Decreased Brain pH Underlies Behavioral and Brain Abnormalities Induced by Chronic Exposure to Glucocorticoids in Mice

Abstract

Depressed patients may exhibit glucocorticoid hypersecretion, suggesting that elevated levels of glucocorticoids may play an important role in the pathophysiology of depression. Some postmortem brain studies have shown decreased pH and increased lactate levels in psychiatric patients, implying involvement of these factors in the pathogenesis. To investigate the effects of glucocorticoids on brain pH and lactate levels, and their roles in depressive symptoms, brain pH and lactate were examined in mice treated with corticosterone (CORT), the major bioactive glucocorticoid in rodents. A single administration of CORT decreased hippocampal pH after 24 h. Three weeks of CORT treatment decreased pH in the prefrontal cortex (PFC), striatum, and hippocampus (HC), whereas intake of pH 9.0 drinking water increased pH in these brain regions. pH and lactate levels were correlated in the PFC and HC of mice treated with CORT for 3 weeks. The suppression of body weight gain and decrease in adrenal weight observed after 3 weeks of CORT treatment were not alleviated by pH 9.0 water. However, an increase in immobility time in the forced swim test and a decrease in neurogenesis in the hippocampus were alleviated. The decrease in brain pH and increase in immobility time in the forced swim test and a decrease in neurogenesis in the hippocampus induced by CORT treatment were abolished by co-treatment with the glucocorticoid receptor (GR) antagonist mifepristone. These findings indicate that decreased brain pH via GRs may be related to glucocorticoid-induced depression-like behavior and decreased hippocampal neurogenesis.

INTRODUCTION

Glucocorticoids are steroid hormones produced in the adrenal glands that play a crucial role in the stress response. Exposure to stress activates the hypothalamus-pituitary-adrenal axis, which is the endocrine system for coping with stress, and causes secretion of glucocorticoids from the adrenal glands. The secreted glucocorticoids cause a variety of responses via mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs), such as regulation of energy metabolism and suppression of immune function to facilitate flight or fight.¹⁾ These biological responses are intended for survival from threats, but persistently elevated levels of glucocorticoids resulting from chronic stress can cause adverse conditions for the organism, such as mood disorders.²⁾

An abnormality in the stress response system is a frequent finding in major depression. Depressed patients often exhibit hypersecretion of cortisol, the major bioactive glucocorticoid in primates. This is closely associated with non-response on the dexamethasone suppression test³⁾ and is reflected in elevated levels of cortisol in the serum and plasma.⁴⁾ To mimic such elevated levels of glucocorticoids, rodents can be chronically treated with corticosterone, the major bioactive glucocorticoid in rodents. This animal model shows behavioral abnormalities, such as increased immobility in the forced swim and tail suspension tests,^5,6) and central nervous system abnormalities such as decreased neurogenesis in the hippocampal dentate gyrus,^7,8) which is pivotal in coping with stress and adapting to the environment.^9,10) This supports the idea that elevated glucocorticoid-induced changes may play an important role in development of depression.

Postmortem brain studies of patients with psychiatric disorders such as schizophrenia and bipolar disorder have shown that the pH is lower in several brain areas, such as the dorsolateral prefrontal cortex and cerebellum, compared to that of control individuals.^11–16) Such a decrease in brain pH is also seen in mice that exhibit symptoms similar to those of psychiatric disorders.¹⁷⁾ Furthermore, increased lactate levels are correlated with lower pH in the brains of psychiatric patients and animal models.^12,17) These studies suggest that lower brain pH associated with elevated lactate is the underlying pathophysiology of psychiatric disorders.

In this study, brain pH and lactate were measured in mice treated with single or chronic administration of corticosterone to investigate the effects of glucocorticoids on brain pH and lactate levels. Furthermore, to investigate whether lower brain pH is involved in depression-like behavior and decreases in hippocampal neurogenesis induced by chronic corticosterone (CORT) treatment, the effects of intake of high pH water on abnormalities induced by CORT were examined.

MATERIALS AND METHODS

Animals

Experimental procedures concerning the use of animals were approved by the Committee for Ethical Use of Experimental Animals at Setsunan University and conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Every effort was made to minimize suffering and to reduce the number of animals used. Six-week-old male ICR mice were obtained from Shimizu Laboratory Supplies (Kyoto, Japan) and housed in cages (24 × 17 × 12 cm³) in groups of 5 mice under controlled environmental conditions (23 ± 1 °C; 12 : 12-h light-dark cycle, humidity of 55%, food and water ad libitum). Separate mice were used in each experiment to avoid the effects of stress from behavioral tests on the brain parameters.

CORT Treatment and Alkaline Water Intake

CORT was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.) and suspended in 0.5% (w/v) carboxymethylcellulose (CMC). CMC or CORT (40 mg/kg) was administered subcutaneously at a fixed volume of 10 mL/kg body weight. CORT doses were based on previous studies.¹⁸⁾ Chronic administration of CORT was performed once daily for 21 consecutive days starting at 6 weeks of age. Tissue collection, behavioral tests, and immunohistochemistry were performed 24 h after the last administration. Spironolactone (30 mg/kg; Tokyo Chemical Industry, Tokyo, Japan), an antagonist for MR, and mifepristone (30 mg/kg; LKT Laboratories, St. Paul, MN, U.S.A.), an antagonist for GRs, were injected subcutaneously at a fixed volume of 10 mL/kg body weight, 30 min before CORT administration. The doses of spironolactone and mifepristone were determined from preliminary studies based on previous studies.^19–21) High pH drinking water was prepared using an alkaline ionized water apparatus (TK-AS30; Panasonic, Kadoma, Japan; purified water set to pH 8.5 or 9.0; measured pH 8.38 ± 0.04 or 9.10 ± 0.05, respectively; n = 3 separate days) was given to mice 24 h before CORT administration. The control group was given purified water (pH 7.5; measured pH 7.59 ± 0.04; n = 3 separate days) produced by the same apparatus. To account for changes in the pH of the drinking water over time, purified water and high pH water were replaced with new water within 2 d. Tap water (measured pH 7.56 ± 0.09; n = 3 separate days) was used for all experiments in which there was no need for specific drinking water.

