data are the means ± SEM.
Inhibitory avoidance (IA) task
The IA training apparatus (length: 33 cm; width: 58 cm; height: 33 cm) was a two-chambered box consisting of a lit safe side and a dark shock side separated by a trap door (Fig. 1A; [22,23]. For training, rats were placed in the lit side of the box, facing the corner opposite the door. After the trap door was opened, the rats could enter the dark box at will. The latency before entering the novel dark box was measured as a behavioral parameter (latency before IA learning, Fig. 1B). Soon after the animals entered the dark side, we closed the door and applied a scrambled electrical foot shock (1.6 mA, 2 s) via electrified steel rods placed in the floor of the box. The rats were kept in the dark compartment for 10 s before being returned to their home cage. Untrained control rats were not moved from their home cages. Moreover, regarding the yoked controls, the unpaired control rats (foot shock only) were housed in the shock cage and subjected to the same scrambled electrical foot shock without any contextual experience. The walk-through control rats were placed in the IA training apparatus and allowed to explore for 1 min, without shock delivery. We confirmed the absence of mEPSC and mIPSC changes in juvenile animals exposed to the untrained, unpaired, or walk-through conditions .
Thirty minutes after the procedure described above, the rats were placed in the lit side of the box. The latency before entering the dark box was measured as an indicator of learning performance (latency after IA learning, Fig. 1B).
Electrophysiological recording of slice-patch clamping
We have previously reported the detailed technical protocol of the slice-patch clamp technique used for analyzing training-induced synaptic plasticity, with a short demonstration movie . Briefly, 1 h after the delivery of the paired foot shock, rats were anesthetized with pentobarbital and acute brain slices were prepared [22,23]. We used naive rats as the untrained group, all of which were injected with the same dose of anesthesia in their home cage. For the whole-cell recordings , the brains were quickly perfused with ice-cold dissection buffer (25.0 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 0.5 mM CaCl2, 7.0 mM MgCl2, 25.0 mM glucose, 90 mM choline chloride, 11.6 mM ascorbic acid, and 3.1 mM pyruvic acid) and gassed with 5% CO2/95% O2. Coronal sections (target CA1 area: AP, −3.8 mm, DV, 2.5 mm, LM, ± 2.0 mm;  were prepared (350 μm, Leica vibratome, VT-1200) in dissection buffer and transferred to physiological solution (22°C–25°C; 114.6 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 10 mM glucose, 4 mM MgCl2, and 4 mM CaCl2, pH 7.4) gassed with 5% CO2/95% O2. The recording chamber was perfused with physiological solution at 22°C–25°C.
Patch recording pipettes (4–7 MΩ) were filled with intracellular solution (127.5 mM cesium methanesulfonate, 7.5 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, and 0.6 mM EGTA at pH 7.25). Whole-cell recordings were obtained from CA1 pyramidal neurons from the rat hippocampus using an Axopatch 700 A amplifier (Axon Instruments). The whole-cell patch-clamp data were collected using a Clampex 10.4 instrument, and the data were analyzed using the Clampfit 10.4 software (Axon Instruments).
Miniature postsynaptic current recordings
We have previously reported the detailed technical protocol of the miniature postsynaptic current recording [22,23]. mEPSCs are thought to correspond to the responses elicited by the presynaptic release of a single vesicle of glutamate. In contrast, mIPSCs are thought to correspond to GABA. Increased mEPSC and mIPSC amplitudes reflect the strengthening of postsynaptic transmission, whereas increased event frequency reflects an increased number of functional synapses or presynaptic release probability.
For the miniature recordings, we added a Na+ channel blocker (0.5 μM tetrodotoxin) to the physiological solution. The mEPSCs (−60 mV holding potential) and mIPSCs (0 mV holding potential) were recorded sequentially for 5 min in the same CA1 neuron. The miniature events were detected using the Clampfit 10.4 software (Axon Instruments), and the events above 10 pA were used in the analysis. We recorded for at least 5 min, to determine the event frequency of mEPSCs or mIPSCs. The amplitudes of the events were averaged to obtain the mean amplitude. Bath application of an AMPA receptor blocker (CNQX, 10 μM) or GABAA receptor blocker (bicuculline methiodide, 10 μM) consistently blocked the mEPSC or mIPSC events, respectively.
