In the present study, we first confirmed that ENR rearing enhances hippocampal gamma oscillation power in mice. We then investigated whether this gamma oscillation enhancement is dependent on astrocytic Ca 2+ signalling by using IP 3 R2‐KO mice. We found that hippocampal gamma oscillations of IP 3 R2‐KO are also larger in ENR than in ISO. Although IP 3 R2 deficiency did not display an obvious phenotype in gamma oscillations, IP 3 R2‐KO mice had impacts in the occurrence and magnitude of sharp wave‐associated ripple events that occur during non‐theta periods.

Similar to all other parts of the brain, the hippocampus is populated with neurons and glia. Amongst glial cell types, astrocytes have been demonstrated to play an enabling role in synaptic plasticity in vitro (Yang et al . 2003 ; Henneberger et al . 2010 ) and in vivo (Takata et al . 2011 ; Chen et al . 2012 ; Navarrete et al . 2012 ; Monai et al . 2016 ) in the cortex and hippocampus. In these studies, astrocytic Ca 2+ elevation has been suggested to be important to mediate synaptic plasticity. Large cytosolic Ca 2+ elevations have indeed been observed in rodent hippocampal and cortical astrocytes in vivo (Stosiek et al . 2003 ; Hirase et al . 2004 ; Kuga et al . 2011 ). The pathway involving inositol trisphosphate receptor type 2 (IP 3 R2), the principal IP 3 receptor in astrocytes, has been recognized as the principal mechanism for astrocytic Ca 2+ elevations because IP 3 R2‐knockout (KO) mice display much compromised cytosolic Ca 2+ elevations in astrocytes (Petravicz et al . 2008 ; Takata et al . 2011 ). The involvement of IP 3 R2‐mediated Ca 2+ signalling in synaptic plasticity and learning, however, remains controversial (Agulhon et al . 2010 ; Petravicz et al . 2014 ).

Enriched environment (ENR) rearing is known to enhance cognitive and memory abilities. In the rodent brain, ENR has been demonstrated to enhance neurogenesis and induce morphological complexity of neurons (van Praag et al . 2000 ; Hirase & Shinohara, 2014 ). Many of these changes have been reported in the hippocampus, which is known to be critical for spatial navigation and episodic memory formation (Andersen et al . 2006 ). The hippocampus operates in distinct network states characterized by distinct local field potential (LFP) patterns. For example, theta and gamma oscillations appear during locomotion, vigilance and rapid‐eye‐movement (REM) sleep, whereas sharp wave‐associated ripples appear during awake immobility and slow wave sleep (Buzsáki, 2002 , 2015 ). These LFP states also appear in urethane anaesthesia, whereby theta and non‐theta states spontaneously alternate, resembling sleep states. We have previously shown that ENR rearing after weaning increases the theta‐associated gamma oscillation power in the hippocampal CA1 than isolated rearing (ISO) in rats (Shinohara et al . 2013 ). This experience‐dependent enhancement of gamma oscillations is NMDA receptor‐dependent and is expressed most prominently in the stratum radiatum , where axons from bilateral CA3 pyramidal cells make synaptic connections.

In a set of experiments, electrodes were coated with DiI and electrode tracks were visualized by fluorescence. After electrophysiological recording, the mouse brain was fixed by transcardial perfusion with 4% paraformaldehyde in 0.1% phosphate buffer after the animal was deeply anaesthetized by additional i.p. injection of urethane (1.5 g kg −1 ). Following post‐fixation (overnight or longer in the fixative), the fixed brain was sliced by a microtome (Pro‐7; Dosaka, Kyoto, Japan) with the thickness set to 60 μm and the sections were mounted on a slide glass. The slide was semi‐dried and sealed with a coverslip using Vectashield mounting medium (H‐1200; Vector Laboratories, Burlingame, CA, USA) as the mounting medium. Sections were examined within 2 days using a standard fluorescence microscope (BX‐60; Olympus, Tokyo, Japan).