Measurement of pH and Lactate Levels in Tissue Homogenates

The measurement of brain pH was based on a previous study.¹⁷⁾ Mice were euthanized by cervical dislocation and the prefrontal cortex (PFC), striatum (STR), and hippocampus (HC) were isolated, frozen in liquid nitrogen, and stored at −80 °C until assay. Each tissue sample was homogenized in distilled water (10 µL per 1 mg of tissue) using an ultrasonic disruptor (UR-21P; Tomy Digital Biology, Tokyo, Japan). pH was measured using a pH meter (AS800; As One, Osaka, Japan) with a micro pH electrode (LME242-3; As One). After pH measurement, the homogenates were immediately frozen and stored at −80 °C until required for measurement of lactate levels. For this measurement, the homogenates were centrifuged at 10000 × g for 5 min at 4 °C. A 10-µL aliquot of the supernatant was injected for HPLC (HTEC-510) using a pre-column (PC-04-CH), a gel column (EICOMPAK LT-GEL), an enzyme column (LT-ENZYMPAK), a platinum electrode (WE-PT) set at +450 mV against a reference electrode, and an electrochemical detector (ECD-100; all Eicom, Kyoto, Japan). The mobile phase contained 100 mM phosphate buffer (pH 7.0) and 200 mg/L tetra-n-hexylammonium bromide.

Forced Swim Test

The forced swim test was performed as previously described,¹⁸⁾ with minor modifications from Porsolt’s method.²²⁾ Briefly, mice were placed individually in a polymethylpentene beaker (height 27 cm, diameter 18 cm) containing water of depth 13 cm at 25 ± 1 °C. The performance of mice for 6 min of swimming was videotaped. The total duration of immobility (no movement of the paws and only minimal movement to keep the head above water) was measured in the final 4 min of the 6-min session by an observer blinded to the treatment conditions.

Open Field Test

Each mouse was placed at the center of a novel open field (50 × 50 cm²) and allowed to explore freely. The total distance traveled by each mouse and time spent in the center area (30 × 30 cm²) was measured for 10 min using ANY-maze video tracking software (Stoelting, Wood Dale, IL, U.S.A.).

Immunohistochemistry

Immunohistochemistry was performed as previously described.²³⁾ Briefly, mice received two injections of 5-bromo-2′-deoxyuridine (BrdU) (100 mg/kg) per day for 3 consecutive days before CORT treatment. Mice were deeply anesthetized and perfused transcardially with saline, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS) 24 h after the last CORT administration. Brains were removed and post-fixed in the same fixative for 2 d. Serial 50-µm coronal sections were cut with a microslicer (DTK-1000, Dosaka EM). Every sixth coronal section between bregma –1.40 and –2.48 mm was prepared for the HC. The free-floating sections were incubated in 50% formamide in saline sodium citrate buffer at 65 °C for 2 h, in 2N HCl at 3 °C for 30 min, and in 0.1 M borate buffer at room temperature for 15 min. For blocking, the free-floating sections were incubated in 1% bovine serum albumin containing 0.3% Triton-X 100 in PBS (PBS-T) or 10% donkey serum in PBS-T. The sections were then incubated at 4 °C overnight with a rat anti-BrdU antibody (1 : 500; Novus Biologicals, Centennial, CO, U.S.A.; RRID: AB_10002608) and a mouse biotin conjugated anti-NeuN antibody (1 : 200; Merck, Kenilworth, NJ, U.S.A.; RRID: AB_177621), followed by incubation at room temperature for 2 h with a donkey anti-rat IgG antibody (1 : 1000; Thermo Fisher Scientific, Waltham, MA, U.S.A.; RRID: AB_2535795) and Alexa Fluor 488-Streptavidin (1 : 1000; Thermo Fisher Scientific). Positive cells were counted manually in every sixth section in the HC using a confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan) by an observer blinded to the treatment conditions. The cell count was multiplied by six to obtain the total number of positive cells in the HC.

Statistical Analysis

For data other than body weight, each value is plotted and the mean ± standard error of the mean (S.E.M.) are shown; for body weight data, means are shown as symbols with the S.E.M. Comparisons of means between two groups were performed by Student t-test (Fig. 1A). Data for three or more groups were analyzed using one-way ANOVA and a post-hoc Tukey HSD test or two-way ANOVA. If the two-way ANOVA showed a significant interaction, each group was considered as an independent group, and a post-hoc Tukey HSD test was performed. If no significant interaction was found, a post-hoc Tukey HSD test was performed for each factor. Data for body weight and amount of water intake were analyzed using two- or three-way repeated measures ANOVA and Bonferroni correction. For correlations, r and p values and a linear regression line were calculated. All analyses were performed using JMP Pro 15 (SAS Institute, Cary, NC, U.S.A.). A value of p < 0.05 was considered to be significant.

Fig. 1. Brain pH at 24 h after a Single Administration of Corticosterone (CORT)

CORT (40 mg/kg) was administered subcutaneously, and the prefrontal cortex (PFC), striatum (STR), and hippocampus (HC) were collected 24 h later, followed by measurement of the pH of the homogenates (A). The correlation between pH and lactate levels was analyzed in the HC homogenates 24 h after CORT administration (B). A mineralocorticoid receptor antagonist, spironolactone (30 mg/kg), or a glucocorticoid receptor antagonist, mifepristone (30 mg/kg), was administered subcutaneously 30 min before CORT administration, and the pH of the HC homogenates was measured 24 h after CORT administration (C). Drinking water was replaced with pH 7.5, 8.5, or 9.0 water 24 h before CORT treatment, and the pH of the HC homogenates was measured 24 h after CORT administration (D). * p < 0.05, ** p < 0.01. n = 10.

