Abstract
Mitochondria are abundant in the fine processes of astrocytes, however, potential roles for astrocyte mitochondria remain poorly understood. In the present study, we performed a systematic examination of the effects of abnormal oxidative phosphorylation in astrocytes on several mouse behaviors. Impaired astrocyte oxidative phosphorylation was produced by astrocyte-specific deletion of the nuclear mitochondrial gene, Cox10, that encodes an accessory protein of complex IV, the protoheme:heme-O-farnesyl transferase. As expected, conditional deletion of the Cox10 gene in mice (cKO mice) significantly reduced expression of COX10 and Cytochrome c oxidase subunit I (MTCO1) of Complex IV, resulting in decreased oxidative phosphorylation without significantly affecting glycolysis. No effects of the deletion were observed on locomotor activity, anxiety-like behavior, nociception, or spontaneous alternation. Cox10 cKO female mice exhibited mildly impaired novel object recognition, while Cox10 cKO male mice were moderately deficient in trace fear conditioning. No group-related changes were observed in conditional place preference (CPP) that assessed effects of morphine on reward. In contrast to CPP, Cox10 cKO mice demonstrated significantly increased aversive behaviors produced by naloxone-precipitated withdrawal following chronic exposure to morphine, that is, jumping and avoidance behavior as assessed by conditional place aversion (CPA). Our study suggests that astrocyte oxidative phosphorylation may contribute to behaviors associated with greater cognitive load and/or aversive and stressful conditions.
Keywords: astrocytes, fear conditioning, mitochondria, morphine withdrawal, naloxone
1 |. INTRODUCTION
Recent physiological investigations demonstrate the presence of mitochondria in the fine processes of astrocytes (Agarwal et al., 2017), however, potential roles for astrocyte mitochondria remain poorly understood (Rose et al., 2017, 2020).
While it is well established that glycolysis is utilized by astrocytes for metabolic support of neuronal activity, the role of astrocytic mitochondria in metabolic support of neurons remains understudied (Agarwal et al., 2017). Prior work showed that impaired oxidative phosphorylation in astrocyte mitochondria had no significant effects on astrocyte morphology, physiology, or survival (Supplie et al., 2017). However, no systematic evaluation of the behavioral consequences of manipulating astrocyte oxidative phosphorylation in mice has been performed. Thus, we assessed the contribution of astrocyte oxidative phosphorylation to a variety of mouse behaviors by deleting cytochrome c oxidase (COX) or Complex IV selectively in astrocytes.
COX is the terminal enzyme of the respiratory chain that, which catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen (Shoubridge, 2001). We deleted the Cox10 gene that encodes for one of the accessory proteins of Complex IV, the protoheme:heme-O-farnesyl transferase, which is involved in the biosynthesis of the heme a responsible for properly keeping the prosthetic group within complex IV (Shoubridge, 2001). We found that astrocyte-specific deletion of Cox10 and resultant deficiency of Complex IV and oxidative phosphorylation attenuated novel object recognition and trace fear conditioning. However, it had no effects on conditional place preference (CPP). In contrast to CPP, selective deletion of Cox10 in astrocytes exacerbated naloxone-precipitated morphine withdrawal. The findings suggest that astrocyte oxidative phosphorylation may contribute to behaviors associated with greater cognitive load and/or stressful conditions.
2 |. METHODS
2.1 |. Animals
To evaluate how oxidative phosphorylation contributes to mouse behaviors, we selectively deleted the Cox10 gene in astrocytes by crossing Aldh1l1-CreERT2 BAC transgenic mice (Srinivasan et al., 2016) with Cox10fl/fl mice. Conditional recombination in astrocytes was induced with intraperitoneal injection of tamoxifen (at the dose of 100 mg/kg of bodyweight daily for 3 days) in one-month-old mice. This approach initiates expression of Cre recombinase at high levels in most astrocytes throughout the brain, with no detectable expression in neurons (Srinivasan et al., 2016).
To study the behavioral, biochemical and metabolic effects of Cox10 deletion in astrocytes, we generated the following groups of male and female 12-week-old mice: heterozygous Cox10 cKO mice (Aldh1l1-Cre/ERT2::Cox10fl/+) and control mice that included a mixture of wild-type, Aldh1l1-Cre/ERT2 and Cox10fl/fl mice. All mice received tamoxifen injections. All mice were C57BL6/j background. Developing mice were housed with their dams until postnatal day 21, and all adult mice were housed in single sex cages (up to five mice per cage) with food and water ad libitum. All procedures were approved by the JHU Animal Care and Use Committee and followed the US National Research Council’s Guide for the Care and Use of Laboratory Animals, the US Public Health Service’s Policy on Humane Care and Use of Laboratory Animals, and Guide for the Care and Use of Laboratory Animals.