For analyses of ripple events, LFP signals in the stratum pyramidale were first resampled to 20 kHz. Then, ripple events were detected automatically. The LFP was band‐pass filtered for the ripple frequency band (80–250 Hz) and the resultant signal was squared and then smoothed with a Hamming window of length 19.2 ms. In the first screening, ripples events were detected as the periods where the smoothed signal exceeds the mean value by seven times the SD with an inter‐ripple interval of 100 ms. In the second screening, the local minima within ± 35 ms of each detected point (i.e. ripple trough) was assigned as the ripple timing and 200 ms ripple‐filtered waveform centered around the ripple timing was extracted for further analyses. After automatic detection, detected ripples were manually confirmed by the coincidence of a sharp wave in the stratum radiatum using NeuroScope (Hazan et al . 2006 ) that marked the detected ripple timings on LFP traces. The peak ripple amplitude was defined as the amplitude of the largest trough of each extracted ripple event and expressed in absolute value. The root‐mean‐square (RMS) amplitude was computed for the ± 25 ms interval from the ripple peak. Cumulative inter‐ripple‐interval distributions were calculated by binning inter‐ripple intervals of less than 20 s into bins of 100 ms width. Ripple occurrence frequency was computed for non‐theta periods, which are complementary to the detected theta‐periods for gamma analyses. Detected times ripples are compared between the left and right hemispheres, and the ripples observed within ± 35 ms on both hemispheres are termed as ‘co‐ripples’.

Data analyses were carried out using MATLAB (MathWorks Inc., Natick, MA, USA). For analyses of theta‐associated gamma oscillations, we employed the same procedure as reported previously (Shinohara et al . 2013 ). Briefly, raw LFP data were resampled to 1.25 kHz and theta periods were automatically detected by computing the spectrogram and finding the periods that satisfy two criteria: (i) the ratio of the peak powers of the theta band (3.5–7 Hz) and the delta (2–3 Hz) band in each bin exceeds 0.6 and (ii) the length of the period is at least 15 s long. The detection was carried out on the LFP recorded from the right CA1 stratum radiatum (150 μm below the stratum pyramidale). Next, the power spectral density was estimated by the Welch periodogram method for the detected theta periods. Theta, and low‐ and high‐gamma powers were calculated by integrating the power spectral densities for 3–6, 30–45 and 55–90 Hz, respectively. The spectral powers were averaged per animal over multiple recording sessions.

LFP recording was performed as reported previously (Sakatani et al . 2007 ; Shinohara et al . 2013 ). Briefly, mice were anaesthetized with an i.p. injection of urethane (1.2–1.4 g kg −1 ) and fixed in a stereotaxic frame. The depth of anaesthesia was determined by movement of the animal when ear bars were inserted and additional urethane was injected if necessary. A craniotomy was performed above each side of the dorsal hippocampus (Bregma: mediolaterial 1.8 mm, anteroposterior −1.8 mm). The dura was surgically removed and a 16‐channel linear silicon probe (inter‐channel distance = 50 μm; Alx15‐5 mim‐50‐177‐A16; NeuroNexus, Ann Arbor, MI, USA) was slowly inserted to the hippocampal CA1 so that the middle channel was located in the stratum pyramidale. A melted mixture of paraffin and paraffin oil was cooled down to ∼35°C (Mishima et al . 2007 ) and applied on the skull and craniotomy to maintain the moisture and temperature of the brain surface. Wideband (0.1–9000 Hz) extracellular field potentials were recorded continuously with a sampling rate of 31 kHz (Digital Lynx; Neuralynx, Bozeman, MT, USA). The body temperature was kept at ∼37°C throughout the surgery and recording sessions by a heat pad with rectal temperature feedback. Mice were supplemented with 0.2 ml of 5% glucose every hour (i.p.).

A , schedule of experiment. B , photographs for the ENR (upper) and ISO (lower) used in the current experiment. C , typical example of PCR‐based genotyping for WT and IP 3 R2‐KO mice. Middle: heterozygous IP 3 R2‐KO mouse (not used in the present study). D , body weight of mice before LFP recording in each combination of genotype (WT, IP 3 R2‐KO) and rearing environment (ENR, ISO). E , schematics of bilateral LFP recording from hippocampal CA1. Below: example of histological verification of electrode location for each hippocampal hemisphere. Inset: magnified to indicate the silicon probe tip location in CA1. F, simultaneous LFP recording example traces from the CA1 strata pyramidale (purple) and radiatum (navy). Statistics for spontaneously occurring theta periods are computed for single theta period ( G ), proportion of theta periods over all recording time ( H ), frequency of theta state ( I ). * P < 0.05, ** P < 0.01.