RESULTS

Brain pH at 24 h after a Single Administration of CORT

First, to determine whether CORT affects brain pH, the pH was measured 24 h after CORT administration. A significant decrease in pH was observed in HC homogenates in CORT-treated mice compared to control mice, but not in PFC or STR homogenates (Fig. 1A). Lactate levels were measured to determine if the CORT-induced decrease in hippocampal pH was due to an increase in lactate. However, there was no significant increase in lactate in the HC of CORT-treated mice (data not shown) and no significant correlation between pH and lactate levels in the HC (r=−0.19, p = 0.41) (Fig. 1B). To identify the receptors involved in the CORT-induced decrease in hippocampal pH, experiments were performed using MR or GR antagonists. One-way ANOVA (F_3,36 = 6.82, p = 0.0009) and a post-hoc Tukey HSD test revealed that pretreatment with mifepristone, a GR antagonist, but not with spironolactone, an MR antagonist, prevented the CORT-induced decrease in hippocampal pH (Fig. 1C). Next, the effect of oral intake of high pH water (pH 8.5 or 9.0) on the CORT-induced decrease in hippocampal pH was examined. Two-way ANOVA revealed significant main effects of CORT treatment (F_1,66 = 28.61, p < 0.0001) and drinking water pH (F_2,66 = 5.16, p = 0.0083), but no significant interaction of CORT treatment × drinking water pH (F_2,66 = 1.41, p = 0.25). A post-hoc Tukey HSD test revealed that the hippocampal pH in mice given pH 9.0 water was significantly higher than that in mice given pH 7.5 water (p = 0.035) (Fig. 1D). These results indicate that hippocampal pH is raised by pH 9.0 water intake.

Brain pH and Lactate in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

Since hippocampal pH was decreased by a single CORT administration and increased by pH 9.0 water intake, the effects of chronic CORT treatment and pH 9.0 water intake on brain pH and lactate levels were examined. For brain pH, two-way ANOVA revealed significant main effects of CORT treatment (F_1,36 = 7.72, p = 0.0086) and drinking water pH (F_1,36 = 9.16, p = 0.0045), and a significant interaction of CORT treatment × drinking water pH (F_1,36 = 6.66, p = 0.014) in the PFC; significant main effects of CORT treatment (F_1,36 = 7.02, p = 0.012) and drinking water pH (F_1,36 = 5.15, p = 0.029), but no significant interaction of CORT treatment × drinking water pH (F_1,36 = 0.012, p = 0.91) in the STR; and significant main effects of CORT treatment (F_1,36 = 12.46, p = 0.0012) and drinking water pH (F_1,36 = 8.60, p = 0.0058), and a significant interaction of CORT treatment × drinking water pH (F_1,36 = 8.25, p = 0.068) in the HC (Fig. 2A). These results indicate that chronic CORT treatment, in contrast to a single administration, decreases pH in all brain regions of the PFC, STR, and HC, and that pH 9.0 water intake raises pH in PFC, STR, and HC, and greatly prevents the pH decrease caused by chronic CORT treatment in the PFC and HC.

Fig. 2. Brain pH and Lactate Levels in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

CORT (40 mg/kg) was administered subcutaneously for 21 d. Drinking water was replaced 24 h before CORT treatment. The PFC, STR, and HC were collected 24 h after the last administration, followed by pH (A) and lactate (B) measurements in the homogenates. ** p < 0.01, *** p < 0.001. n = 10.

For brain lactate levels, two-way ANOVA revealed a significant main effect of CORT treatment (F_1,36 = 14.85, p = 0.0005), but no significant main effect of drinking water pH (F_1,36 = 0.20, p = 0.66), and no significant interaction of CORT treatment × drinking water pH (F_1,36 = 0.027, p = 0.87) in the PFC; significant main effects of CORT treatment (F_1,36 = 4.25, p = 0.047) and drinking water pH (F_1,36 = 5.27, p = 0.028), but no significant interaction of CORT treatment × drinking water pH (F_1,36 = 0.70, p = 0.41) in the STR; and significant main effects of CORT treatment (F_1,36 = 6.94, p = 0.012) and drinking water pH (F_1,36 = 4.49, p = 0.041), and a significant interaction of CORT treatment × drinking water pH (F_1,36 = 3.26, p = 0.079) in the HC (Fig. 2B). These results indicate that chronic CORT treatment, in contrast to a single administration, increase lactate levels in all brain regions of the PFC, STR, and HC, and that that pH 9.0 water intake decreases lactate levels in STR, and HC, but not PFC.

Correlation analysis of brain pH and lactate levels revealed significant correlations in the PFC (r=−0.75, p = 0.000092) and HC (r=−0.70, p = 0.00031), but not in the STR (r = 0.013, p = 0.95) in mice given pH 7.5 water (Fig. 3A); and significant correlations in the HC (r=−0.45, p = 0.038), but not in the PFC (r=−0.083, p = 0.71) or STR (r=−0.33, p = 0.14) in mice given pH 9.0 water (Fig. 3B). For amount of water intake, three-way repeated measures ANOVA revealed no effects of CORT treatment (F_1,16 = 3.01, p = 0.12), drinking water pH (F_1,16 = 1.39, p = 0.27), or days (F_2,16 = 0.68, p = 0.52); or any significant interaction between these factors (CORT treatment × drinking water pH: F_1,16 = 0.45, p = 0.52; drinking water pH × days: F_2,16 = 0.08, p = 0.92; days × CORT treatment: F_2,16 = 0.16, p = 0.85; CORT treatment × drinking water pH × days: F_2,16 = 0.74, p = 0.49) (Table 1).