2.2 |. Tamoxifen injections
Tamoxifen (T5648, Sigma) was dissolved in sunflower seed oil (S5007, Sigma), and Aldh1l1-Cre/ERT2::Oprm1fl/+ and control mice were injected intraperitoneally (i.p.) with 3 doses of tamoxifen (100 mg/kg) for 3 consecutive days starting at P30.
2.3 |. Morphine injections
Morphine Sulfate CII (Cat: #1448005, USP) was dissolved in saline (Cat: #114-055-101, Quality Biological) for i.p. or subcutaneous (s.c.) injections. Animals received i.p. injections in the course of conditional place tests and s.c. injection in the course of analgesic tests. Naloxone was dissolved in normal saline and administrated by s.c. at conditional place aversion test. For all treatments, the dose volume was 10 ml/kg.
2.4 |. Behavioral testing
Four weeks following tamoxifen injection, mice were tested for locomotor activity in the open field test, anxiety-like behaviors in the elevated plus maze, and spontaneous alternation in the Y maze test followed by tests for learning and memory: novel object recognition test (NORT), novel place recognition test (NPRT) and trace fear conditioning (TFC) tests. All tests were performed as previously described (Jouroukhin et al., 2018; Jouroukhin et al., 2019; Pletnikov et al., 2008; Shevelkin et al., 2020).
2.5 |. Conditional place preference (CPP)
Separate cohorts of control and Cox10 cKO 12-week-old mice were tested in CPP as previously described (Li et al., 2002) (Figure S1). Each experiment was carried out for 12 consecutive days and was divided into three phases: preconditioning, conditioning and postconditioning tests. During the preconditioning test, the initial preference of the animals to one of two compartments connected by a bridge was tested. Mice were placed in the bridge connecting the two compartments, which had different floor textures and wall patterns. Animals were able to move freely to explore both compartments for 15 min each day for three consecutive days. The time spent in each compartment was recorded by Any Maze software (Stoelting). The compartment in which the mouse spent less time by the third day was designated as the less preferred side and was chosen to be paired with morphine during the conditioning phase. During the conditioning phase (8 days), the animals were given morphine or saline on alternate days (4 days of each treatment) and paired with a single closed compartment for 30 min. Mice were confined to their less preferred side immediately after morphine injection (10 mg/kg, i.p.), while they were confined to their more preferred side when given saline the following day. The control animals received saline in both compartments. Twenty-four hours after the last conditioning session, the animals were placed in the bridge with open access to both compartments. Time spent in each compartment during a 15 min period was recorded as during the preconditioning test. CPP score was calculated as the time (%) spent in the morphine-paired compartment minus the time (%) spent in the same compartment on the preconditioning day.
2.6 |. Conditional place aversion (CPA)
Three to four weeks after the basic behavioral tests, control and Cox10 cKO mice were subjected to CPA, a test that elicits strong aversion, behavioral and physical symptoms of a negative state (Gomez-Milanes et al., 2012) (Figure S2). Mice were injected i.p. with escalating doses of morphine sulfate (20, 40, 60, 80 mg/kg) or saline twice a day for 4 days (at 9 a.m. and 5 p.m.). On day 5, mice received a single injection of morphine (100 mg/kg) or saline in their home cages. Five hours after the morning injection on day 3, mice were allowed free exploration of the CPA apparatus for 15 min (preconditioning). Place conditioning was performed on days 4 (saline) and 5 (naloxone 0.25 mg/kg, s.c.), 5 h after the morning injection. Place conditioning was tested on day 6, 24 h after the naloxone injection. The CPA score was calculated as the time (%) spent in the naloxone-paired compartment minus the time spent in the same compartment on the preconditioning day.
2.7 |. Analgesic effects
2.7.1 |. Hot plate test
The Hot Plate test was conducted using the Hot Plate Analgesia Meter (Columbus Instruments, OH). Mice were placed on the plate surface, which was heated to 55°C, and the time when animals first lift their paws and lick them (paw-lick response) was recorded as the basal response time. The maximal time of the test was 30 s (cut off time). The procedure was repeated 30 min after injection of morphine (5 mg/kg, s.c.), and the antinociceptive morphine response was calculated as a percentage of maximal possible effect using the formula %MPE = 100% × (morphine response time − basal response time)/(cutoff time − basal response time). The MPEs were compared by Mann–Whitney test.
2.7.2 |. Hargreaves test
The Hargreaves test was conducted using the IITC Plantar Analgesia Meter (IITC Life Science Inc., CA). Briefly, mice were acclimated in individual enclosures on a glass platform heated to 25°C for 30 min. The average time taken to withdraw the rear left and right paws from the heat stimulus was recorded as the basal response time. The parameters of the light source were adjusted to 1% for idle intensity, 13% for active intensity and 30 sec for cut off time. The procedure was repeated 30 min after injection of morphine (5 mg/kg, s.c.) and the antinociceptive morphine response was calculated as a percentage of maximal possible effect using the formula %MPE = 100% × (morphine response time − basal response time)/(cutoff time − basal response time). The MPEs were compared by Mann–Whitney test.
