Altered Expression of Astrocytic ATP Channels and Ectonucleotidases in the Cerebral Cortex and Hippocampus of Chronic Social Defeat Stress-Susceptible BALB/c Mice

Abstract

The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5′nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.

INTRODUCTION

Some, but not all, subjects exposed to psychological, physical and/or social stress transiently or for a certain period of time, develop mood disorders such as depressive disorder, adjustment disorder, bipolar disorder, etc., and the number of patients with mood disorders has been increasing year-by-year worldwide. In addition to distress and a decrease of QOL of patients with mood disorders, their social disability is a serious socio-economic problem, and thus development of preventive and eradicable therapeutic strategies is an urgent issue, to attain this, elucidation of causable mechanisms for stress susceptibility and/or resiliency being necessary.

Patients with depressive disorder are well-known to exhibit lost or reduced sociability and motivation, anxiety, etc., resulting in social disability. As a mechanism underlying development of depressive phenotypes, dysfunction of the hypothalamic-pituitary axis is widely accepted recently.¹⁾ In this scenario, psychophysical stress induces gut inflammation with dysbiosis, and the derived inflammatory factors such as cytokines, stress-related hormones, etc. cause neuroinflammation via microglial activation in the brain, resulting in development of depressive disorder.^2–4)

It is noted that ATP derived from astrocytes is a molecule responsible for development of depressive-like behavior^5–7) via microglial activation.^8,9) ATP is released from astrocytes via ATP channels such as connexin 43, pannexin-1, calcium homeostasis modulator 2 (CALHM2), P2X7 receptors and maxi anion channels,^10–12) in addition to lysosome exocytosis.¹³⁾ As for alteration of ATP release under stress-exposed conditions, there are controversial reports that the extracellular ATP levels in hippocampus and cortex were increased^8,14) or decreased.^{6,10,15–20)} Regarding this discrepancy, Iwata et al.⁸⁾ indicate that just after stress-exposure, ATP release from astrocytes is increased, and then extracellular ATP is immediately degraded by ectonucleotidases, resulting in a decrease in its extracellular level. Ectonucleotidases are expressed by astrocytes, in which ecto-nucleoside triphosphate diphosphohydrase 2 (E-NTPD2), ecto-nucleoside pyrophosphate/phosphodiesterase 1 (E-NPP1), E-NPP2 and ecto-5′nucleotidase (E-NT) are functionally expressed^21,22) and metabolize ATP into nucleosides such as adenosine, which also plays roles in regulation of neuronal activity,^23–26) and then the nucleosides were taken up mainly into astrocytes via nucleoside transporters.²⁷⁾ Judging from these findings, functional expression of ATP channels and ectonucleotidases in astrocytes have crucial roles in fine tuning of extracellular ATP levels in the brain, suggesting that their imbalance might be involved in development of depressive phenotypes. However, to our limited knowledge, whether or not expression of astrocytic ATP channels and ectonucleotidases is altered on development of depressive phenotypes remains to be unveiled.

Here, we investigated expression of ATP channels and ectonucleotidases in astrocytes derived from cerebral cortices and hippocampi of mice with a chronic social defeat stress (CSDS)-induced decrease of sociability.

MATERIALS AND METHODS

Animals

Six-week-old male BALB/cCrSlc (BALB/c, total 35 mice) and 8-weeks-old male ICR (total 50 mice) mice purchased from Japan SLC (Hamamatsu, Japan) were individually housed in cages with food (AIN-93G for BALB/c mice and MF for ICR mice) and water ad libitum, and were acclimated to housing environments (22 ± 1 °C with 12/12 h light/dark cycle) in a specific pathogen-free facility for 1 week before initiation of experiments. All experiments were performed strictly according to ARRIVE guidelines and were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University (authorization number: DEB-20-001, 2020-2024). The number of mice were kept to the minimum necessary for meaningful interpretation of the data, and animal discomfort was minimized. Among 36 BALB/c mice, 7 were excluded due to abnormal body weight gain during the acclimation period, and thus the control and CSDS groups consisted of 12 and 16 mice, respectively.