Fig. 3. Correlations between Brain pH and Lactate Levels in the PFC) STR, and HC in CORT Chronically Treated Mice Given pH 7.5 (A) or pH 9.0 (B) Water

Data for brain pH and lactate levels are from Figs. 2A and B, respectively. n = 10.

Table 1. Amount of Water Intake

Mouse group		Drinking water intake (mL/5 mice/d)
CORT treatment	Drinking water pH	Day 1	Day 8	Day 15
Control	pH 7.5	33.2 ± 4.1	32.3 ± 3.9	33.3 ± 4.2
Control	pH 9.0	38.3 ± 7.3	27.7 ± 1.5	29.3 ± 4.3
CORT	pH 7.5	41.5 ± 4.3	36.7 ± 6.7	37.3 ± 7.2
CORT	pH 9.0	33.3 ± 3.3	32.7 ± 4.3	36.7 ± 3.3

Body and Adrenal Gland Weights in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

Since the decrease in brain pH caused by chronic CORT treatment was alleviated by pH 9.0 water intake, the effects of pH 9.0 water intake on suppression of body weight gain and adrenal atrophy caused by chronic CORT treatment were examined. For body weight, three-way repeated measures ANOVA revealed significant main effects of CORT treatment (F_1,720 = 17.57, p = 0.0002) and days (F_20,720 = 153.55, p < 0.0001) and a significant interaction of CORT treatment × days (F_20,720 = 8.08, p < 0.0001), but no effect of drinking water pH (F_1,720 = 0.11, p = 0.74) or any other significant interaction (CORT treatment × drinking water pH: F_1,720 = 0.68, p = 0.41; drinking water pH × days: F_1,720 = 0.56, p = 0.94; CORT treatment × drinking water pH × days: F_1,720 = 0.41, p = 0.99) (Fig. 4A). For adrenal gland weight, two-way ANOVA revealed a significant main effect of CORT treatment (F_1,36 = 19.55, p = 0.0025), but not drinking water pH (F_1,36 = 0.013, p = 0.91), and no significant interaction of CORT treatment × drinking water pH (F_1,36 = 0.059, p = 0.81) (Fig. 4B). These results indicate that chronic CORT treatment suppresses weight gain and atrophies the adrenal glands, but pH 9.0 water intake does not prevent these effects.

Fig. 4. Body (A) and Adrenal Gland (B) Weights in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

CORT (40 mg/kg) was administered subcutaneously for 21 d (Days 1–21). Drinking water was replaced 24 h before CORT treatment. The adrenal glands were collected 24 h after the last administration. n = 10.

Behaviors in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

The effects of pH 9.0 water intake on abnormal behaviors in mice chronically treated with CORT were examined. For the forced swim test, two-way ANOVA revealed no significant main effect of CORT treatment (F_1,36 = 3.97, p = 0.054) or drinking water pH (F_1,36 = 5.23, p = 0.28), but a significant interaction of CORT treatment × drinking water pH (F_1,36 = 19.57, p < 0.0001). A post-hoc Tukey HSD test revealed that the immobility time was significantly higher in CORT chronically treated mice compared to controls in mice given pH 7.5 water (p = 0.0003), and significantly lower in CORT chronically treated mice given pH 9.0 water compared to those drinking pH 7.5 water (p = 0.0002) (Fig. 5A).

Fig. 5. Behaviors in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

CORT (40 mg/kg) was administered subcutaneously for 21 d. Drinking water was replaced 24 h before CORT treatment. The immobility time in the forced swim test (A) and the total distance traveled by each mouse (B) and time spent in the center area (C) in the open field test were measured 24 h after the last administration. * p < 0.05, *** p < 0.001. n = 10.

For total distance traveled in the open field test, two-way ANOVA revealed no significant main effect of CORT treatment (F_1,36 = 0.087, p = 0.77) or drinking water pH (F_1,36 = 0.17, p = 0.68) and no significant interaction of CORT treatment × drinking water pH (F_1,36 = 0.83, p = 0.37) (Fig. 5B). For time spent in the center area in the open field test, two-way ANOVA revealed no significant main effect of CORT treatment (F_1,36 = 7.68, p = 0.0088) or drinking water pH (F_1,36 = 5.20, p = 0.029) and no significant interaction of CORT treatment × drinking water pH (F_1,36 = 0.89, p = 0.35) (Fig. 5C). These results indicate that chronic CORT treatment and pH 9.0 water intake do not affect spontaneous locomotor activity, but decrease time spent in the center area.

Hippocampal Neurogenesis in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

The effects of pH 9.0 water intake on decreased hippocampal neurogenesis were examined in mice chronically treated with CORT (Fig. 6A). For the number of BrdU-positive cells, two-way ANOVA revealed a significant main effect of CORT treatment (F_1,44 = 4.07, p = 0.0499), but not of drinking water pH (F_1,44 = 0.22, p = 0.64), and no significant interaction of CORT treatment × drinking water pH (F_1,44 = 0.58, p = 0.45) (Fig. 6B). These results indicate that chronic CORT treatment decreases the number of BrdU-positive cells, but pH 9.0 water intake does not prevent these effects.

