Short-Term Preexposure to Novel Enriched Environment Augments Hippocampal Ripples in Urethane-Anesthetized Mice

Abstract

Learning and memory are affected by novel enriched environment, a condition where animals play and interact with a variety of toys and conspecifics. Exposure of animals to the novel enriched environments improves memory by altering neural plasticity during natural sleep, a process called memory consolidation. The hippocampus, a pivotal brain region for learning and memory, generates high-frequency oscillations called ripples during sleep, which is required for memory consolidation. Naturally occurring sleep shares characteristics in common with general anesthesia in terms of extracellular oscillations, guaranteeing anesthetized animals suitable to examine neural activity in a sleep-like state. However, it is poorly understood whether the preexposure of animals to the novel enriched environment modulates neural activity in the hippocampus under subsequent anesthesia. To ask this question, we allowed mice to freely explore the novel enriched environment or their standard environment, anesthetized them, and recorded local field potentials in the hippocampal CA1 area. We then compared the characteristics of hippocampal ripples between the two groups and found that the amplitude of ripples and the number of successive ripples were larger in the novel enriched environment group than in the standard environment group, suggesting that the afferent synaptic input from the CA3 area to the CA1 area was higher when the animals underwent the novel enriched environment. These results underscore the importance of prior experience that surpasses subsequent physical states from the neurophysiological point of view.

INTRODUCTION

Learning and memory are primarily, but not exclusively, supported by neural activity in the hippocampus and neocortex and dependent on prior experience, contributing to behavioral individuality and flexibility. This adaptive process necessitates short-term or long-term modifications at the molecular, cellular, circuit, and system-wide levels.¹⁾ To experimentally address an effect of prior experience on the subsequent adaptive process, researchers have often modified environments where animals are reared or exploring. For example, animals are allowed to behave with a variety of objects and toys and multiple conspecifics, facilitating complex movements and interactions. This empirical condition is called enriched environment (EE)^2–4); note that the genuine effects of EE-exposure is differentiated from those of facilitated motor activity.⁵⁾

At the network level, EE-exposure alters the function of inhibitory interneurons and lends changes in balance of excitation and inhibition (i.e., E/I balance), favoring increased neural activity.^6,7) In terms of behavioral electrophysiology, neural representation of spatial contexts in the hippocampus is selectively modified by EE-exposure.⁸⁾ Consistent with these studies, EE-exposure alters neural plasticity and improves spatial memory formation,^9–11) highlighting the significance of neural networks in the hippocampus and neocortex in flexible adaptation relying on prior experience.

In the course of memory formation, the neural ensemble activated during learning is believed to form strong connections, a process called consolidation.¹²⁾ In this consolidation stage usually during slow-wave sleep, hippocampal neurons activated during previous exploration are reactivated in a temporally compressed manner in ripples, high-frequency extracellular oscillations observed in the hippocampus.^13–24) Hippocampal ripples are repeatedly generated in a naturally sleeping rodent after it has been actively running in the EE.²⁵⁾

Naturally occurring sleep shares characteristics in common with general anesthesia to some extent in consciousness levels and extracellular oscillations.^26,27) Although the two stages can be clearly distinguished,²⁸⁾ the similarities between the two have allowed us to investigate neural activity in a slow-wave-sleep-like state using anesthetized animals.^26,27) However, it remains largely unknown whether and how preexposure to novel EE affects hippocampal ripples under subsequent general anesthesia.

To address this question, we allowed mice to undergo their standard environment (SE) or novel EE and anesthetized them by intraperitoneal injection of urethane. We then recorded the local field potentials (LFPs) in the hippocampal CA1 area and neocortex and compared the features of hippocampal ripples between the SE and EE groups.

MATERIALS AND METHODS

Ethical Approvals

Animal experiments were performed with the approval of the Animal Experiment Ethics Committee at the University of Tokyo (Approval Number: P4-4) and according to the University of Tokyo guidelines for the care and use of laboratory animals. These experimental protocols were carried out in accordance with the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities of the Academic Research Institutions (Ministry of Education, Culture, Sports, Science and Technology, Notice No. 71 of 2006), the Standards for Breeding and Housing of and Pain Alleviation for Experimental Animals (Ministry of the Environment, Notice No. 88 of 2006) and the Guidelines on the Method of Animal Disposal (Prime Minister’s Office, Notice No. 40 of 1995). While our experimental protocols have a mandate to humanely euthanize animals if they exhibit any signs of pain, prominent lethargy, and discomfort, such symptoms were not observed in any of the mice tested in this study. All efforts were made to minimize the animals’ suffering.