2.8 |. RNAscope experiment
In situ hybridization was performed using the “BaseScope” method (Wang et al., 2012) using a kit supplied by ACD (Newark, California, USA), and 15 μm fresh frozen sagittal mouse brain sections. Custom “3ZZ” probes were designed by ACD and were targeted to heme A:farnesyltransferase cytochrome c oxidase assembly factor mRNA (Cox10, GenBank accession NM_178379), nucleotides 788-934 and aldehyde dehydrogenase 1 family, member L1 (Aldh1l1, NM_027406), nucleotides 1256-2112. Aldh1l1 hybridization was used as a marker for astrocytes and was detected by precipitation of Fast Red. Cox10 hybridization was detected by precipitation of Fast Green. Images were acquired with a Leica DM 6B upright microscope and a×40 oil immersion objective using Leica LAS X acquisition software.
Expression was evaluated using ImageJ 1.53c (Schneider et al., 2012). Cox10 expression was assessed by placing random regions of interest (ROI; defined by a 30 μm circle) over a field that contained at least one red signal suggesting that it was within an astrocyte. Pixels containing blue Cox10 staining were then counted within the ROI. 15 ROIs were evaluated within each 312 μm × 234 μm image from various brain regions. Images were uniformly adjusted for brightness, contrast, and color balance.
2.9 |. Immunofluorescent staining and image analysis
Mice were deeply anesthetized with Forane (isoflurane USP, NDC 10019-360-60, Baxter Healthcare Corporation, Deerfield, IL, USA) followed by transcardiac perfusion with ice cold 0.1 M phosphate buffer (PBS; pH 7.4) with heparin (10,000 U/L) and 4.0% paraformaldehyde in 0.1 M PBS. The brains were dissected out and post-fixed in 4.0% paraformaldehyde in 0.1 M PBS for 24 h at 4°C. After cryoprotection in 30% sucrose in 0.1 M PBS for 48 h, the brains were cut into 40 μm thick parasagittal sections.
Sections were stained with chicken anti-GFAP (1:1000, Aves Labs, Cat: #GFAP) and rabbit anti-TOM20 (1:50, Proteintech, Cat: #11802-1-AP). Briefly, after incubating brain sections in the blocking solution for 1 h at room temperature (RT), the sections were incubated for 48 h at 4°C with the primary antibodies. Afterwards, the sections were incubated for 2 h at RT with the corresponding Alexa 488- and 568-labeled species-specific secondary antibodies (1:1000, Invitrogen, Cat: #A-11001; A-11075). After 3 × 5-min PBS washes and 30 min drying sections were mounted on Superfrost plus slides (Thermo Fisher Scientific, Waltham, MA; Cat: #22-037-246) with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Cat: #H-1200) and stored at 4 °C until imaging. Z-stack images were acquired using a Leica laser-scanning confocal microscope (Leica TCS SP8 STED/FLIM/Lightsheet Confocal Microscope) equipped with the white light laser (the Confocal Microscope and Flow Cytometry Facility at the Jacobs School of Medicine and Biomedical Sciences, SUNY, University at Buffalo). All images were acquired using a×100 oil immersion objective with a ×1.5 digital zoom factor. Acquisition settings were as follows: 1024 × 1024 frame size, 8-bit image resolution, and a 0.3 μm step size. Z-stacks were typically in the range of 40–50 μm. Care was taken to ensure that selected astrocytes were complete through the x, y, and z planes, and non-overlapping. Acquired Z-stacks were exported to BitPlane Autoquant deconvolution software. Deconvolved 3D z-stacks were analyzed with Imaris software, using the colocalization module. Colocalization analysis was performed with Bitplane Imaris 9.8.0 3D image analysis software (Bitplane AG, Zurich, Switzerland) following deconvolution. Experimenter was blind to the group’s identity for all quantifications.
2.10 |. Biochemical assays
Brain astrocytes were isolated from P150 adult Oprm1 cKO and control mice. Whole brains were dissected and placed in PBS with calcium/magnesium. Astrocytes were isolated according to the “Isolation and cultivation of astrocytes from adult mouse brain” protocol (Miltenyi Biotec) using Adult Brain Dissociation Kit (Cat: #130-107-677) and Anti-ACSA-2 MicroBead Kit (Cat: #130-097-678). After magnetic sorting, cells were centrifuged for 5 min at 300g at 4°C. Pellets were dissolved in the AstroMACS medium (Miltenyi Biotec) for mitochondrial assays, or in the cell lysis buffer for Western blot (Cell Signaling Technology, #9803).