Fig. 6. Hippocampal Neurogenesis in CORT Chronically Treated Mice Given pH 7.5 or 9.0 Water

CORT (40 mg/kg) was administered subcutaneously for 21 d. Drinking water was replaced 24 h before CORT treatment and 5-bromo-2′-deoxyuridine (BrdU) (100 mg/kg) was given for 3 consecutive days before CORT treatment. Brains were removed 24 h after the last CORT administration and sections containing the hippocampus were immunostained with an anti-BrdU antibody and an anti-NeuN antibody (A). The number of BrdU-positive cells (B) and percentage of BrdU/NeuN double-positive cells among BrdU-positive cells (C) were measured. Scale bars for the broad image and the magnified image are 100 and 50 µm, respectively. ** p < 0.01, *** p < 0.001. n = 12.

For the percentage of NeuN-positive cells among BrdU-positive cells, two-way ANOVA revealed significant main effects of CORT treatment (F_1,44 = 9.61, p = 0.034) and drinking water pH (F_1,44 = 8.97, p = 0.045), and a significant interaction of CORT treatment × drinking water pH (F_1,44 = 4.89, p = 0.032). A post-hoc Tukey HSD test showed that this percentage was significantly lower in CORT chronically treated mice compared to control mice given pH 7.5 water (p = 0.0027), and higher in CORT chronically treated mice given pH 9.0 water compared to those drinking pH 7.5 water (p = 0.0034) (Fig. 6C).

Involvement of MR and GR in Abnormalities Observed in CORT Chronically Treated Mice

Finally, to identify the involvement of MRs or GRs in the decreased brain pH and increased lactate levels, behavioral abnormalities, and suppression of hippocampal neurogenesis observed in CORT chronically treated mice, the antagonists were administered every 30 min prior to CORT administration. For brain pH, one-way ANOVA (F_3,36 = 19.81, p < 0.0001 in the PFC; F_3,36 = 1.71, p = 0.18 in the STR; F_3,36 = 27.23, p < 0.0001 in the HC) and a post-hoc Tukey HSD test revealed that the decrease in pH caused by chronic CORT treatment was abolished by the GR antagonist, but not by the MR antagonist, in the PFC and HC. No significant changes in pH were observed in the striatum under these conditions (Fig. 7A). For brain lactate levels, one-way ANOVA (F_3,36 = 4.55, p = 0.0084 in the PFC; F_3,36 = 2.47, p = 0.07 in the STR; F_3,36 = 3.57, p = 0.023 in the HC) and a post-hoc Tukey HSD test revealed that the increase in lactate levels caused by chronic CORT treatment was not significantly prevented by either the MR antagonist or the GR antagonist in any brain region (Fig. 7B).

Fig. 7. Brain pH and Lactate Levels in CORT Chronically Treated Mice Co-treated with MR or GR Antagonist

CORT (40 mg/kg) was administered subcutaneously for 21 d. Spironolactone (30 mg/kg), an MR antagonist, and mifepristone (30 mg/kg), a GR antagonist, were treated 30 min before CORT treatment. The PFC, STR, and HC were collected 24 h after the last administration, followed by pH (A) and lactate (B) measurements in the homogenates. * p < 0.01, ** p < 0.01, *** p < 0.001. n = 10.

For body weight, two-way repeated measures ANOVA revealed significant main effects of treatment (F_3,720 = 5.42, p = 0.0035) and days (F_20,720 = 150.87, p < 0.0001) and a significant interaction of treatment × days (F_60,720 = 8.62, p < 0.0001). Bonferroni correction after the repeated measure ANOVA revealed that CORT-induced suppression of weight gain was not prevented by either the MR antagonist or the GR antagonist (Fig. 8A). For adrenal gland weight, one-way ANOVA (F_3,36 = 12.68, p < 0.0001) and a post-hoc Tukey HSD testrevealed that CORT-induced adrenal atrophy was prevented by the GR antagonist, but not by the MR antagonist (Fig. 8B).

Fig. 8. Body (A) and Adrenal Gland (B) Weights in CORT Chronically Treated Mice Co-treated with MR or GR Antagonist

CORT (40 mg/kg) was administered subcutaneously for 21 d (Days 1–21). Spironolactone (30 mg/kg), an MR antagonist, and mifepristone (30 mg/kg), a GR antagonist, were treated 30 min before CORT treatment. The adrenal glands were collected 24 h after the last administration. ** p < 0.01, *** p < 0.001. n = 10.

For the forced swim test, one-way ANOVA (F_3,36 = 5.05, p = 0.0050) and a post-hoc Tukey HSD test revealed that CORT-induced immobility was prevented by the GR antagonist, but not by the MR antagonist (Fig. 9A). For total distance traveled in the open field test, one-way ANOVA (F_3,36 = 5.65, p = 0.0028) and a post-hoc Tukey HSD test revealed that spontaneous locomotor activity was decreased in mice treated with CORT and the MR antagonist or the GR antagonist compared to control mice. (Fig. 9B). For time spent in the center area in the open field test, one-way ANOVA (F_3,36 = 1.94, p = 0.14) revealed that there is no significant difference in the time spent in the central area for either mouse under these conditions (Fig. 9C).

Fig. 9. Behaviors in CORT Chronically Treated Mice Co-treated with MR or GR Antagonist

CORT (40 mg/kg) was administered subcutaneously for 21 d. Spironolactone (30 mg/kg), an MR antagonist, and mifepristone (30 mg/kg), a GR antagonist, were treated 30 min before CORT treatment. The immobility time in the forced swim test (A) and the total distance traveled by each mouse (B) and time spent in the center area (C) in the open field test were measured 24 h after the last administration. ** p < 0.01, *** p < 0.001. n = 10.

For the number of BrdU-positive cells, one-way ANOVA (F_3,36 = 7.77, p = 0.0004) and post-hoc Tukey HSD test revealed that decrease in the number of BrdU-positive cells by chronic CORT treatment was not prevented by either the MR antagonist or the GR antagonist (Figs. 10A, B). For the percentage of NeuN-positive cells among BrdU-positive cells, one-way ANOVA (F_3,36 = 38.02, p < 0.0001) and post-hoc Tukey HSD test revealed that CORT-induced decrease in the percentage was prevented by the GR antagonist, but not by the MR antagonist (Figs. 10A, C).