2.3.2 Paired-pulse stimulation
We have previously reported the detailed technical protocol of the paired-pulse stimulation . To analyze presynaptic plasticity at excitatory synapses, we added 0.1 mM picrotoxin and 4 μM 2-chloroadenosine to the physiological solution and performed paired-pulse stimulation at −60 mV. To analyze presynaptic plasticity at inhibitory synapses, we added 10 μM CNQX to the perfusate and performed paired-pulse stimulation at 0 mV. To evaluate the paired-pulse ratio from the EPSC or IPSC average, 50–100 sweeps were recorded with paired stimuli at 100-ms intervals. We placed the stimulation electrode in either the stratum oriens (basal) or stratum radiatum (apical), to record evoked somatic currents. The ratio of the second amplitude to the first amplitude was calculated as the paired-pulse ratio [23,70].
Based on the Shannon entropy, we quantified the synaptic diversity by measuring the population differences in mE(I)PSC amplitude and frequency compared with untrained rats [24,25]. We used a standard spreadsheet software (Excel 2010, Microsoft Co., Redmond, WA, USA) to calculate the self-entropy per neuron. First, we obtained four miniature parameters (i.e., mean mEPSC amplitude, mean mIPSC amplitude, mean mEPSC frequency, and mean mIPSC frequency) in individual CA1 pyramidal neurons. Subsequently, we determined the distribution of the appearance probability of four miniature parameters separately using one-dimensional kernel density analysis. The geometric/topographic features of the appearance probability were calculated using a kernel density analysis. Let X1, X2,…, Xn denote a sample of size n from real observations. The kernel density estimate of P at the point x is given by the following equation:
where K is a smooth function called the Gaussian kernel function and h > 0 is the smoothing bandwidth that controls the amount of smoothing. We chose Silverman’s reference bandwidth or Silverman’s rule of thumb [71,72], which is given by the following equation:
h = 0.9 An−1/5,
where A = min (standard deviation, interquartile range/1.34). By normalizing the integral value in untrained controls, we identified the distribution of the appearance probability at any point. Subsequently, we calculated the appearance probability at selected points. All data points for probability in untrained and trained rats were converted to self-entropy (bits) using the Shannon entropy concept, as defined in the Information Theory .
To perform calculations using the spreadsheet software, the data for four miniature parameters were summarized in four different sheets, and we obtained the bandwidth (h) of individual parameters in the untrained group using the following formula: [= 0.9 STDEV (neuron 1, neuron 2,… neuron N) / COUNT (neuron 1, neuron 2,… neuron N) ^ (1/5)]. Then, using the data from the untrained group, we calculated the distribution of the appearance probability as follows:
- The probability distribution of the first datum for a parameter (neuron 1) was calculated using the formula [= EXP (−(((data of neuron 1 − any point) / h) ^ 2 / 2)) / SQRT (2 × PI())].
- Moreover, the probability distribution of the second datum for the parameter (neuron 2) was calculated using the formula [= EXP (−(((data of neuron 2 − any point) / h)^2 / 2)) / SQRT(2 × PI())].
- Similarly, the probability distribution of the N datum for the parameter (neuron N) was calculated using the formula [= EXP (−(((data of neuron N − any point) / h)^2 / 2)) / SQRT(2 × PI())].
- All probability distributions from neurons 1 to N were summed, and the integral value was normalized to 1.
Based on the probability distribution, we calculated the individual appearance probability of all recorded neurons. Subsequently, the appearance probability of the neuron was converted to the self-entropy using Shannon’s formula (= −LOG [appearance probability of the neuron, 2]) (Fig. 3A, B). For graphic expression, the distribution was visualized two-dimensionally in the R software environment (R Foundation for Statistical Computing, Vienna, Austria) (Figs 2B, C and 3A, B).