2.10.1 |. Measurements of mitochondrial function
The mitochondrial function was evaluated in isolated astrocytes using an XF96 Extracellular Flux Analyzer (Agilent). Isolated astrocytes were plated at a density of 50 × 103/well in XF96 cell-culture microplates pre-coated with poly-D-lysine (Sigma P6407). The cultures were incubated for 5 days prior to assay. On the fifth day, medium was replaced with Seahorse XF medium supplemented with 10 mM glucose (Sigma, G8769), 1 mM L-glutamine (Life Technologies, 25030081) and 1 mM sodium pyruvate (Life Technologies, 11360070) and Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were measured by the Seahorse Analyzer using Seahorse XF Cell Mito Stress test after injections of the mitochondrial electron transport chain (ETC) inhibitors: oligomycin (an inhibitor of complex V), carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple of the proton gradient and rotenone, an inhibitor of complex I. The OCR and ECAR measurements were recorded following an 11-min equilibration, with a 1-min mix and a 1-min wait period. All measurements were normalized to total protein concentration in each well.
2.10.2 |. Lactate assay
Lactate was measured in medium using an L-lactate assay kit (Lactate-Glo™ assay, Promega, J5021). Cells were seeded at a density of 20,000 cells per well in 96-well plates pre-coated with poly-d-lysine. The next day, the medium was replaced with FBS-free medium (Life Technologies, 11,965,092). After 3 h of incubation, the medium was collected and centrifuged at 10,000g for 5 min. The secreted l-lactate concentration in the supernatant was determined by measurement of luminescent signal.
2.10.3 |. Western blotting
Isolated astrocytes were sonicated for 30 s in cell lysis buffer (Cell Signaling Technology, #9803) containing 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF and 1 μg/ml leupeptin. Cell lysates were spun down at 10,000g for 10 min at 4°C. The resulting supernatant was processed by SDS/PAGE, and the separated proteins were transferred onto a nitrocellulose membrane. The membrane was washed in Tris-buffered saline solution containing 0.05% Tween 20 (TBST) and was blocked for 1 h at RT in TBST containing 5% non-fat dry milk (Cell Signaling Technology, #9999). The membrane was incubated overnight at 4°C with anti-COX10 rabbit antibody (1:1000, Proteintech, 10611-2-AP) and HRP-conjugated anti-rabbit secondary antibody (1:1000, NA934, Cytiva) or mouse OxPhos Rodent antibody cocktail that contains a clone against MTCO1 (1;1000, Invitrogen, 45-8099) followed by HRP-conjugated anti-mouse secondary antibody (1:1000, NA931, Cytiva) The immunoblots were visualized on Blu-Ray autoradiography films (NDA8803, Next Day Science) using Super Signal West Pico Chemiluminescent Substrate (34080, Thermo Fisher Scientific). The optical density of protein bands on each digitized image was normalized to the optical density of β-actin band (A5441, Millipore-Sigma) using ImageJ software (version 1.49v).
2.11 |. Statistical analysis
All data are presented as mean ± SEM. Behavioral, biochemical, and metabolic data were analyzed with unpaired Student’s t-test, Mann–Whitney test or two-way ANOVA with or without repeated measures. Significant main effects and interactions were further investigated with Fisher LSD or Bonferroni post-hoc tests. Sigmaplot 14.0 software (Systat Software) was used for data analysis and GraphPad Prism 9.0 (GraphPad Software) was used for graph generation. Significance was set at p < .05. The Bonferroni correction was used for multiple behavioral tests on the same mice. All raw data are available to the scientific community upon request.
3 |. RESULTS
3.1 |. Decreased levels of COX10 in astrocytes
Control and Cox10 cKO mice were sacrificed, and astrocytes were isolated from the brain as previously described (Philips et al., 2021). Compared to control samples, there was a significant decrease in protein levels of COX10 in astrocytes (Figure 1a,b). Prior studies reported that decreased expression of COX10 leads to degradation of Cytochrome c oxidase subunit I (MTCO1), another subunit of Complex IV (Diaz et al., 2006). Consistent with previous findings, we found significantly reduced levels of MTCO1 in astrocytes of Cox10 cKO mice (Figure 1a,c). To assess regional changes in expression of Cox10 mRNA, we used BaseScope in situ hybridization and found an approximately 50%-decrease in expression of Cox10 in the dentate gyrus of the hippocampus, the CA1-CA2 areas of the hippocampus and the caudate putamen (Figure S3). These regions were selected because of the maximal activity of the promoter as previously reported (Srinivasan et al., 2016). In addition, we evaluated expression of a major marker of mitochondrial integrity, TOM20, and found no significant group-related differences (Figure S4).
FIGURE 1.