CSDS Exposure

Following the protocol reported previously,^28,29) a BALB/c mouse was defeated by introducing it to and keeping it in the home cage of a resident aggressor ICR mouse for 10 min daily for 10 consecutive days (days 0–10), and then was housed in the transparent perforated plastic divider-separated compartment of the home cage of the ICR mouse during the rest period (Fig. 1a). During the 10-d CSDS period, a mouse in the CSDS group was euthanized ethically on day 7 by deep anesthesia with isoflurane because of a 25 or more % decrease in body weight.

Fig. 1. Induction of Depressive-Like Behavior in CSDS-Susceptible Mice

Panel a shows the social defeat procedures for the CSDS protocols. (b) After 10-d exposure of mice to CSDS, their sociability was determined by SI tests. The body weight (c), food consumption (d), and water intake (e) of mice were measured daily, and body weight change is given as % of day 0. Data are shown as means ± S.D. In both the control and susceptible groups, the number of mice was 11. *, ** and *** p < 0.05, 0.01 and 0.001 significantly different from the value in the control group. (b) Student’s t-test gave t = 5.60 and p < 0.001. Repeated measures of two-way ANOVA gave (c) a significant main effect of stress at F_(1,20) = 205 and p < 0.001, no significant main effect of day at F_(9,180) = 1.87 and p = 0.0581, and a significant interaction between stress and day at F_(9,180) = 3.93 and p < 0.001, (d) a significant main effect of stress at F_(1,20) = 4.08 and p = 0.0448, a significant main effect of day at F_(9,180) = 3.24 and p = 0.0011, and a significant interaction between stress and day at F_(9,180) = 2.84 and p = 0.0036, and (e) a significant main effect of stress at F_(1,20) = 144 and p < 0.0001, a significant main effect of day at F_(9,180) = 3.00 and p = 0.0022, and a significant interaction between stress and day at F_(9,180) = 2.30 and p = 0.0177.

Social Interaction (SI) Test

To evaluate development of depressive-like behavior in CSDS-subjected mice, their sociability was determined by SI testing on day 10 (Fig. 1a) as reported previously,^28,29) and SI ratios, as an index of sociability of mice, were calculated as 100 × (the time spent in the interaction zone in the presence of a social target (ICR mouse))/(the time spent in the interaction zone in the absence of a social target), CSDS-subjected mice with SI ratios of <100% being defined as CSDS-susceptible mice. In the control and CSDS groups, 1 and 2 mice, respectively, were excluded from the following experiments because their SI ratios could not be determined due to no movement in the arena during the entire test period.

Sorting of Astrocytes

Just after the SI test on day 10, mice were transcardially perfused with saline under deep anesthesia with intraperitoneal injection of a mixture of medetomidine (0.75 mg/kg), midazolam (4 mg/kg), and butophanol (5 mg/kg), and then their brains were obtained (Fig. 1a). Cerebral cortices and hippocampi were dissected out in ice-cold phosphate buffered saline containing 2 mM ethylenediaminetetraacetic acid, and then were treated with 0.2 mg/mL collagenase and 2.5 µg/mL deoxyribonuclease in RPMI (0.5 mL/a tissue) at 37 °C for 30 min, followed by pipetting dissociation to obtain single cell suspensions. After filtration using a cell strainer with 70 µm pores, the filtrates were centrifuged at 300 × g for 10 min at 4 °C and the supernatants were discarded. Sorting of astrocytes was performed using an Anti-ACSA-2 MicroBead Kit (130-097-678, Miltenyi Biotec B. V. & Co. KG, Bergisch Gladbach, Germany) based on magnetic activated cell sorting (MACS) technology. According to the manufacturer’s protocol, the suspension (10⁷ cells/80 µL) was treated sequentially with 10 µL FcR Blocking Reagent for 10 min and 10 µL Anti-ACSA-2 Microbeads for 15 min, and then was centrifuged at 300 × g for 10 min at 4 °C, followed by suspension with 500 µL RPMI. The suspension was applied onto the MACS Column in the magnetic field, and the column was washed with 3 mL RPMI 3-times to remove unlabeled cells. After the column was removed from the magnetic field, ACSA2-positive astrocytes were obtained by flushing the column with 1 mL RPMI. Pan and Wan demonstrated that the purity of astrocytes in the MACS-sorted ACSA2-positive cells was high enough to be defined as isolated astrocytes despite negligible contamination by some endothelial and oligodendrocyte cell types.³⁰⁾