Adult IP 3 R2‐KO mice (Futatsugi et al . 2005 ) (background strain: C57BL/6J) and littermates (wild‐type; WT) were used. Genotyping was made by PCR using a mixture of three primers (5′‐ to 3′): B2: AGAGACACGATGTCCCCAATGTAG; Z2: GATGTGCTGCAAGGCGATTAAG; F2: CCAGGAACAGGAAACCTACTTCTG. A 237 bp band (B2–F2) is amplified in the WT allele, whereas a 311 bp band (Z2–F2) is amplified in the IP 3 R2‐KO allele. After weaning (postnatal 20 days), mice were subjected to either ISO or ENR rearing for the following 6–7 weeks (Fig. 1 A ). In the ISO condition, mice were kept singly in a standard cage (length 30 cm, width 20 cm, height 18 cm) without further enrichment. In the ENR condition, three to five mice were kept in a 3D‐enhanced cage (length 32 cm, width 23 cm, height 25 cm) with a ladder, two running wheels, and tunnels (Fig. 1 B ). In addition, one novel object with different texture, shape or colour was replaced with a familiar object in the ENR cage every 2 days, with its location changed on the second day. In either ISO or ENR condition, animals were given access to food and water ad libitum . Body weight was measured before conducting the electrophysiological recording.

The procedures involving animal care, surgery and sample preparation were approved by the Animal Experimental Committee of RIKEN Brain Science Institute [Animal: H27‐2‐230(6); DNA: 2016‐038(1)] and were performed in accordance with the guidelines of the Animal Experimental Committee of RIKEN Brain Science Institute. All efforts were made to minimize the animals’ pain and suffering and to reduce the number of animals used in the study. We understand the ethical principles under which The Journal of Physiology operates and our work complies with the journal's animal ethics checklist (Grundy, 2015 ).

A , scatter plot to show a positively correlated relationship between ripple RMS amplitude and ripple peak amplitude in a WT‐ENR mouse. B , contour plots representing the distributions of ripple RMS amplitude vs . ripple peak amplitude for WT‐ENR, WT‐ISO, IP 3 R2‐KO‐ENR and IP 3 R2‐KO‐ISO groups. C , box plots showing individual variabilities of ripple RMS amplitude. Dashed lines represent the range for upper and lower quartile. The median of each distribution is marked by the horizontal line in the box. D and E , RMS amplitude is separately analysed for co‐ripples ( D ) and unilaterally significant ripples ( E ). * P < 0.05.

Examination of the extracted ripple waveforms suggested variable profiles (Fig. 3 A ). We computed two measures that represent the magnitude of a ripple: the peak ripple amplitude and the RMS amplitude. We found that they are positively correlated with correlation coefficients of >0.8 in all groups (WT‐ENR = 0.85; WT‐ISO = 0.80; IP 3 R2‐KO‐ENR = 0.82; IP 3 R2‐KO‐ISO = 0.85) (Fig. 4 A and B ). Distributions of RMS ripple amplitude for all mice are shown in box plots in Fig. 4 C . We investigated ripple amplitudes and whether their variability is affected by the environmental or genetic factors. We compared ripple amplitudes by taking the median of RMS amplitude values as the representative value for each mouse. RMS amplitudes were plotted separately for co‐ripples and unilaterally significant ripples for the right hemisphere in Fig. 4 D and E . Interestingly, the RMS ripple amplitude was affected by the genetic deletion of IP 3 R2 (co‐ripples: P = 0.082, unilaterally significant ripples: P = 0.035). Of note, neither rearing environment, nor genotype had significant effects in RMS ripple amplitudes in the left hemisphere. To address the variability of ripple amplitudes, the coefficient of variation was calculated for each mouse. Accordingly, we found that neither rearing environment, nor genotype had a significant effect on the variability of ripple amplitude.

Most hippocampal ripples were observed synchronously in both hemispheres, satisfying ripple detection criteria on each side (‘co‐ripples’; see Methods). Occasionally, ripples on the contralateral side did not satisfy the detection criteria even though high‐frequency oscillations were discernible. We refer to these ripple events as unilaterally significant ripples. The proportion of co‐ripple occurrence over all detected ripple events on the right hemisphere was 0.70, 0.74, 0.73 and 0.76 for WT‐ENR, WT‐ISO, IP 3 R2‐KO‐ENR and IP 3 R2‐KO‐ISO, respectively. Similar values were calculated for the left hemisphere. Two‐way ANOVA analysis showed that there is a significant effect for rearing environment (right: P = 0.042; left: P = 0.0005) (Fig. 3 D ), whereas there is a weak effect for genotype (right: P = 0.25; left: P = 0.099). These analyses showed that ripples are probably more bilaterally detectable in ISO mice. Conversely, the likelihood of unilaterally significant ripple occurrence was significantly higher in WT‐ENR mice (right: 6.30 ± 0.46 events min –1 ) than in IP 3 R2‐KO‐ISO (3.88 ± 0.45 events min –1 , Tukey–Kramer post hoc test, P = 0.04245) (Fig. 3 E ). We have varied the ripple detection threshold from 6 SD to 8 SD and found that ripple occurrence and magnitude have similar statistical tendency as the default threshold of 7 SD (see Supporting information, Fig. S1).