Fig. 10. Hippocampal Neurogenesis in CORT Chronically Treated Mice Co-treated with MR or GR Antagonist

CORT (40 mg/kg) was administered subcutaneously for 21 d. Spironolactone (30 mg/kg), an MR antagonist, and mifepristone (30 mg/kg), a GR antagonist, were treated 30 min before CORT treatment and 5-bromo-2′-deoxyuridine (BrdU) (100 mg/kg) was given for 3 consecutive days before CORT treatment. Brains were removed 24 h after the last CORT administration and sections containing the hippocampus were immunostained with an anti-BrdU antibody and an anti-NeuN antibody (A). The number of BrdU-positive cells (B) and percentage of BrdU/NeuN double-positive cells among BrdU-positive cells (C) were measured. Scale bars for the broad image and the magnified image are 100 and 50 µm, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 10.

DISCUSSION

This study revealed that a single administration of CORT decreases the pH in the HC. This CORT-induced decrease in hippocampal pH was inhibited by pretreatment with the GR antagonist mifepristone, indicating that the action is mediated by GRs. Given that a single administration of CORT did not decrease pH in the PFC or STR, the HC seems to be the brain region that is particularly susceptible to CORT in terms of a decrease in pH. The reason for this is not clear, but it may be related to differences in the distribution of GRs, as HC is a brain region with relatively high GR expression.²⁴⁾ In contrast to findings in some psychiatric patients and animal models,^12,17) the decrease in hippocampal pH at 24 h after a single administration of CORT was not correlated with lactate levels. This suggests that this decrease in pH is lactate-independent and may be distinct from the pathophysiology in patients with psychiatric disorders.

In contrast to mice treated with a single administration of CORT, those chronically treated with CORT showed a decrease in pH in the PFC and STR, as well as in the HC. Similarly to the correlation between brain pH and lactate levels in psychiatric patients, brain pH and lactate levels were significantly correlated in the PFC and HC, but not in the STR. These findings suggest that the decrease in pH and increase in lactate levels in the PFC and HC may be similar to the pathophysiology in patients with psychiatric disorders, and that the PFC and HC are more prone than the STR to lactate accumulation upon chronic and elevated glucocorticoid exposure. The brain regions that correlate with pH and lactate levels and those that do not may be due to differences in energy metabolism. Lactate is produced mainly in astrocytes in the brain through glucose metabolism, but the glucose metabolism differs greatly depending on the brain regions.²⁵⁾ The cerebral cortex and hippocampus have a high ATP/ADP ratio and are prone to lactate accumulation due to fasting, suggesting that these regions have different characteristics in energy metabolism from other brain regions. Furthermore, the decrease in pH in the PFC and HC caused by chronic CORT treatment was prevented by the GR antagonist mifepristone, but the increase in lactate levels was not significantly prevented. These results indicate that GR is involved in the decrease in brain pH caused by chronic treatment with CORT as well as by a single administration of CORT, and that lactate is not the only factor contributing to the decrease in brain pH.

Since the electrical and biochemical mechanisms of synaptic transmission in the brain are sensitive to pH,²⁶⁾ it is assumed that brain pH homeostasis is robustly maintained. However, several studies have shown that intraperitoneal administration of sodium bicarbonate (NaHCO₃) increases brain pH.^27,28) The current study also showed that brain pH was slightly, but significantly, higher in mice given pH 9.0 water compared to mice given pH 7.5 water. Based on this finding, we investigated the involvement of brain pH in the various abnormalities observed in CORT chronically treated mice. Although we expected that pH 9.0 water would alleviate the decrease in pH in each brain region induced by chronic CORT, it was surprising that pH 9.0 water also alleviated the increase in lactate levels in the STR and HC. Furthermore, pH and lactate levels were significantly correlated in the HC of mice given pH 9.0 water. These results suggest that changes in pH due to external factors may affect lactate levels in some brain regions. There may be an interaction between pH and lactate, such that a decrease in pH may cause an increase in lactate levels, while an increase in lactate may cause a decrease in pH. Since this phenomenon has not been reported before, the mechanism by which a decrease in pH causes an increase in lactate levels is unclear, but it is thought that a decrease in intracellular and extracellular pH may cause a change in the function of proteins involved in lactate metabolism. Monocarboxylate transporters, a lactate transporter, co-transport lactate and protons, and it is known that their function changes depending on the difference in pH between the intracellular and extracellular environments,²⁹⁾ so they may be involved in the accumulation of lactate due to changes in pH. A decrease in the intracellular and extracellular pH may also change the function of the synthetic enzymes. To clarify the detailed mechanism, it will be necessary to measure intracellular pH and extracellular pH separately in the future.

Intake of pH 9.0 water did not affect peripheral abnormalities such as suppression of body weight gain or adrenal atrophy in CORT chronically treated mice, suggesting that the decrease in brain pH is not involved in these peripheral abnormalities. In contrast to the periphery, pH 9.0 water intake prevented depression-like behavior characterized by increased immobility in the forced swim test in CORT chronically treated mice. Furthermore, pH 9.0 water intake alleviated the decreased percentage of NeuN-positive cells among BrdU-positive cells, but not the decreased number of BrdU-positive cells, in the HC, suggesting that hippocampal pH affects differentiation and maturation of neural stem/progenitor cells into neurons. The mechanism by which brain pH regulates neurogenesis is unclear, but it is thought to involve factors that respond to intracellular and extracellular pH. There are several signal pathways in cells that change depending on the intracellular and extracellular pH. Wnt/β-catenin pathway is an intracellular signaling pathway that orchestrates differentiation in neural stem cells in the adult hippocampus,³⁰⁾ and its function is regulated by intracellular pH.^31,32) Acid-sensing ion channels that open in response to extracellular pH are also involved in the differentiation of neural stem cells.³³⁾ Given that extracellular pH is known to affect differentiation and proliferation of stem cells,^34,35) the current findings indicate that depression-like symptoms caused by chronic exposure to elevated levels of glucocorticoids may involve a decrease in brain pH.