Real-Time PCR

Extraction, reverse transcription of total RNA and real-time PCR of cDNA obtained from MACS-sorted astrocytes were performed as reported previously.³¹⁾ The target genes focused on in this study were Gja1 (connexin 43), Panx1 (pannexin-1), Calhm2 (CALHM2), P2rx7 (P2X7 receptor), and Slco2a1 (maxi anion channel) as ATP channels,^10–12) and Entpd2 (E-NTPD2), Enpp1 (E-NPP1), Enpp2 (E-NPP2), and Nt5e (E-NT) as ectonucleotidases,^21,22) all of which were reported to be expressed by astrocytes. Their primer sets were as follows: Gja1: forward, 5′-TGCTTCCTCTCACGTCCCAC-3′, reverse, 5′-CGCGATCCTTAACGCCCTTG-3′; Panx1: forward, 5′-TGACCCCATGCTACTCCTGA-3′, reverse, 5′-CTTGGTCTGTAGGGACGTGG-3′; Calhm2: forward, 5′-TTCAAGAGCAAGGATGTGATG-3′, reverse, 5′-CAGTCCATACAGGTAGTTCCG-3′; P2rx7: forward, 5′-ACACCGTGCTTACAGGTGCTA-3′, reverse, 5′-GCAACAGCTGGGCAGAATGG; Slco2a1: forward, 5′-CCGCTCGGTCTTCAACAACA-3′, reverse, 5′-AAGAACTGGAGAGCCCAAAGC-3′; Entpd2: forward, 5′-AGACAGATATGCCAGCACTC-3′, reverse, 5′-GGCACCACGGAAGTCAAAGG-3′; Enpp1: forward, 5′-GAATGGCGTCAATGTTGTCAG-3′, reverse, 5′-GACTGCGGATGACTCTGGTG-3′; Enpp2: forward, 5′-CCACTACTACAGCATCATCACCAG-3′, reverse, 5′-GATGAAAGAAGACACAGAGAGA-3′; and Nt5e: forward, 5′-TCAGAAAGTTCGAGGTGTGGA-3′, reverse, 5′-GTCCATCATCTGCGGTGACTA-3′. Following the manufacturer’s protocol for a KAPA SYBR FAST qPCR Master Mix (2X) kit (Kapa Biosystems, Wilmington, MA, U.S.A.), PCR amplification was performed under the cycling conditions of 94 °C for 3 min, and 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 1 s. The expression levels of target genes were normalized as to the corresponding levels of mRNA for Actb (forward, 5′-CCTGAGGAGCACCCTG-3′, reverse, 5′-TCCGGAGTCCATCACA-3′).

Statistical Analysis

All data are given as means ± standard deviation (S.D.). The statistical significance in Figs. 1b, 2a, b, 3 and Figs. 1c–e was evaluated by Student’s t-test and two-way repeated measures ANOVA followed by Tukey–Kramer’s post hoc multiple comparison test, respectively, using Bellcurve for Excel (version 3.21; Social Survey Research Information Co., Ltd., Tokyo, Japan), p-values of 0.05 or less being considered to be statistically significant.

RESULTS

Induction of Depressive-Like Behavior

In the CSDS group, the number of CSDS-susceptible mice was 11, and their SI ratio (39.1 ± 30.0%) was significantly lower than that of control mice (138.8 ± 50.8%, N = 11) (Fig. 1b). The body weight change of CSDS-susceptible mice was significantly lower than in the case of control mice (F_(1,20) = 205, p < 0.001, Fig. 1c), and this decrease of body weight gain was partially due to a decrease of daily food consumption (F_(1,20) = 4.08, p = 0.0448, Fig. 1d). The increased daily water intake in CSDS-susceptible mice (Fig. 1e) was similar to the finding in our previous study,²⁹⁾ and was considered to be due to stress-induced over-hydration.