A , example CA1 stratum pyramidale LFP traces during non‐theta periods from a WT‐ENR mouse (left) and a IP 3 R2‐KO‐ISO mouse (right) from the right (upper) and left (lower) hemispheres. Bandpass filtered traces (80–250 Hz) for ripple detection is displayed in the middle. Detected ripples are highlighted in the filtered trace and coloured according to the detected hemisphere (right: red, left: blue). The ripple waveforms are extracted are magnified in the vicinity. B , cumulative plot for inter‐ripple intervals for WT‐ENR (red), WT‐ISO (brown), IP 3 R2‐KO‐ENR (blue) and IP 3 R2‐KO‐ISO (green). C , comparison of inter‐ripple‐interval of 75% quartile. D , proportion of bilaterally detectable co‐ripples are compared. E , occurrence of unilaterally significant ripples is increased in WT‐ENR compared to IP 3 R2‐KO‐ISO. * P < 0.05, ** P < 0.01.

Large irregular activities are observed in CA1 stratum radiatum LFPs during non‐theta periods. Transient ripple oscillations appear in the stratum pyramidale as sharp waves occur in the stratum radiatum (Fig. 1 F ). We next investigated whether ripple occurrence and magnitude are influenced by rearing environment or by IP 3 R2. As shown in Fig. 3 A , ripples appear multiple times during 5 s of non‐theta periods in WT and IP 3 R2‐KO mice. To examine ripple occurrence, we plotted the group‐averaged inter‐ripple interval cumulative distribution for each combination of the rearing environment and genotype (Fig. 3 B ). Strikingly, the distribution for IP 3 R2‐KO‐ISO was shifted to the right, indicating a relative abundance of longer inter‐ripple intervals. Indeed, the upper quartile (75th percentile) value of the inter‐ripple‐intervals was significantly affected by both the genotype and rearing environment (Genotype: F = 4.50, P = 0.008; Environment: F = 3.10, P = 0.0239, for ripples detected in the right CA1) (Fig. 3 C ) and IP 3 R2‐KO‐ISO mice had significantly higher upper quartile inter‐ripple interval values than WT‐ENR mice (Tukey–Kramer post hoc test, P = 0.005749).

Next, the same analyses were performed for IP 3 R2‐KO mice (Fig. 2 F and G ). We find that slow gamma power also tended to increase by ENR rearing on both sides. Group comparison by two‐way ANOVA for slow gamma power on the right side shows that the main effect is the rearing environment (Environment: F = 6.35, P = 0.018; Genotype: F = 0.07, P = 0.7984) (Fig. 2 G ). A similar outcome was computed for the left‐side slow gamma power (Environment: F = 4.98, P = 0.0342; genotype: F = 0.21, P = 0.6472) (Fig. 2 G ). Rearing condition‐induced fast gamma power changes were not remarkable in IP 3 R2‐KO mice, resembling WT. These results suggest that slow gamma oscillations have higher power (i.e. magnitude) in ENR mice than in ISO mice regardless of IP 3 R2.

A , example CA1 stratum radiatum LFP traces during theta periods from three individual WT‐ENR mice (upper, purple) and three WT‐ISO (lower, brown) mice. B , power spectral densities of CA1 stratum radiatum LFP of WT‐ENR and WT‐ISO mice. Shaded areas represent the frequency ranges for slow (35–45 Hz) and fast (55–90 Hz) gamma oscillations. C , interhemispheric coherence spectrum of CA1 stratum radiatum LFP. D and E , slow and fast gamma powers are compared in WT mice in the left (blue) and right (red) hemispheres. F and G , gamma power comparisons in IP 3 R2‐KO mice. H , comparison of slow gamma power by rearing condition in the left and right CA1 stratum radiatum. * P < 0.05.