Behavioral test battery
Behavioral tests were performed in the following sequence: open field, object recognition, social preference, social recognition task, social interaction, object-in-place task, light-dark box test, visual placing response test, hanging wire test, Y-maze spontaneous alternation, contextual fear conditioning, and flinch and jump. We used different sampling rats in social preference, social recognition task and social interaction, and different object in object recognition and object-in-place task. The between tests interval was at least 60 min. Rats were habituated to the testing room 30 min prior to testing and the apparatus was cleaned with 70% ethanol between each trial.
Visual placing response test
To evaluate the sense of sight, we administered the visual placing response test [74,75]. In this test, the rat was suspended by its tail and then lowered toward a black foam plate placed on the front side of its head, without any contact to the vibrissae. Normally, when the head of a rat is lowered to near the edge of the plastic plate, the animal turns its head and trunk and extends its forelimbs to place them on the plate. The success ratio was calibrated to whether the rat successfully placed its forelimbs on the plate .
Hanging wire test
To evaluate basic motor function, we administered the hanging wire test. Rats were placed on a meshed wire, and the wire was turned upside down. The latency was tracked from the beginning of the test until the rats fell to the ground.
Open field test
To evaluate emotional state and spontaneous locomotor activity, we used the open field test. The center area (diameter 36 cm) and the peripheral area (diameter 60 cm) of the gray circle floor were lined. After the audio-visual recording, we measured the time spent in the center area and the distance traveled over 5 min .
Flinch and jump test
To evaluate pain sensibility, we performed the flinch and jump test . Rats were placed individually in the fear-conditioning chamber. The conditioning chamber (length: 25 cm; width: 31 cm; height: 42 cm) was constructed of clear Plexiglass on the top, front, and back. The floor had 18 stainless steel bars (4 mm in diameter; 15-mm spacing), to deliver the scrambled shocks produced by a stimulator (LE100-26 Shocker, Panlab, Cornellà, Spain). After a 3-min period of habituation to the test box, shock titrations continued to increase in a stepwise manner (0.05 mA increments; range, 0.05–0.6 mA). In this way, the “flinch” and “jump” thresholds (in mA) were defined for each rat. The interval between shocks was 2 min, and each animal was tested only once at each intensity. The behavior of each rat was recorded through a front digital video recording camera. The “flinch” threshold was defined as the lowest shock intensity that elicited a detectable response. The “jump” threshold was defined as the lowest shock intensity that elicited the simultaneous removal of at least three paws (including both hind paws) from the grid . The “vocalization” threshold was defined as the lowest shock intensity that led to a detectable audible vocalization in response to shock stimuli.
Light–dark box test
To evaluate anxiety and the exploration of a novel environment, we administered the light–dark box test [79,80,81]. The light–dark box (length: 48 cm; width: 20 cm; height: 23 cm) was constructed of light and dark compartments separated by a sliding door (width: 7 cm; height: 8 cm). The rats were placed in the center of the dark box and allowed to explore for 5 min. Then, the sliding door was opened, and they explored both boxes for 5 min (Fig. 5G).
Social interaction test
To evaluate the social interaction with an unfamiliar partner (stranger; same strain, sex, and age), we used the social interaction test . Prior to the task, we habituated the rats to an empty open field arena (diameter: 45 cm; height: 45 cm) for 5 min. Five minutes later, the rats were placed in the center of the arena again. One minute later, we placed an unfamiliar partner in the center of the arena, to assess social interaction for 5 min (Fig. 5H). Sniffing behavior was defined as the animal directing its nose toward or touching the partner.
Social preference test
To evaluate social preference, we used a U-field two-choice box [83,84,85]. The U-field box consisted of two symmetrical rectangular fields that were defined by partitioning an open field (length: 45 cm; width: 45 cm; height: 45 cm) with a wall (length: 20 cm; height: 45 cm). Prior to the task, we habituated the rats to the U-field two-choice box containing two circular wire empty cages (8 cm in diameter) for 5 min. Five minutes later, the rat was placed in the center of the box again and allowed to freely explore a wire cage containing a social target (stranger; same strain, sex, and age) or an empty wire cage for 5 min (Fig. 5I).