These findings indicated that CSDS-susceptible mice exhibited disabled sociability as a depressive-like behavior, and because of only limited amounts of cDNAs obtained from ACSA2-positive astrocytes sorted from their cerebral cortices and hippocampi, a total 11 mice in both the control and CSDS-susceptible groups were divided into 6 and 5, and 5 and 6 mice for determination of mRNA expression of ATP channels and ectonucleotidases, respectively. As for the CSDS-resilient mice with SI ratios of 100 or more, their number was just 2, of which the SI ratios were 125 and 101 (average: 113). These two CSDS-resilient mice were subjected to mRNA expression analyses on ATP channels, but were not included in the comparison between experimental groups.

Expression of ATP Channels and Ectonucleotidases

As shown in Figs. 2a and b, cortical astrocytes of CSDS-susceptible mice exhibited significantly greater expression of mRNAs for Gja1, P2rx7 and Slco2a1 than those of control ones, while mRNA expression of Panx1 in hippocampal astrocytes of CSDS-susceptible mice was significantly lower than in those of control ones. As to the astrocytic expression of ATP channels in control, and CSDS-resilient and -susceptible mice, the expression levels of mRNAs for Gja1 and P2rx7 in cerebral cortex were correlated inversely with the corresponding SI ratios (Figs. 2c, d).

Fig. 2. Expression of mRNAs for ATP Channels in Astrocytes Isolated from Cerebral Cortex and Hippocampus of Control and CSDS-Susceptible Mice

After 10-d exposure of mice to CSDS, mRNA expression of Gja1 (connexin 43), Panx1 (pannexin 1), Calhm2 (Calhm 2), P2rx7 (P2X7R), and Slco2a1 (maxi anion channel) was determined in astrocytes isolated from cerebral cortex (a) and hippocampus (b) by real-time PCR. Each column represents the mean ± S.D. for 6 and 5 mice in the control and susceptible groups, respectively. *, ** and *** p < 0.05, 0.01 and 0.001 significantly different from the value in the control group. Student’s t-test gave t = −7.59 and p < 0.001 (Gja1 in cerebral cortex), t = −5.74 and p < 0.001 (P2rx7 in cerebral cortex), t = −5.35 and p = 0.0031 (Slco2a1 in cerebral cortex), and t = 2.84 and p = 0.0251 (Panx1 in hippocampus). Panels c and d show correlation of mouse sociability with expression levels of mRNAs for Gja1 and P2rx7, respectively, in astrocytes isolated from cerebral cortex of control (n = 6), and CSDS-resilient (n = 2) and -susceptible (n = 5) mice. Their correlation was found to be significant by a simple linear regression analysis, r² and p values being shown in each panel.

As for ectonucleotidases (Fig. 3), in astrocytes of cerebral cortex, there was lower and greater expression of mRNAs for Endpd2 and Nt5e, respectively, in CSDS-susceptible mice than in control ones, and hippocampal astrocytes of CSDS-susceptible mice exhibited lower and greater expression of mRNAs for Enpp2 and Nt5e, respectively, than those of control ones.

Fig. 3. Expression of mRNAs for Ectonucleotidases in Astrocytes Isolated from Cerebral Cortex and Hippocampus of Control and CSDS-Susceptible Mice

After 10-d exposure of mice to CSDS, mRNA expression of Entpd2 (E-NTPD2), Enpp1 (E-NPP1), Enpp2 (E-NPP2), and Nt5e (E-NT) was determined in astrocytes isolated from cerebral cortex (a) and hippocampus (b) by real-time PCR. Each column represents the mean ± S.D. for 5 and 6 mice in the control and susceptible groups, respectively. * and ** p < 0.05 and 0.01 significantly different from the value in the control group. Student’s t-test gave t = 4.29 and p = 0.0020 (Entpd2 in cerebral cortex), t = 3.96 and p = 0.0033 (Nt5e in cerebral cortex), t = 2.86 and p = 0.0188 (Enpp2 in hippocampus), and t = 3.06 and p = 0.0136 (Nt5e in hippocampus).

DISCUSSION

In this study, we demonstrated that the SI ratios, as an index of depressive-like behavior, significantly correlated inversely with astrocytic expression of mRNAs for connexin 1 and P2X7 receptors in cerebral cortex. Therefore, it is suggested that astrocytic ATP channels in cerebral cortex might be potential targets for development of therapeutic/preventive approach for depressive disorder.