Decreased expression of COX10, MTCO1 and oxidative phosphorylation in Cox10 cKO mice. (a) Representative Western blot images of the protein bands corresponding to endogenous mouse COX10 (~48 kDa), cytochrome c oxidase subunit I (C-IV-MTCO1; 40 kDa). Actin (42 kDa) was used as a loading control. (b, c) Quantitative analysis revealed significantly decreased expressions of mouse (B) COX10 (males: t = 2.68; p = .027; females: t = 2.56; p = .033) and (C) C-IV-MTCO1 (males: t = 4.08; p = .003; females: t = 2.96; p = .017), two-tailed Student test; *Denotes p <.05, **denotes p < .01. (d) Astrocyte-restricted deletion of Cox10 decreased mitochondrial respiration in brain astrocytes. Representative OCR for control (solid circle, n = 4) and Cox10 cKO (open circle, n = 4) astrocytes; each culture was measured in duplicates. Two-way repeated measures ANOVA revealed a significant effect of the genotype on the basal respiration (males: F(1,6) = 17.65, p = .005; females: F(1,6) = 7.02; p = .038), respiratory capacity after oligomycin injection (males: F(1,6) = 7.44; p = .034; females: F(1,6) = 7.23; p = .036) and on the maximal respiratory capacity measured following CCCP injection (males: F(1,6) = 9.5; p = .021; females: F(1,6) = 15.93; p = .007). Bonferroni post-hoc test shows that basal respiration, respiration after oligomycin injection and maximal respiration rate measured after CCCP injection and non-mitochondrial respiration were significantly lower in Cox10 cKO astrocytes compared to control ones; *Denotes p <.05, **denotes p < .01, ***denotes p < .001. (e) ECAR for control (solid circle, n = 5) and Cox10 cKO (open circle, n = 4) astrocytes; each culture was measured in duplicates. Two-way repeated measures ANOVA revealed no significant group effects. (f) Lactate measurement in primary astrocytes. No significant group-related changes were observed
3.2 |. Decreased oxidative phosphorylation in Cox10 deficient astrocytes
Since COX10 plays a critical role in oxidative phosphorylation (Diaz et al., 2006), we further examined the effects of Cox10 deletion on mitochondrial function in astrocytes using the Seahorse Mito stress test. Astrocytes were isolated from the whole brain of control and Cox10 cKO mice. Compared to the control group, Cox10 cKO astrocytes had significantly decreased basal respiration or CCCP-induced maximal respiration (Figure 1d), but no significant changes in glycolysis as assessed by the ECAR test or production of lactate (Figure 1e,f). Collectively, our data confirm that astrocyte-restricted deletion of Cox10 significantly reduces mitochondrial oxidative phosphorylation in these cells.
3.3 |. Behavioral phenotypes of Cox10 cKO mice
We found no genotype- or sex-dependent differences between control and cKO mice in the initial battery of behavioral tests in which we measured: general locomotor activity in the open field test (Figure S5a,b), anxiety-like behaviors in the elevated plus maze test (Figure S5c,d), and spatial working memory as assessed by spontaneous alternations in the Y maze test (Figure S5e,f) and in the novel (object location) place recognition test (NPRT) (Figure S5g,h). Cox10 cKO female, but not male, mice showed a modest decrease in preference for a novel object (Figure 2a,b).
FIGURE 2.
Impaired novel object recognition and trace fear conditioning in Cox10 cKO mice. (a,b) Novel object recognition test (NORT). The Y-axes depict the preference (%); the X-axes depict the experimental groups: Control—control mice; Cox10 cKO—Aldh1l1-Cre/ERT2::Cox10fl/+ mice. Compared to controls, Cox10 cKO mice exhibited the significantly decreased preference for a novel object. Two-way ANOVA revealed a significant effect of the genotype, F(1,55) = 19.26, p < .001. Fisher LSD post-hoc test showed that Cox10 cKO female mice exhibited significantly less preference compared to their respective controls, *denotes p < .05. (c) Compared to controls, Cox10 cKO male mice exhibited significantly decreased freezing during presentation of the cue (sound). Student’s t-test showed that Cox10 cKO males had significantly decreased freezing compared to control males (t = 3.19; p = .003), n = 22 for control; n = 9 for Cox10 cKO; *Denotes p < .05. (d) No effects on TFC were observed in Cox10 cKO female mice. (e) Compared to controls, Cox10 cKO male mice exhibited the significantly decreased freezing behavior during the inter-tone intervals. Student t-test showed that Cox10 cKO males had significantly decreased freezing compared to control males (t = 3.51; p = .001); n = 22 for control; n = 9 for Cox10 cKO, *Depicts p < .05. (f) No effects on freezing during the inter-tone intervals in Cox10 cKO female mice; n = 18 for control; n = 10 for Cox10 cKO
However, in a more difficult and somewhat stressful cognitive test, trace fear conditioning, that is dependent on the interaction between the prefrontal cortex and dorsal hippocampus (Lugo et al., 2014; Terrillion et al., 2017), Cox10 cKO male, but not female, mice exhibited deficient cue-dependent memory manifested in decreased freezing behavior during tone presentation (Figure 2c,d) and between tone intervals (Figure 2e,f). Freezing times remain comparable between two groups before the first tone presentation of the cue during the cue test (Figure S6a,b) or exposure to the context, i.e., context-dependent freezing (Figure S6c,d).