Animals

A total of 42 male 5- to 7-week-old ICR mice (Japan SLC, Japan) were reared and group-housed in a home cage (i.e., a cage measuring 14 × 20 × 10 cm) in the vivarium under conditions of controlled temperature and humidity (22 ± 1 °C, 55 ± 5%) and maintained on a 12 : 12-h light/dark cycle (lights on from 6:00 a.m. to 6:00 p.m.) with ad libitum access to food and water unless otherwise specified.

In Vivo Electrophysiology

On the recording day, a given mouse was randomly picked up and assigned to the SE or EE group (described below) in an alternating manner. The mouse was transferred from the vivarium to the experimental room for the first time and allowed to individually explore (i) another cage identical in shape and size to the home cage (i.e., SE group) without any toys inside or (ii) a larger cage measuring 28 × 40 × 25 cm in which several novel toys such as a running wheel, a cup, a file stand, and a bumpy box were placed (i.e., EE group) for 30 min before surgery and recording.²⁹⁾ The mice in the EE group moved around more actively than those in the SE group. After this preexposure, each mouse was anesthetized by intraperitoneal injection of 2.25 g/kg urethane.^29–33) Anesthesia was confirmed by the absence of paw and tail withdrawal from pinches, whisker movement, and eyeblink reflexes. The skin was subsequently removed from the head, and a metal head-holding plate was fixed to the skull.^34,35) A craniotomy (1.5 × 1.5 mm²) was performed, which was centered at 1.5 mm posterior to the bregma and 1.5 mm ventrolateral to the sagittal suture. The exposed cranial window was covered with 2.0% agar at a thickness of 2.0 mm. The surgery was completed 60–90 min after the confirmation of anesthesia.

LFPs were recorded from the dorsal hippocampal CA1 region and the neocortex right above the hippocampus using tungsten electrodes (UEWMGCSEKNNM, FHC, U.S.A.) coated with 9% (w/v) 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) solution (i.e., a crystalline powder of DiI dissolved in a cocktail solvent of acetone and methanol at a ratio of 1 : 1) for approximately 30 min. Neural signals were amplified using an AC amplifier (DAM80, World Precision Instruments (WPI), U.S.A.) and digitized at a sampling rate of 20 kHz using a digitizer (Digidata 1440A, Molecular Devices, U.S.A.) that was controlled by pCLAMP 10.3 software (Molecular Devices).^36–39) The experiments (including the surgery and measurements) were done from noon to 6 p.m. in the light period.

Histology

Following each experiment, the electrode was carefully withdrawn from the hippocampus and neocortex. No anesthetics were additionally administered to the mice before the following perfusion because they remained deeply anesthetized confirmed again by the absence of paw and tail withdrawal from pinches, whisker movement, and eyeblink reflexes. The anesthetized mice were transferred to another experimental room and transcardially perfused with 0.1 M phosphate-buffered saline (PBS) and 4% paraformaldehyde (in 0.1 M PBS), followed by overnight postfixation.^40–44) The brains were coronally sectioned at a thickness of 100 µm using a vibratome.^45–49) The sections were incubated with 0.2% NeuroTrace 435/455 blue fluorescent Nissl stain (N21479; Thermo Fisher Scientific, U.S.A.) and 0.3% Triton X-100 dissolved in 0.1 M PBS for 1.5–3 h. In some experiments, 0.3% Triton X-100 was replaced with 1 mg/mL digitonin (D0540, Tokyo Chemical Industry, Japan) to preserve more DiI signals.⁵⁰⁾ The location of LFP electrodes was visually detectable via DiI fluorescence and lesions. Fluorescence images were acquired using a confocal microscope (A1 HD25, Nikon, Japan) and were subsequently merged using ImageJ.⁵¹⁾ The experimental data were discarded if the LFP electrode tip location was out of the hippocampal CA1 area (including the stratum oriens, stratum pyramidale, and stratum radiatum) or unidentified.

Data Analysis

All data analyses were performed offline using custom-made MATLAB routines (R2021a, MathWorks, U.S.A.). The summarized data in the text are reported as the mean ± the standard error of the mean. The significance level was set at 0.05, and the null hypothesis was statistically rejected when p < 0.05 based on two-tailed tests unless otherwise specified.

To detect hippocampal ripples, LFP traces were first downsampled to 2 kHz by averaging and bandpass-filtered between 150 and 250 Hz. Ripples were detected at a threshold of 4 × standard deviation (S.D.) of the baseline noise in the filtered signals.⁵²⁾ The detected events were subsequently scrutinized by eye and manually rejected if the detection was erroneous. For each ripple event, the onset and offset were defined as times when the filtered signals were above and below the threshold for the first time, respectively. For a given ripple event, its duration and amplitude were determined as the difference between the onset and offset and the maximum of absolute values of bandpassed traces, respectively.