It is known that astrocytes play crucial roles in release of ATP into extracellular space via ATP channels in addition to exocytotic ATP release, and expression levels of ATP channels regulate extracellular ATP levels.¹⁰⁾ We found the strict inverse correlation between mouse sociability and the expression level of connexin 43 in cortical astrocytes. Connexin 43 is highly expressed by astrocytes, through which released ATP is a major gliotransmitter in the neuron-glia interaction,^32,33) and also plays a critical role in development of depressive-like behavior via P2X7 receptor-mediated microglial activation.^8,9) P2X7 receptor is also expressed by astrocytes and its expression in cortical astrocytes of CSDS-susceptible mice was inversely correlated with mouse sociability. Under pathological conditions, astrocytic P2X7 receptors play critical roles in release of ATP and proinflammatory cytokines, resulting in the exacerbation of neuronal injury.^34–36) Thus, P2X7 receptor expressed by microglia and astrocytes is a potential therapeutic target for depressive disorder. In addition, blockade of astrocytic connexin 43 is also suggested to be an attractive therapeutic approach for depressive disorder. Based on the recent literature, however, the function and expression of connexin 43 in astrocytes increases and decreases in the early and prolonged phases of depression, respectively, and in a chronic unpredictable stress model, the former phase is up to 2 weeks and the latter one is 5 or more weeks.^13,17,37,38) Lu et al. demonstrate that in medial prefrontal cortex (mPFC) under stressed conditions, astrocyte activation, which stimulates ATP release via ATP channels, depends on activation of glucocorticoid receptors, and its activity is increased and decreased in the early and chronic phases, respectively, of stress exposure.¹³⁾ Although cerebral cortex used in this study included mPFC and thus, their findings not being directly comparable to ours, we suggested that astrocytic expression of connexin 43 and P2X7 receptors in the cerebral cortex might be increased in an early phase of depression, implying the necessity of a disease phase-specific therapeutic approach for ATP channels expressed by cortical astrocytes.

As for astrocytic ATP catabolism, under physiological pH, E-NTPDs and E-NTs are dominant ectonucleotidases,²¹⁾ and hydrolyze ATP to ADP/AMP and AMP to adenosine, respectively. In cortical astrocytes of CSDS-susceptible mice, expression of E-NTPD2 and E-NT was decreased and increased, respectively. Together with increased expression of ATP channels, decreased expression of E-NTPD2 in cortical astrocytes of CSDS-susceptible mice might contribute to increased extracellular ATP levels. Furthermore, as in the case of P2X7 receptors, overfunction of neuronal adenosine A_2A receptors is considered to be involved in mood dysfunction,²²⁾ and thus elevation of the extracellular adenosine level in the cortex through the increased expression of astrocytic E-NT might also play roles in development of depressive-phenotypes and might be relevant for an effective therapeutic approach for depressive disorder.

In hippocampal astrocytes of CSDS-susceptible mice, on the other hand, pannexin 1 expression was decreased, and expression of E-NPP2, which hydrolyzes ATP to AMP, and E-NT was decreased and increased, respectively, suggesting that a decrease of the extracellular ATP level in the hippocampus may be involved in development of depressive-like behavior.

Based on our findings on astrocytic expression of ATP channels and ectonucleotidases, the estimated alteration profile of extracellular ATP in hippocampi is opposite that in cerebral cortices, but we have no reasonable explanation for this. Recent accumulating evidence demonstrates that PFC in cerebral cortex plays crucial roles in regulation of cognition, motivation and emotion, and its dysfunction is implicated in neuropsychiatric disorders including depressive disorder.³⁹⁾ It is known that information on contexts and environmental conditions is input into PFC,^40,41) is integrated with information memorized in other brain regions, and then provides instructions for context-specific responses of different subcortical regions including the lateral habenula, dorsal raphe nucleus, etc., which assess the response information referring with memories in other brain regions such as the hippocampus, etc. for output for adoptive responses.^42–45) The hippocampus is a key brain region responsible for advanced functions such as learning, memory and emotion, and sends nerve afferents to the frontal cortex, amygdala, and other emotion-related brain areas, and its dysfunction is also implicated in development of depressive disorders.⁴⁶⁾ Overall, we think that it is reasonable that the effects of psychosocial stress exposure are different between the cerebral cortex and hippocampus, and purinergic regulation of activity of the cerebral cortex including PFC might play a primary role in control of flexible behavior. In addition to confirm protein levels of the altered expression of mRNAs found in this study, in future, we will examine the regulatory mechanisms underlying extracellular ATP levels in astrocytes in mPFC of CSDS-susceptible mice.