3.4 |. The effects on drug-induced behaviors
We next assessed the effects of conditional deletion of the Cox10 gene in astrocytes on performance in tests that evaluate reward (Li et al., 2002) or aversion produced by psychotropic drugs, namely, conditional place preference (CPP) or conditional place aversion (CPA). We chose to use morphine as astrocytes express opioid receptors (Nam et al., 2018). In addition, prior in vitro studies suggested a link between astrocyte opioid receptors and astrocyte mitochondrial functions (Zhang et al., 2016).
3.4.1 |. No effects on CPP
A separate cohort of 12-week-old male and female control and Cox10 cKO mice were used to evaluate the effects of conditional Cox10 deletion on morphine-produced CPP (Figure S1). We did not find any effect of Cox10 cKO in male or female mice in the CPP paradigm (Figure 3).
FIGURE 3.
No change of conditioned place preference (CPP) in Cox10 cKO mice. The Y-axes depict the CPP score calculated as the % time spent in the morphine-paired compartment minus the % of time spent in the same compartment on the preconditioning day. No effects on CPP in cKO male (a) or female (b) mice. Two-way ANOVA revealed a significant effect of morphine (males: F(1,28) = 36.83, p < .001; females: F(1,35) = 41.64; p < .001). Fisher LSD post-hoc tests showed that both morphine-treated control and Cox10 cKO mice exhibited significantly increased CPP scores compared to the respective saline-treated groups; **Denotes p < .01, ***denotes p < .001
3.4.2 |. Naloxone-precipitated aversion
Another cohort of mice was tested for naloxone-precipitated morphine withdrawal using CPA (Figure S2). In addition, we measured naloxone-produced aversion by monitoring the frequency of jumping immediately following naloxone administration (Garcia-Carmona et al., 2015; Kest et al., 2001). We found that compared to control mice, Cox10 cKO mice exhibited increases in naloxone-precipitated withdrawal jumping (Figure 4a,b) and decreases in time spent in the naloxone-paired compartment (Figure 4c,d). Extinction of CPA produced by naloxone-precipitated morphine withdrawal was tested after 3 or 6 weeks after. Compared to the respective control groups, the CPA score was still significantly greater after 3 weeks in Cox10 cKO mice (Figure 5a,b); after 6 weeks, the score was elevated only in Cox10 cKO males (Figure 5c,d).
FIGURE 4.
Cox10 cKO mice showed exacerbated aversion. (a) Jumping, as a physical sign of aversion, was significantly increased in morphine-dependent Cox10 cKO male mice compared to controls. Two-way ANOVA revealed the significant effects of morphine, F(1,44) = 89.78; p < .001, and the genotype, F(1,44) = 6.02; p = .018. Fisher LSD post-hoc test showed that both morphine-treated control and Cox10 cKO mice jumped more than saline-treated animals (p < .001); morphine-treated Cox10 cKO mice jumped more than morphine-treated controls did (p = .001); **Denotes p < .01, ***denotes p < .001. (b) Number of jumps was significantly increased in morphine-dependent Cox10 cKO female mice compared to controls. Number of jumps was significantly higher in morphine-treated Cox10 cKO female mice compared to control female mice. Two-way ANOVA revealed the significant effects of morphine, F(1,36) = 58.66, p < .001, and the genotype, F(1,36) = 6.16; p = .018. Fisher LSD post-hoc test showed that both morphine-treated control and Cox10 cKO female mice jumped more than saline-treated animals (p ≤ .001 for both groups); morphine-treated Cox10 cKO mice jumped more than morphine-treated controls did (p < .001); **Denotes p < .01, ***denotes p < .001. (c,d) The Y-axes depict the CPA score calculated as the % time spent in the morphine-paired compartment minus the % of time spent in the same compartment on the preconditioning day. (c) Morphine-dependent Cox10 cKO male mice demonstrated significantly increased aversion (i.e., the decreased CPA scores) compared to controls. Two-way ANOVA revealed the significant effects of morphine, F(1,44) = 21.69; p < .001, and the genotype, F(1,44) = 4.62; p = .037. Fisher LSD post-hoc test showed that both morphine-treated control and Cox10 cKO mice had the significantly decreased CPA scores (i.e., increased aversion) compared to saline-treated groups (p = .031 for controls and p < .001 for Cox10 cKO mice); morphine-treated Cox10 cKO mice had the significantly decreased CPA scores compared to morphine-treated controls (p = .015); *Denotes p < .05, ***denotes p < .001. (d) Morphine-dependent Cox10 cKO female mice demonstrated significantly increased aversion (i.e., the decreased CPA scores) compared to morphine-dependent controls. Two-way ANOVA revealed the significant effects of morphine, F(1,36) = 29.97; p < .001, and the borderline effect of genotype, F(1,36) = 4.05, p = .052; and the genotype by morphine interaction, F(1,36) = 4.05; p = .049. Fisher LSD post-hoc test showed that both morphine-treated control and Cox10 cKO mice had the significantly decreased CPA scores (i.e., increased aversion) compared to saline-treated groups (p = .020 for controls and p < .001 for Cox10 cKO mice); morphine-treated Cox10 cKO mice had the significantly decreased CPA scores compared to morphine-treated controls (p = .007); *Denotes p < .05, **denotes p < .01, ***denotes p < .001
FIGURE 5.