Raw neocortical LFP traces were downsampled to 2 kHz in the same manner as the hippocampal LFPs. The downsampled LFP traces were transformed into a frequency domain. The “peak frequency” of neocortical slow oscillations was defined as a frequency (between 2 and 4.5 Hz) that produced the maximum value of the oscillatory power.

RESULTS

After allowing mice to freely explore the SE or novel EE, we anesthetized them and recorded LFPs from the dorsal hippocampal CA1 area and neocortex right above the hippocampus of the anesthetized mice (n = 23 and 19 mice in the SE and EE group, respectively) (Fig. 1, Supplementary Fig. 1). We observed at least one ripple from 9 and 13 mice in the SE and EE, respectively, during the 30-min recording period (p = 0.61, χ² = 0.26, chi-squared test).

Fig. 1. Simultaneous Recording of Hippocampal and Neocortical LFPs in Urethane-Anesthetized Mice That Undergo Short-Term Preexposure to the Novel Enriched Environment

A, Photographs of the standard environment (SE, left) and novel enriched environment (EE, right) explored by mice prior to electrophysiological recordings. The cage for the novel EE is equipped with different kinds of toys and larger than the cage for the SE. B, LFP recordings from the hippocampus and neocortex of urethane-anesthetized mice after preexposure to the novel EE. C, Post-hoc histological verification of the sites (red) of DiI-coated recording electrodes in the hippocampus on the Nissl-stained section (blue). D, Representative traces of the hippocampal and neocortical LFPs. Triangles (teal) denote times when hippocampal ripples occur. Abbreviations: SE, standard environment; EE, enriched environment; LFP, local field potential; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate.

Extracellular slow oscillations in the neocortex are composed of Up and Down states. Since the slow transition between the two states characterizes the deep anesthesia and vice versa,⁵³⁾ we computed the peak frequency of neocortical slow oscillations in the SE and EE groups (see Materials and methods; Supplementary Fig. 2A). The peak frequency in the EE group did not significantly differ from that in the SE group (3.40 ± 0.15 Hz (SE) vs. 3.04 ± 0.14 Hz (EE), p = 0.17, U = 549.5, z = 1.38, Mann–Whitney U test, n = 23 (SE) and 19 (EE) mice; Supplementary Fig. 2B), suggesting almost the same depth of anesthesia in both groups. Moreover, the occurrence of hippocampal ripples was not significantly correlated with the depth of anesthesia in either group (r=−0.05, p = 0.82, t₂₁=−0.22, t-test for the correlation coefficient, n = 23 (SE); r = 0.17, p = 0.48, t₁₇ = 0.72, t-test for the correlation coefficient, n = 19; Supplementary Fig. 2C), indicating that the hippocampal ripples were frequently generated irrespective of anesthesia in any case. Furthermore, we investigated whether the hippocampal ripples were generated independently of the elapsed time. We did not find any significant temporal (e.g., increasing, decreasing, or zigzag) tendency in the ripple occurrence in either group (p = 0.053, χ² = 9.32 (SE), Friedman test; p = 0.47, χ² = 3.54 (SE), Friedman test; Supplementary Fig. 3), suggesting the random appearance of hippocampal ripples.

We found that hippocampal ripples occurred more frequently in the EE group than in the SE group (0.05 ± 0.07 Hz (SE) vs. 0.20 ± 0.16 Hz (EE), p = 2.5 × 10⁻³, U = 381, z − 3.02, Mann–Whitney U test, n = 23 (SE) and 19 (EE) mice; Fig. 2A). A similar result was replicated even when we excluded mice in which ripples were not observed during the recording period (0.13 ± 0.05 Hz (SE) vs. 0.29 ± 0.09 Hz (EE), p = 1.1 × 10⁻³, U = 54, z − 3.27, Mann–Whitney U test, n = 9 (SE) and 13 (EE) mice). Moreover, compared with the SE group, the ripples in the EE group were significantly larger (0.08 ± 0.03 mV (SE) vs. 0.12 ± 0.05 mV (EE), p = 0.038, U = 72, z=−2.07, Mann–Whitney U test, n = 9 (SE) and 13 (EE) mice; Fig. 2B). In contrast, the duration of ripples was not significantly different between the two groups (29.1 ± 6.2 ms (SE) vs. 32.6 ± 5.1 ms (EE), p = 0.12, U = 80, z=−1.54, Mann–Whitney U test, n = 9 (SE) and 13 (EE) mice; Fig. 2C). These results suggested that hippocampal ripples were augmented even under anesthesia after exposure to the novel EE.