CONCLUSION

In this study, we demonstrated that in CSDS-subjected mice, expression of mRNAs for connexin 43 and P2X7 receptors as ATP channels in cortical astrocytes was correlated inversely with mouse sociability, while there was no significant correlation between them in hippocampal astrocytes. Together with the findings obtained in the previous studies, in addition to the disease stage, the target brain region should be taken into account for the development of a preventive and therapeutic strategy for depressive disorder.

Acknowledgments

We thank N. J. Halewood for the helpful and critical proofreading of this manuscript.

Funding

A part of this study was financially supported by a Grant-in-aid from the Japan Society for the Promotion of Science (JSPS), KAKEN 22K06590 (K. N.).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Evrensel A, Unsalver BO, Ceylan ME. Neuroinflammation, gut-brain axis and depression. Psychiatry Investig., 17, 2–8 (2020).
• 2) Eyre H, Baune BT. Neuroimmunological effects of physical exercise in depression. Brain Behav. Immun., 26, 251–266 (2012).
• 3) Franco R, Fernandez-Suarez D. Alternatively activated microglia and macrophages in the central nervous system. Prog. Neurobiol., 131, 65–86 (2015).
• 4) Iwata M, Ota KT, Duman RS. The inflammasome: pathways linking psychological stress, depression, and systemic illnesses. Brain Behav. Immun., 31, 105–114 (2013).
• 5) Cao X, Li LP, Wang Q, Wu Q, Hu HH, Zhang M, Fang YY, Zhang J, Li SJ, Xiong WC, Yan HC, Gao YB, Liu JH, Li XW, Sun LR, Zeng YN, Zhu XH, Gao TM. Astrocyte-derived ATP modulates depressive-like behaviors. Nat. Med., 19, 773–777 (2013).
• 6) Ma J, Qi X, Yang C, Pan R, Wang S, Wu J, Huang L, Chen H, Cheng J, Wu R, Liao Y, Mao L, Wang FC, Wu Z, An JX, Wang Y, Zhang X, Zhang C, Yuan Z. Calhm2 governs astrocytic ATP releasing in the development of depression-like behaviors. Mol. Psychiatry, 23, 883–891 (2018).
• 7) Xiong W, Cao X, Zeng Y, Qin X, Zhu M, Ren J, Wu Z, Huang Q, Zhang Y, Wang M, Chen L, Turecki G, Mechawar N, Chen W, Yi G, Zhu X. Astrocytic epoxyeicosatrienoic acid signaling in the medial prefrontal cortex modulates depressive-like behaviors. J. Neurosci., 39, 4606–4623 (2019).
• 8) Iwata M, Ota KT, Li XY, Sakaue F, Li N, Dutheil S, Banasr M, Duric V, Yamanashi T, Kaneko K, Rasmussen K, Glasebrook A, Koester A, Song D, Jones KA, Zorn S, Smagin G, Duman RS. Psychological stress activates the inflammasome via release of adenosine triphosphate and stimulation of the purinergic type 2X7 receptor. Biol. Psychiatry, 80, 12–22 (2016).
• 9) Yue N, Huang H, Zhu X, Han Q, Wang Y, Li B, Liu Q, Wu G, Zhang Y, Yu J. Activation of P2X7 receptor and NLRP3 inflammasome assembly in hippocampal glial cells mediates chronic stress-induced depressive-like behaviors. J. Neuroinflammation, 14, 102 (2017).
• 10) Illes P, Verkhratsky A, Tang Y. Pathological ATPergic signaling in major depression and bipolar disorder. Front. Mol. Neurosci., 12, 331 (2020).
• 11) Karasawa A, Michalski K, Mikhelzon P, Kawate T. The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition. eLife, 6, e31186 (2017).
• 12) Taruno A. ATP release channels. Int. J. Mol. Sci., 19, 808 (2018).
• 13) Lu CL, Ren J, Mo JW, Fan J, Guo F, Chen LY, Wen YL, Li SJ, Fang YY, Wu ZF, Li YL, Gao TM, Cao X. Glucocorticoid receptor-dependent astrocytes mediate stress vulnerability. Biol. Psychiatry, 92, 204–215 (2022).