Cox10 cKO mice showed decreased extinction of aversion. (a) Attenuated extinction of CPA in morphine-dependent Cox10 cKO male mice 3 weeks after naloxone-precipitated withdrawal. Two-way ANOVA revealed significant effects of morphine, F(1,44) = 19.87, p < .001, a borderline effect of the genotype, F(1,44) = 3.48; p = .069. Fisher LSD post-hoc test showed that both morphine-treated control and Cox10 cKO mice had significantly decreased CPA scores compared to saline-treated groups (p = .027 for controls and p < .001 for Cox10 cKO); morphine-treated Cox10 cKO mice had significantly decreased CPA scores compared to morphine-treated controls (p = .039); *Denotes p < .05, ***denotes p < .001. (b) Attenuated extinction of CPA in morphine-dependent Cox10 cKO female mice 3 weeks after naloxone-precipitated withdrawal. Two-way ANOVA revealed a significant effect of morphine, F(1,36) = 27.07, p < .001. Fisher LSD post-hoc test showed that both morphine-treated control and Cox10 cKO mice had significantly decreased CPA scores compared to saline-treated groups (p = .013 for controls and p < .001 for Cox10 cKO); morphine-treated Cox10 cKO mice had significantly decreased CPA scores compared to morphine-treated controls (p = .043); *Denotes p < .05, ***denotes p < .001. (c) Attenuated extinction of CPA in morphine-dependent Cox10 cKO male mice 6 weeks after naloxone-precipitated withdrawal. Two-way ANOVA revealed a significant effect of morphine (F(1,44) = 10.47; p = .002). Fisher LSD post-hoc test showed that morphine-treated Cox10 cKO mice had significantly decreased CPA scores compared to saline-treated Cox10 cKO mice (p = .001); morphine-treated Cox10 cKO mice had significantly decreased CPA scores compared to morphine-treated controls (p = .042); *Denotes p < .05, **denotes p < .01. (d) No effects on extinction of CPA in morphine-dependent Cox10 cKO female mice 6 weeks after naloxone-precipitated withdrawal
3.5 |. Acute analgesic effects of morphine
Using a separate cohort of 12-week-old mice, we also evaluated the acute analgesic effects of morphine (5 mg/kg, s.c.) in the hot plate and Hargreaves tests. Mildly decreased pain inhibition was observed in the Hargreaves test in male cKO mice only (Figure S7).
4 |. DISCUSSION
Our data demonstrate that astrocyte-restricted deficiency in oxidative phosphorylation impaired trace fear conditioning and exacerbated physical and behavioral consequences of naloxone-precipitated morphine withdrawal in mice. The data suggest that astrocyte oxidative phosphorylation may contribute to behaviors associated with greater cognitive load and/or aversive and stressful conditions.
Although astrocytes have been viewed as key supporters of neuronal metabolism, we still know little about the exact mechanisms of metabolic coupling between astrocytes and neurons in physiological and pathophysiological conditions (Bolanos, 2016; Rose & Chatton, 2016; Takahashi, 2020). In the present study we tested various behaviors in mice with altered mitochondrial function using non-stressful tests and those associated with aversion produced by electric shock or naloxone-precipitated morphine withdrawal.
To disrupt mitochondrial oxidative phosphorylation in astrocytes, we selectively deleted Cox10 gene that encodes Cytochrome c Oxidase (COX) 10 in astrocytes. Complex IV or COX is the last electron acceptor of the respiratory chain, involved in the reduction of O2 to H2O (Mansilla et al., 2018; Zong et al., 2018). As an accessory protein, COX10 is involved in building a functional complex IV (Diaz et al., 2006). In mouse models, selective deletion of Cox10 in different tissues led to variable phenotypes depending on the cell type in which the gene was deleted (Diaz et al., 2005; Supplie et al., 2017). Similarly, deleting Cox10 in neurons produced molecular alterations and behavioral abnormalities that depended on the type of neurons in which deletion took place (Fukui et al., 2007).