There was no increase in the immobility time in the forced swim test or decrease in the percentage of NeuN-positive cells among BrdU-positive cells in the dentate gyrus of the hippocampus caused by chronic CORT treatment in the GR antagonist-co-treated mice whose pH dose not decrease by CORT, as there was not in mice given pH 9.0 water. These results support the hypothesis that a decrease in brain pH via GRs is involved in depression-like behavior and a decrease in hippocampal neurogenesis. In contrast, the atrophy of the adrenal glands caused by chronic CORT treatment was prevented by co-treatment with the GR antagonist, but not by the intake of pH 9.0 water, suggesting that suggests that GRs is involved in the adrenal atrophy, but not in pH. In discussing the involvement of GRs based on the results of this study, it is important to note that mifepristone blocks the progesterone receptor as well as GRs. Although the current results do not completely rule out the involvement of progesterone receptors, the fact that the changes caused by CORT loading are blocked by mifepristone suggests that GRs are likely to be involved. Further investigation is needed to rule out the possibility of involvement of the progesterone receptors.

In contrast to the depression-like behavior characterized by increased immobility in the forced swim test, the anxiety-like behavior characterized by decreased time spent in the central area of the open field test was not affected by the intake of pH 9.0 water. Although it is necessary to examine multiple behavioral tests to fully evaluate anxiety-like behavior, it is speculated that some of the behavioral abnormalities caused by chronic CORT treatment are dependent on brain pH, while others are not, based on the current data. This difference in behaviors may be due to whether or not they are dependent on hippocampal neurogenesis.

The purpose of giving mice pH 9.0 water in this study is to alleviate the decrease in brain pH caused by CORT, and it is used as an experimental tool. This study did not investigate whether the intake of high pH water alleviates depression. Therefore, current results should not be easily extrapolated to humans, that is, they do not support the efficacy of pH 9.0 water intake for depression.

In conclusion, brain pH fluctuates due to external stimuli such as glucocorticoids and drinking water, and these pH fluctuations may affect brain function. Further studies are needed to clarify whether mechanisms regulating brain pH are therapeutic targets for mood disorders.