Fig. 2. Properties of Individual Hippocampal Ripples in Urethane-Anesthetized Mice That Undergo Preexposure to the Novel Enriched Environment

A, Rates of ripple events in the SE (gray) and EE (red) groups (n = 23 and 19 mice in the SE and EE groups, respectively). B, Amplitude of hippocampal ripples in the two groups (n = 9 and 13 mice in the SE and EE groups, respectively). Each dot denotes the average amplitude calculated from all amplitudes of ripples recorded from a mouse. C, Duration of ripples in the two groups (n = 9 and 13 mice in the SE and EE groups, respectively). Each dot represents the average duration calculated from all durations of ripples recorded from a mouse. * p < 0.05, Mann–Whitney U test. Abbreviations: SE, standard environment; EE, enriched environment.

In addition to differences in the characteristics of individual ripples, we investigated how the individual ripples were associated with neighboring ripples. Based on inter-ripple intervals, we confirmed that ripples in the SE group occurred more intermittently than in the EE group (5.0 ± 17.1 s (SE) vs. 3.8 ± 15.8 s (EE), p = 7.2 × 10⁻²¹, D = 0.084, Kolmogorov–Smirnov test; Fig. 3A), raising the possibility that ripples in the EE group were generated in a bursty fashion. To verify this possibility, we detected a ripple event over 300 ms apart from neighboring ones and defined it as a sporadic event; the rest of the ripples were included in successive events.^29,54–56) We found significantly more successive ripples in the EE group, compared with the SE group (1302 events (successive, SE), 6625 events (successive, EE), 1328 events (sporadic, SE), 3526 events (sporadic, EE), p = 8.1 × 10⁻⁵⁰, χ² = 220.23, chi-squared test; Fig. 3B). These results suggest that prior experience of exploring the EE modulates not only the properties of individual ripples but also the collective dynamics of hippocampal ripples.

Fig. 3. Collective Behavior of Hippocampal Ripples in Urethane-Anesthetized Mice Preexposed to the Novel Enriched Environment

A, Inter-ripple intervals are shorter in the EE group (red) than in the SE group (black). B, Successive ripples occur more frequently in the EE group (pink) than in the SE group (gray); sporadic ripples occur less frequently in the EE group. Numerical values superimposed on the bar indicate the number of sporadic or successive ripples. Abbreviations: EE, enriched environment; SE, standard environment.

DISCUSSION

Here, we allowed mice to freely explore the SE or novel EE, after which we treated them intraperitoneally with urethane to anesthetize them. We recorded LFPs in the hippocampus of the anesthetized mice in the SE and EE groups and found that individual ripples occurred in the hippocampus more frequently in the EE group than in the SE group. Amplitude and duration of individual ripples were larger and longer in the EE group even under urethane-anesthesia. Moreover, there were more successive ripple events in the EE group than in the SE group.

In our experimental paradigm, we captured hippocampal ripples from 9 out of 23 mice (39%) in the SE group and from 13 out of 19 mice (68%) in the EE group; the success rate especially in the SE group is seemingly low. Based on the average ripple occurrence (approximately 0.1 Hz) in the SE group (Fig. 2A), we consider that our recording time of 30 min was long enough to capture at least one ripple. Another explanation for the moderate success rate should be the electrode location. Since each hippocampal ripple is composed of perisomatic fast inhibitory currents and synchronous population spikes,^57–60) indicating the ripple amplitude is not large enough to detect when the recording electrode is located far from the stratum pyramidale, or in the stratum oriens.^24,58) According to the post-hoc histological verification of the electrode tip (Fig. 1B, Supplementary Fig. 1), we did not reject the experimental data in which the tip was inside the stratum oriens (see Materials and methods), but practically, some electrodes were out of “hot spots,” where large-amplitude (i.e., detectable) ripples were generated. Moreover, more ripple candidates (including small-amplitude ripples) could be probed offline if our threshold for the ripple detection was lowered.

Drug pretreatment per se affects hippocampal ripples in vivo through various receptors.^61–63) For example, general anesthetics, widely used in animal experiments, act on various neurotransmitter systems.⁶⁴⁾ Among a variety of general anesthetics, urethane potentiates γ-aminobutyric acid (GABA) receptors⁶⁵⁾ and inhibits N-methyl-D-aspartate (NMDA) and α-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors⁶⁵⁾ and spontaneous glutamate release from presynaptic sites,⁶⁶⁾ leading to alteration of the E/I balance. The urethane-induced E/I imbalance modulates hippocampal ripples.⁶¹⁾ Urethane-treatment almost vanishes hippocampal ripples, but a few ripples are still generated under anesthesia,⁶¹⁾ consistent with our observation (Fig. 1).