To assess social approach, we measured the time spent touching the social target or empty wire cage in a test phase. Touching behavior was defined as the animal directing its nose toward or its forelimbs touching the wire cage. The apparatus was cleaned with 70% alcohol and air-dried prior to each trial.
Contextual fear conditioning
To evaluate the longer retention of contextual memory, we administered contextual fear conditioning using the conditioning chamber described above. Under audio-visual recording (IXY3, Canon Inc, Tokyo, Japan), rats were allowed to explore for 3 min. Then, as the aversive unconditioned stimulus, we delivered foot shocks three times (0.8 mA, 2 s duration). Subsequently, the rats were allowed to recover for 30 s in the conditioning chamber and returned to their home cage. Twenty-four hours later, the rats were again placed in the conditioning chamber, and spontaneous behaviors were monitored for a 5-min period. To assess conditioning, we measured the time spent freezing per every 30 s of the testing period. The time spent freezing in the chamber was considered the measure of contextual learning. Freezing behavior was defined as cessation of all but respiratory movements .
Object recognition task
In the habituation phase, rats were placed in the center of an empty open field box (length: 45 cm; width: 45 cm; height: 45 cm) and allowed to explore the box for 5 min. In the sample phase, we placed two identical objects in the box. We placed the rats in the center of the open field box again and allowed them to explore for 5 min. Five minutes after the sample phase, we exchanged one of the familiar objects with a new object. In the testing phase, we placed the rats in the center of the open field box and allowed them to explore for 5 min. The apparatus was cleaned with 70% alcohol and air-dried prior to the commencement of each trial for each rat. To assess novel object memory, we measured the time spent touching a novel/familiar object during the test phase. Touching behavior was defined as the animal directing its nose toward or its forelimbs touching the object. Any other touching behavior, such as resting against the object, was not considered as touching [9,86].
Prior to the task, the rats were habituated to the empty open field box for 5 min. In the sample phase, we placed four different objects (A, B, C, and D) in the corners of the arena, respectively. The rats were then placed in the center of the arena and allowed to explore for 5 min. During the 5-min delay period, all of the objects were cleaned with alcohol, to remove olfactory cues. In the test phase, the positions of two of the four objects were exchanged, and the rats were allowed to explore for 5 min. The time spent touching the exchanged objects was compared with the time spent touching the unexchanged objects. We chose the exchanged objects randomly.
Social recognition task
Prior to the task, the rats were habituated to the U-field two-choice box for 5 min. Then, in the sample phase, each rat was placed in the center of the box and allowed to explore an unfamiliar target (same strain, age, and sex) placed in one side. Five minutes later, the rat was placed in the box again and allowed to freely explore the same target (familiar) or a stranger (novel) for 5 min. To assess social recognition memory, we measured the time spent touching a familiar or novel social target in test phase (Fig.6D). Touching behavior was defined as the animal directing its nose toward or its forelimbs touching the object.
Y-maze spontaneous alternation
To evaluate spatial working memory, we used a Y-maze apparatus (Fig.6E, F). The maze consisted of three arms made of gray plastic joined in the middle to form a “Y” shape (MY-10, Shinfactory, Japan). The walls of the arms had an outside slope of 76° (12 cm high), allowing the rat to see distal spatial landmarks. There were no intermaze cues inside the arms.
Prior to the experiment, the rats were allowed to explore the maze for 5 min. Then, 24 h after the habituation, the rats were placed in one arm again and their spontaneous behavior was recorded for 5 min. By analyzing the number and sequence of arms entered, we calculated the score as the number of alternations divided by the total alternations .
We used unpaired t-tests to analyze the data for mEPSCs, mIPSCs, and self-entropy. Because the self-entropy data had large variations within the group, we performed log (1 + x) transformation prior to the analysis . To analyze the recognition memory tasks (object recognition, object-in-place, social preference, and social recognition tasks), we used paired t-tests to compare the time spent in touching novel and familiar targets in test phase. To analyze other behavioral tasks (hanging wire, visual, open field, pain threshold, light–dark box, social interaction, and Y-maze spontaneous alternation tests), we used a one-way factorial ANOVA in which the between-group factors were the individual development stages. Significance was set at P < 0.05.