We first analysed gamma oscillations during theta states. As shown by the example traces in Fig. 2 A , stratum radiatum theta LFPs from WT‐ENR mice encompass gamma oscillations. In WT‐ISO mice, gamma oscillations appear to be diminished. Slow and fast gamma oscillation (30–45 Hz and 55–90 Hz, respectively) power was computed by spectral analyses. Comparison of the mean power spectral density between WT‐ENR and WT‐ISO mice showed general increases of spectral power around the slow gamma range (Fig. 2 B ) in WT‐ENR mice. On the other hand, the interhemispheric gamma coherence was low (<0.2) and was not significantly affected by rearing condition (Fig. 2 C ). Spectral powers of WT mice were compared between WT‐ENR and WT‐ISO for slow gamma and fast gamma (Fig. 2 D and E ). There was a general tendency that slow gamma power was elevated in ENR group in both hemispheres; however, the increase reached statistical significance only on the right side ( P = 0.0157). Fast gamma power did not show obvious changes depending on rearing condition in either hemisphere.

We then recorded LFPs from bilateral dorsal hippocampal CA1 with 16‐channel silicon probes (Fig. 1 E ) under anaesthesia. As reported previously (Wolansky et al . 2006 ), the hippocampal LFP spontaneously alternated between theta and non‐theta patterns (Fig. 1 F ). Theta states were automatically detected by an algorithm based on the theta/delta power ratio of stratum radiatum LFP (see Methods). The average duration of a theta state was similar across genotypes and rearing environments (Genotype: F = 3.56, P = 0.07; Environment: F = 1.63, P = 0.21; Interaction: F = 1.06, P = 0.31) (Fig. 1 G ), although there was a trend for longer theta state duration in IP 3 R2‐KO‐ISO ( P = 0.152, post‐hoc Tukey–Kramer test against WT‐ENR) (Fig. 1 G ). The proportion of theta states also showed similar tendencies across genotypes and rearing environments as single theta state duration (Genotype: F = 1.98, P = 0.17; Environment: F = 0.87, P = 0.36; Interaction: F = 3.05, P = 0.09) (Fig. 1 H ). On the other hand, the frequencies of LFP state transition in all groups were similar ( P > 0.4 for post‐hoc Tukey–Kramer tests of all combinations) (Fig. 1 I ).

To investigate whether rearing environment affects neuronal activity, we reared mice in two distinct rearing environments for 6–7 weeks after weaning: isolated (ISO) and enriched (ENR) environments (Fig. 1 A and B ). In addition to WT mice, we performed experiments with IP 3 R2‐KO mice to examine whether astrocytic IP 3 /Ca 2+ signalling plays a role in rearing experience‐dependent neural activity changes (Fig. 1 C ). The numbers of mice used in the present study are: WT‐ENR, n = 9; WT‐ISO, n = 9; IP 3 R2‐KO‐ENR, n = 7; and IP 3 R2‐KO‐ISO, n = 6. Body weight was significantly larger in ENR mice regardless of IP 3 R2 deficiency (Genotype: F = 0.75, P = 0.39; Environment: F = 24.28, P < 0.0001) (Fig. 1 C ).

Discussion

ENR rearing has been reported to enhance cognitive and memory abilities. In rodents, it has been demonstrated that ENR rearing enhances learning and memory (Nithianantharajah & Hannan, 2006), as well as facilitates hippocampal LTP and neuronal excitability (Malik & Chattarji, 2012). In the present study, we first aimed to confirm the relative increase of hippocampal gamma power in ENR mice compared to that in ISO mice, which we have previously reported in rats (Shinohara et al. 2013). Second, we investigated whether astrocytic IP 3 /Ca2+ signalling is involved in such experience‐dependent LFP changes. Although urethane affects multiple neurotransmitter systems (Hara & Harris, 2002) and potentially elicits quasi‐natural oscillatory patterns, we performed LFP recordings under urethane anaesthesia because: (i) both synchronized (theta) and de‐synchronized (non‐theta) states are expressed during urethane anaesthesia, resembling slow wave and rapid‐eye‐movement sleep states, respectively (Wolansky et al. 2006; Clement et al. 2008); (ii) a silicon probe implant that lasts for 6 weeks from the onset of environmental exposure (i.e. at weaning) for chronic recording would be technically difficult; and (iii) identical recording conditions can be imposed for all mice.