In addition to the pharmacological intervention, preexisting physical states also modify hippocampal ripples in living animals.⁶⁷⁾ Nonpharmacological sleep deprivation increases ripple occurrence called ripple rebound during the subsequent slow-wave sleep periods.⁶³⁾ Moreover, chronic restraint stress depresses the occurrence and magnitude of hippocampal ripples, but this attenuation is rescued by physical exercise in a cage with running wheels.⁶⁸⁾ Surprisingly, we demonstrated that short-term preexposure to the novel EE increased the frequency of ripple events in urethane-anesthetized mice, although most hippocampal ripples are likely to be abolished under anesthesia. Chronic exposure of mice to the EE modulates sharp wave-ripple complexes under in vitro conditions,⁶⁹⁾ but few studies have explicitly reported an effect of short-term EE-exposure on hippocampal ripples; note that acute EE-exposure adds to ripples during subsequent recording in the awake state.²⁵⁾ Comparable with this previous literature,²⁵⁾ we observed frequent ripples in urethane-anesthetized mice that had experienced the novel EE. Regarding the cage for preexploration, we should note that the large cage for the EE group was not only enriched but also novel for animals during exploration, compared with the standard cage for the SE group. In this sense, we cannot necessarily attribute the difference in the ripple properties under anesthesia between the two groups solely to the environmental richness; the increased physical activity or hyperarousal in the novel EE may also be influential.

At the microscopic level, transient exposure to the novel EE may induce short-term plasticity in the hippocampal CA1 area and acutely modulate the hippocampal activity,²⁹⁾ overwhelming urethane-induced suppression of the hippocampal network activity⁶¹⁾; note again that the environmental novelty also modulates neural activity in the hippocampus through synaptic plasticity.⁷⁰⁾ Specifically, we found that the ripple amplitude was larger in mice that had previously explored the novel EE than ones had undergone the SE. Larger amplitude of ripples reflects higher synchronicity of neural activity in the hippocampus,^60,71) consistent with an intrinsic emergent property of the urethane-induced slow oscillations in the hippocampus and neocortex,⁷²⁾ both of which are considered to be independent of respiration.

We further observed more successive ripples in the EE group than in the SE group (Fig. 3B). Of the intrahippocampal and extrahippocampal sources of the main afferent projection to the CA1 area (i.e., the CA3 area and entorhinal cortex), the entorhinal cortex is believed to trigger successive ripples when animals are quietly awake, whereas it is dispensable for generating successive ripples during slow-wave sleep.^55,56) Since we recorded neural activity from mice under urethane-anesthesia that induced slow-wave-sleep-like activity,^26,27) the increased number of successive ripples in the novel EE condition may mirror modifications in the afferent synaptic inputs from the CA3 subregion, rather than the medial entorhinal cortex, to the CA1 subregion through the Schaffer collaterals. From the functional viewpoints, the CA3 area contributes to spatial representation in the CA1 area, spatial navigation and memory compared to the entorhinal cortex, whereas the entorhinal cortex conveys relatively sensory and multimodal (i.e., nonspatial) information to the CA1 area directly and indirectly via the temporoammonic pathway and the trisynaptic circuit, respectively.^73–75) In this regard, our results suggest ‘mild’ consolidation of spatial memory even under anesthesia. Nevertheless, we have not found direct evidence for novel EE-induced changes in synaptic inputs from the CA3 area. In vivo measurements of membrane potentials such as whole-cell recordings and voltage dye imaging simultaneously with LFPs will provide us with the answer.⁷⁶⁾

Acknowledgments

The authors sincerely thank Hiroyuki Mizuno, Asako Noguchi, and Miki Nakashima for their technical assistance. This work was supported by JSPS Grants-in-Aid for Scientific Research (18H05525) and Grants from JST ERATO (JPMJER1801) and Institute for AI and Beyond of the University of Tokyo to Y. Ikegaya; JSPS Grants-in-Aid for Scientific Research (20K15926) and Grants from Konica Minolta Science and Technology Foundation (Konica Minolta Imaging Science Encouragement Award), the Public Foundation of Chubu Science and Technology Center, Hachiro Honjo Ocha Foundation, the Pharmacological Research Foundation, Tokyo, KOSÉ Cosmetology Research Foundation, and Yamazaki Spice Promotion Foundation to N. Matsumoto.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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