• 14) Dias L, Pochmann D, Lemos C, Silva HB, Real JI, Goncalves FQ, Rial D, Goncalves N, Simoes AP, Ferreira SG, Agostinho P, Cunha RA, Tome AR. Increased synaptic ATP release and CD73-mediated formation of extracellular adenosine in the control of behavioral and electrophysiological modifications caused by chronic stress. ACS Chem. Neurosci., 14, 1299–1309 (2023).
• 15) Cai W, Xue C, Sakaguchi M, Konishi M, Shirazian A, Ferris HA, Li ME, Yu R, Kleinridders A, Pothos EN, Kahn CR. Insulin regulates astrocyte gliotransmission and modulates behavior. J. Clin. Invest., 128, 2914–2926 (2018).
• 16) Cui QQ, Hu ZL, Hu YL, Chen X, Wang J, Mao L, Lu XJ, Ni M, Chen JG, Wang F. Hippocampal CD39/ENTPD1 promotes mouse depression-like behavior through hydrolyzing extracellular ATP. EMBO Rep., 21, e47857 (2020).
• 17) Huang D, Li C, Zhang W, Qin J, Jiang W, Hu C. Dysfunction of astrocytic connexins 30 and 43 in the medial prefrontal cortex and hippocampus mediates depressive-like behaviours. Behav. Brain Res., 372, 111950 (2019).
• 18) Kuang XJ, Zhang CY, Yan BY, Cai WZ, Lu CL, Xie LJ, Li SJ, Kong PL, Fan J, Pan SM, Guo T, Cao X. P2X2 receptors in pyramidal neurons are critical for regulating vulnerability to chronic stress. Theranostics, 12, 3703–3718 (2022).
• 19) Lin S, Huang L, Luo ZC, Li X, Jin SY, Du ZJ, Wu DY, Xiong WC, Huang L, Luo ZY, Song YL, Wang Q, Liu XW, Ma RJ, Wang ML, Ren CR, Yang JM, Gao TM. The ATP level in the medial prefrontal cortex regulates depressive-like behavior via the medial prefrontal cortex-lateral habenula pathway. Biol. Psychiatry, 92, 179–192 (2022).
• 20) Ni M, He JG, Zhou HY, Lu XJ, Hu YL, Mao L, Wang F, Chen JG, Hu ZL. Pannexin-1 channel dysfunction in the medial prefrontal cortex mediates depressive-like behaviors induced by chronic social defeat stress and administration of mefloquine in mice. Neuropharmacology, 137, 256–267 (2018).
• 21) Langer D, Hammer K, Koszalka P, Schrader J, Robson S, Zimmermann H. Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res., 334, 199–217 (2008).
• 22) Zimmermann H. History of ectonucleotidases and their role in purinergic signaling. Biochem. Pharmacol., 187, 114322 (2021).
• 23) Agostinho P, Madeira D, Dias L, Simoes AP, Cunha RA, Canas PM. Purinergic signaling orchestrating neuron-glia communication. Pharmacol. Res., 162, 105253 (2020).
• 24) Cunha RA. Regulation of the ecto-nucleotidase pathway in rat hippocampal nerve terminals. Neurochem. Res., 26, 979–991 (2001).
• 25) Zimmermann H, Zebisch M, Strater N. Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal., 8, 437–502 (2012).
• 26) IJzerman AP, Jacobson KA, Muller CE, Cronstein BN, Cunha RA. International union of basic and clinical pharmacology. CXII: adenosine receptors: a further update. Pharmacol. Rev., 74, 340–372 (2022).
• 27) Nagai K, Nagasawa K, Fujimoto S. Transport mechanisms for adenosine and uridine in primary-cultured rat cortical neurons and astrocytes. Biochem. Biophys. Res. Commun., 334, 1343–1350 (2005).
• 28) Kamimura Y, Kuwagaki E, Hamano S, Kobayashi M, Yamada Y, Takahata Y, Yoshimoto W, Morimoto H, Yasukawa T, Uozumi Y, Nagasawa K. Reproducible induction of depressive-like behavior in C57BL/6J mice exposed to chronic social defeat stress with a modified sensory contact protocol. Life Sci., 282, 119821 (2021).