Several studies have attempted to delete Cox10 in glial cells. Funfschilling et al. (Funfschilling et al., 2012) crossed the Cox10flox/flox line with Cnp1Cre/+ or Dhh-Cre or Plp1-CreERT2 mice to target Schwann cells or oligodendrocytes. They reported that deletion of Cox10 in the peripheral nervous system led to neuropathy, abnormal Remak bundles, muscle atrophy and paralysis without any signs of glial cell death. In contrast, no demyelination, axonal degeneration, or inflammation were observed in the central nervous system. When using GLASTCreERT2 mice, the same group found no changes in astrocytes (i.e., Bergman glia) even 1 year after deletion of Cox10. It was proposed that deficient mitochondrial bioenergetics did not affect astrocyte survival or produce any signs of neurodegeneration (Supplie et al., 2017). The present study sought to further evaluate the effects of deletion of Cox10 in astrocytes using a Cre line with more ubiquitous expression in astrocytes (Srinivasan et al., 2016). We found that deficient oxidative phosphorylation in astrocytes had no effects on locomotor activity or anxiety but impaired trace fear conditioning and produced exacerbated naloxone-precipitated aversion in morphine-dependent mice. Thus, impaired oxidative phosphorylation in astrocytes led to alterations in mouse behaviors associated with greater cognitive load under stressful conditions and/or aversive states. Future studies will address this intriguing phenomenon in greater detail.
Our data suggest that there could be sex-dependent effects of deletion of Cox10 in astrocytes on some mouse behaviors. Most previous studies on mouse models of conditional deletion of Cox10 in various tissues either provide no information on the sex of mice used or used one sex only (Diaz et al., 2012; Peralta et al., 2016; Supplie et al., 2017). However, female mice lacking Cox10 in skeletal muscle were shown more susceptible to myopathy and died at younger age than males (Diaz et al., 2005). Interestingly, in some mitochondrial genetic diseases, such as Leber hereditary optic neuropathy, which is associated with mutations in genes encoding complex I subunit of ETC, approximately 50% of males and only 10% of females with mutation actually develop the disease (Yu-Wai-Man et al., 2002). In this study, we evaluated the effects of Cox10 deletion on mitochondria respiration and glycolysis and behaviors in male and female mice. We found no significant sex-related differences in mitochondria respiration or lactate production. Male and female mice showed some differences in the novel object recognition test, trace fear conditioning test and extinction of the CPA 6 but not 3 weeks after morphine withdrawal. This outcome appears consistent with the growing appreciation of the role of estrogens in influencing glia function (Chisholm & Sohrabji, 2016; Crespo-Castrillo & Arevalo, 2020; Martin-Jimenez et al., 2019; Wang et al., 2014). Future studies will address possible sex-related outcomes of mitochondrial deficiency in astrocytes.
One limitation of the current work is that Cox10 was deleted from astrocytes throughout the brain, precluding assessment of which regions mediate the observed changes. More research is needed to understand whether the behavioral phenotypes observed in the present study could be mediated by astrocytes in selected brain regions. The mechanisms whereby deficient astrocyte oxidative phosphorylation could contribute to cognitive functions and/or lead to aversion remain obscure. Chronic exposure to opioids has been shown to impair glutamate uptake due to long-lasting downregulation of expression of the glutamate transporter, GLT-1 (Roberts-Wolfe & Kalivas, 2015; Wang et al., 2019). Prior reports demonstrated the physical proximity of glutamate transporters and mitochondria in astrocytes (Bauer et al., 2012; Genda et al., 2011; Robinson et al., 2020; Shevelkin et al., 2020) and interaction of glutamate transporters with the Na+/K+ ATPase (Rose et al., 2009). It is tempting to speculate that decreased oxidative phosphorylation in astrocytes might alter the Na+/K+ ATPase functioning, affecting glutamate transporters and leading to decreased uptake of glutamate. For example, it was shown that inhibition of mitochondrial function by fluorocitrate in astrocytes increased glutamate excitotoxicity in neuron-astrocyte co-cultures (Voloboueva et al., 2007). Future studies will need to address these hypothetical mechanisms in detail.
In conclusion, our findings provide evidence that astrocyte oxidative phosphorylation contributes to behaviors associated with greater cognitive load and/or aversive and stressful states and support the hypothesis that astrocyte mitochondria could be involved in physiological and pathological conditions.
Supplementary Material
ACKNOWLEDGMENT
We would like to thank Dr. Yavin Shaham (NIDA) for his expert advice on testing morphine-produced behaviors. We are also grateful to Ms. Olga Groser Gluzman and Ms. Olga Mychko for their expert technical assistance in genotyping, colony breeding, Western blotting, and their help with behavioral tests and data analysis. The Table of Contents Image was adapted from “Brain Callout (Mouse)”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates.
Funding information
This work was supported by the Pilot Grant from the JHU Drug Abuse Center (Y.J. and J.M.B.); the R01NS110598, R01NS117761 grants (Y.G.), the MH-083728 grant (D.E.B. and M.V.P.), and the R01DA041208, MH-094268 grants (M.V.P.). Dr. Huseynov was supported by the US Fulbright Visiting Scholar Program-IIE ID: PS00303489.
Footnotes
CONFLICT OF INTEREST
The authors declare no biomedical financial interests or potential conflicts of interest.
SUPPORTING INFORMATION
Additional supporting information may be found in the online version of the article at the publisher’s website.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Supplementary Materials
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.