Acknowledgments

We thank Mr. Mutsuki Asanuma, Ms. Saki Nishiura, Ms. Yuka Niizeki, Ms. Moe Tomita, and Ms. Ramu Nishida for their technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev., 21, 55–89 (2000).
• 2) McEwen BS. Physiology and neurobiology of stress and adaptation: central role of the brain. Physiol. Rev., 87, 873–904 (2007).
• 3) Nelson JC, Davis JM. DST studies in psychotic depression: a meta-analysis. Am. J. Psychiatry, 154, 1497–1503 (1997).
• 4) Sahu MK, Dubey RK, Chandrakar A, Kumar M, Kumar M. A systematic review and meta-analysis of serum and plasma cortisol levels in depressed patients versus control. Indian J. Psychiatry, 64, 440–448 (2022).
• 5) Ago Y, Yano K, Araki R, Hiramatsu N, Kita Y, Kawasaki T, Onoe H, Chaki S, Nakazato A, Hashimoto H, Baba A, Takuma K, Matsuda T. Metabotropic glutamate 2/3 receptor antagonists improve behavioral and prefrontal dopaminergic alterations in the chronic corticosterone-induced depression model in mice. Neuropharmacology, 65, 29–38 (2013).
• 6) Gourley SL, Wu FJ, Kiraly DD, Ploski JE, Kedves AT, Duman RS, Taylor JR. Regionally specific regulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depression. Biol. Psychiatry, 63, 353–359 (2008).
• 7) Wong EY, Herbert J. The corticoid environment: a determining factor for neural progenitors’ survival in the adult hippocampus. Eur. J. Neurosci., 20, 2491–2498 (2004).
• 8) Wong EY, Herbert J. Raised circulating corticosterone inhibits neuronal differentiation of progenitor cells in the adult hippocampus. Neuroscience, 137, 83–92 (2006).
• 9) Anacker C, Luna VM, Stevens GS, Millette A, Shores R, Jimenez JC, Chen B, Hen R. Hippocampal neurogenesis confers stress resilience by inhibiting the ventral dentate gyrus. Nature, 559, 98–102 (2018).
• 10) Surget A, Belzung C. Adult hippocampal neurogenesis shapes adaptation and improves stress response: a mechanistic and integrative perspective. Mol. Psychiatry, 27, 403–421 (2022).
• 11) Guillozet-Bongaarts AL, Hyde TM, Dalley RA, Hawrylycz MJ, Henry A, Hof PR, Hohmann J, Jones AR, Kuan CL, Royall J, Shen E, Swanson B, Zeng H, Kleinman JE. Altered gene expression in the dorsolateral prefrontal cortex of individuals with schizophrenia. Mol. Psychiatry, 19, 478–485 (2014).
• 12) Halim ND, Lipska BK, Hyde TM, Deep-Soboslay A, Saylor EM, Herman MM, Thakar J, Verma A, Kleinman JE. Increased lactate levels and reduced pH in postmortem brains of schizophrenics: medication confounds. J. Neurosci. Methods, 169, 208–213 (2008).
• 13) Iwamoto K, Bundo M, Kato T. Altered expression of mitochondria-related genes in postmortem brains of patients with bipolar disorder or schizophrenia, as revealed by large-scale DNA microarray analysis. Hum. Mol. Genet., 14, 241–253 (2005).
• 14) Mistry M, Gillis J, Pavlidis P. Meta-analysis of gene coexpression networks in the post-mortem prefrontal cortex of patients with schizophrenia and unaffected controls. BMC Neurosci., 14, 105 (2013).
• 15) Ryan MM, Lockstone HE, Huffaker SJ, Wayland MT, Webster MJ, Bahn S. Gene expression analysis of bipolar disorder reveals downregulation of the ubiquitin cycle and alterations in synaptic genes. Mol. Psychiatry, 11, 965–978 (2006).
• 16) Shao L, Vawter MP. Shared gene expression alterations in schizophrenia and bipolar disorder. Biol. Psychiatry, 64, 89–97 (2008).
• 17) Hagihara H, Catts VS, Katayama Y, Shoji H, Takagi T, Huang FL, Nakao A, Mori Y, Huang KP, Ishii S, Graef IA, Nakayama KI, Shannon Weickert C, Miyakawa T. Decreased brain pH as a shared endophenotype of psychiatric disorders. Neuropsychopharmacology, 43, 459–468 (2018).
• 18) Araki R, Tachioka H, Kita A, Fujiwara H, Toume K, Matsumoto K, Yabe T. Kihito prevents corticosterone-induced brain dysfunctions in mice. J. Tradit. Complement. Med., 11, 513–519 (2021).
• 19) Grobe JL, Grobe CL, Beltz TG, Westphal SG, Morgan DA, Xu D, de Lange WJ, Li H, Sakai K, Thedens DR, Cassis LA, Rahmouni K, Mark AL, Johnson AK, Sigmund CD. The brain renin-angiotensin system controls divergent efferent mechanisms to regulate fluid and energy balance. Cell Metab., 12, 431–442 (2010).
• 20) Meir Drexler S, Wolf OT. The role of glucocorticoids in emotional memory reconsolidation. Neurobiol. Learn. Mem., 142 (Pt/.A), 126–134 (2017).
• 21) Fiancette JF, Balado E, Piazza PV, Deroche-Gamonet V. Mifepristone and spironolactone differently alter cocaine intravenous self-administration and cocaine-induced locomotion in C57BL/6J mice. Addict. Biol., 15, 81–87 (2010).
• 22) Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature, 266, 730–732 (1977).
• 23) Nishida S, Araki R, Baba A, Asari S, Tachibana S, Nakajima Y, Iwakumo A, Yabe T. Post-weaning folate deficiency induces a depression-like state via neuronal immaturity of the dentate gyrus in mice. J. Pharmacol. Sci., 143, 97–105 (2020).
• 24) Morimoto M, Morita N, Ozawa H, Yokoyama K, Kawata M. Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study. Neurosci. Res., 26, 235–269 (1996).
• 25) Kleinridders A, Ferris HA, Reyzer ML, Rath M, Soto M, Manier ML, Spraggins J, Yang Z, Stanton RC, Caprioli RM, Kahn CR. Regional differences in brain glucose metabolism determined by imaging mass spectrometry. Mol. Metab., 12, 113–121 (2018).
• 26) Sinning A, Hubner CA. Minireview: pH and synaptic transmission. FEBS Lett., 587, 1923–1928 (2013).
• 27) Ziemann AE, Allen JE, Dahdaleh NS, Drebot II, Coryell MW, Wunsch AM, Lynch CM, Faraci FM, Howard MA 3rd, Welsh MJ, Wemmie JA. The amygdala is a chemosensor that detects carbon dioxide and acidosis to elicit fear behavior. Cell, 139, 1012–1021 (2009).
• 28) Schuchmann S, Schmitz D, Rivera C, Vanhatalo S, Salmen B, Mackie K, Sipila ST, Voipio J, Kaila K. Experimental febrile seizures are precipitated by a hyperthermia-induced respiratory alkalosis. Nat. Med., 12, 817–823 (2006).
• 29) Kobayashi M, Narumi K, Furugen A, Iseki K. Transport function, regulation, and biology of human monocarboxylate transporter 1 (hMCT1) and 4 (hMCT4). Pharmacol. Ther., 226, 107862 (2021).
• 30) Kuwabara T, Hsieh J, Muotri A, Yeo G, Warashina M, Lie DC, Moore L, Nakashima K, Asashima M, Gage FH. Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nat. Neurosci., 12, 1097–1105 (2009).
• 31) Czowski BJ, White KA. Intracellular pH regulates beta-catenin with low pHi increasing adhesion and signaling functions. bioRxiv, (2024).
• 32) Oginuma M, Harima Y, Tarazona OA, Diaz-Cuadros M, Michaut A, Ishitani T, Xiong F, Pourquie O. Intracellular pH controls WNT downstream of glycolysis in amniote embryos. Nature, 584, 98–101 (2020).
• 33) Ge H, Zhou T, Zhang C, Cun Y, Chen W, Yang Y, Zhang Q, Li H, Zhong J, Zhang X, Feng H, Hu R. Targeting ASIC1a promotes neural progenitor cell migration and neurogenesis in ischemic stroke. Research (Wash. D. C.), 6, 0105 (2023).
• 34) Kim N, Minami N, Yamada M, Imai H. Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells. Reprod. Med. Biol., 16, 58–66 (2016).
• 35) Chen X, Chen A, Woo TL, Choo AB, Reuveny S, Oh SK. Investigations into the metabolism of two-dimensional colony and suspended microcarrier cultures of human embryonic stem cells in serum-free media. Stem Cells Dev., 19, 1781–1792 (2010).