• 29) Takahata Y, Yoshimoto W, Kuwagaki E, Yamada Y, Nagasawa K. Alteration of sweet taste receptor expression in circumvallate papillae of mice with decreased sweet taste preference induced by social defeat stress. J. Nutr. Biochem., 107, 109055 (2022).
• 30) Pan J, Wan J. Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain. J. Immunol. Methods, 486, 112834 (2020).
• 31) Iwamura M, Honda R, Nagasawa K. Elevation of the blood glucose level is involved in an increase in expression of sweet taste receptors in taste buds of rat circumvallate papillae. Nutrients, 12, 990 (2020).
• 32) Chever O, Lee CY, Rouach N. Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission. J. Neurosci., 34, 11228–11232 (2014).
• 33) Roux L, Madar A, Lacroix MM, Yi C, Benchenane K, Giaume C. Astroglial connexin 43 hemichannels modulate olfactory bulb slow oscillations. J. Neurosci., 35, 15339–15352 (2015).
• 34) Anderson CM, Bergher JP, Swanson RA. ATP-induced ATP release from astrocytes. J. Neurochem., 88, 246–256 (2004).
• 35) Franke H, Gunther A, Grosche J, Schmidt R, Rossner S, Reinhardt R, Faber-Zuschratter H, Schneider D, Illes P. P2X7 receptor expression after ischemia in the cerebral cortex of rats. J. Neuropathol. Exp. Neurol., 63, 686–699 (2004).
• 36) Lovatt D, Sonnewald U, Waagepetersen HS, Schousboe A, He W, Lin JH, Han X, Takano T, Wang S, Sim FJ, Goldman SA, Nedergaard M. The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex. J. Neurosci., 27, 12255–12266 (2007).
• 37) Guo J, Qiu T, Wang L, Shi L, Ai M, Xia Z, Peng Z, Zheng A, Li X, Kuang L. Microglia loss and astrocyte activation cause dynamic changes in hippocampal [¹⁸F]DPA-714 uptake in mouse models of depression. Front. Cell. Neurosci., 16, 802192 (2022).
• 38) Sun JD, Liu Y, Yuan YH, Li J, Chen NH. Gap junction dysfunction in the prefrontal cortex induces depressive-like behaviors in rats. Neuropsychopharmacology, 37, 1305–1320 (2012).
• 39) Pizzagalli DA, Roberts AC. Prefrontal cortex and depression. Neuropsychopharmacology, 47, 225–246 (2022).
• 40) Sánchez-Bellot C, AlSubaie R, Mishchanchuk K, Wee RWS, MacAskill AF. Two opposing hippocampus to prefrontal cortex pathways for the control of approach and avoidance behaviour. Nat. Commun., 13, 339 (2022).
• 41) Spellman T, Rigotti M, Ahmari SE, Fusi S, Gogos JA, Gordon JA. Hippocampal-prefrontal input supports spatial encoding in working memory. Nature, 522, 309–314 (2015).
• 42) Baker PM, Oh SE, Kidder KS, Mizumori SJ. Ongoing behavioral state information signaled in the lateral habenula guides choice flexibility in freely moving rats. Front. Behav. Neurosci., 9, 295 (2015).
• 43) Baker PM, Raynor SA, Francis NT, Mizumori SJ. Lateral habenula integration of proactive and retroactive information mediates behavioral flexibility. Neuroscience, 345, 89–98 (2017).
• 44) Kawai T, Yamada H, Sato N, Takada M, Matsumoto M. Roles of the lateral habenula and anterior cingulate cortex in negative outcome monitoring and behavioral adjustment in nonhuman primates. Neuron, 88, 792–804 (2015).
• 45) Warden MR, Selimbeyoglu A, Mirzabekov JJ, Lo M, Thompson KR, Kim SY, Adhikari A, Tye KM, Frank LM, Deisseroth K. A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature, 492, 428–432 (2012).
• 46) Battaglia FP, Benchenane K, Sirota A, Pennartz CM, Wiener SI. The hippocampus: hub of brain network communication for memory. Trends Cogn. Sci., 15, 310–318 (2011).