The gamma oscillations that we investigated were recorded during theta states. Theta states are associated with medial septal cholinergic activity that projects to the hippocampus (Green & Arduini, 1954; King et al. 1998; Yoder & Pang, 2005). We did not observe statistically significant changes in the proportion of theta state periods by rearing condition or by IP 3 R2 deficiency, which is consistent with the study by Cao et al. (2013), who reported similar sleep patterns in WT and IP 3 R2 KO. A recent study by Foley et al. (2017) reported longer REM sleep in transgenic mice in which astrocytic IP 3 signalling is attenuated by overexpression of an IP 3 ‐degrading enzyme. Such an approach to block IP 3 signalling is potentially more powerful because it also interferes the signalling through IP 3 R1 and IP 3 R3 receptors. Indeed, sparse yet discernible IP 3 R3‐dependent astrocytic Ca2+ elevations have been reported recently (Sherwood et al. 2017). Nevertheless, we found trends for prolonged theta state period and larger portion of theta states over a recorded period for IP 3 R2‐KO‐ISO mice (Fig. 1G). These results lend credence to the notion that IP 3 R2 phenotypes are expressed in stressful conditions. Notably, abnormal REM sleep patterns, including a shorter latency from the onset of slow wave sleep and longer period, have been reported in depressed patients (Berger & Riemann, 1993). Given these connections, further investigation of astrocytic IP 3 /Ca2+ signalling may lead to potential therapeutic interventions for mood disorders caused by external environmental stress.

We found that slow gamma oscillation power is higher in ENR mice than in ISO mice (Fig. 2B), showing a similar tendency to our previous study with rats (Shinohara et al. 2013). Indeed, there is a broad enhancement of spectral power density over the slow gamma range in ENR mice compared to the power spectral density distribution of ISO mice. The higher gamma powers in ENR mice are consistent with previously reported associations between gamma oscillations and memory or cognitive abilities in rodents and humans (Sederberg et al. 2006; Tort et al. 2009; Lu et al. 2011; Igarashi et al. 2014; Staresina et al. 2016). We note that the relative increase of slow gamma power of ENR to that of ISO gamma power was more modest in mice (left: 33%; right: 44%) than in rats (left: 51%; right: 103%) (Shinohara et al. 2013). Recently, Valero‐Aracama et al. (2015) reported a critical time window of 40 days for ENR‐induced facilitation of dorsal CA1 neuronal excitability in C56BL/6 female mice. It is possible that the ENR rearing length used in the current experiment (6 weeks) was close to the end of this plastic window, and that some of the ENR‐induced (or ISO‐induced) circuit plasticity had already reverted by the time of recording.

We also noted that the interhemispheric coherence at gamma frequencies is considerably lower in mice than in rats. Furthermore, gamma coherence was generally low (<0.1) under the urethane‐anaesthetized condition. The low interhemispheric gamma coherence recorded during anaesthesia was similar between ISO and ENR mice. This result was in contrast to our previous rat experiments, in which marked gamma coherence increases were observed in a urethane‐anaesthetized condition (Shinohara et al. 2013). The low gamma coherence is probably related to the anaesthesia because highly coherent interhemispheric gamma activity has been reported in mice running in a wheel (Buzsaki et al. 2003). It is not clear how interhemispheric gamma desynchronization occurs in urethane‐anaesthetized mice, whereas urethane‐anesthetized rats, especially those raised in an enriched environment, display a significant gamma coherence (∼0.5) (Shinohara et al. 2013). Indeed, it appears to be counterintuitive because the relative sizes of both cortical and hippocampal commissures are larger in the mouse than the rat (Bishop & Wahlsten, 1999). The exact mechanism of urethane anaesthesia remains to be clarified; however, urethane has been known to enhance GABA A ‐, glycine‐ and nACh‐ receptor‐mediated currents and inhibit NMDA‐ and AMPA‐receptor‐mediated currents (Hara & Harris, 2002). Although we are unaware of a comprehensive transcriptome database that allows inter‐species comparison of receptors and channels in distinct brain regions, it is speculated that differential expression of these urethane‐sensitive receptors might help understand the gamma coupling differences.

As noted earlier, theta states are associated with cholinergic input to the hippocampus. Hippocampal astrocytes have been shown to respond to synaptically released acetylcholine with intracellular Ca2+ elevations (Araque et al. 2002; Navarrete et al. 2012; Pabst et al. 2016). Prior studies have shown cholinergically activated astrocytes play a role in glutamatergic synaptic plasticity (Takata et al. 2011; Chen et al. 2012; Navarrete et al. 2012; Papouin et al. 2017). The presence of ENR‐dependent gamma enhancement in IP 3 R2‐KO mice suggests that astrocytic IP 3 /Ca2+ signalling is not an essential component of this modulation. The results of the present study, however, do not exclude the possibility that residual Ca2+ astrocytic activities observable in IP 3 R2‐KO mice (Kanemaru et al. 2014; Srinivasan et al. 2015; Agarwal et al. 2017; Stobart et al. 2017), albeit with reduced occurrence, have a role.