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Dopamine-induced astrocytic Ca2+ signaling in mPFC is mediated by MAO-B in young mice, but by dopamine receptors in adult mice
Molecular Brain volume 15, Article number: 90 (2022)
Dopamine (DA) plays a vital role in brain physiology and pathology such as learning and memory, motor control, neurological diseases, and psychiatric diseases. In neurons, it has been well established that DA increases or decreases intracellular cyclic AMP (cAMP) through D1-like or D2-like dopamine receptors, respectively. In contrast, it has been elusive how astrocytes respond to DA via Ca2+ signaling and regulate synaptic transmission and reward systems. Previous studies suggest various molecular targets such as MAO-B, D1R, or D1R–D2R heteromer to modulate astrocytic Ca2+ signaling. However, which molecular target is utilized under what physiological condition remains unclear. Here, we show that DA-induced astrocytic Ca2+ signaling pathway switches during development: MAO-B is the major player at a young age (5–6 weeks), whereas DA receptors (DARs) are responsible for the adult period (8–12 weeks). DA-mediated Ca2+ response in the adult period was decreased by either D1R or D2R blockers, which are primarily known for cyclic AMP signaling (Gs and Gi pathway, respectively), suggesting that this Ca2+ response might be mediated through Gq pathway by D1R–D2R heterodimer. Moreover, DAR-mediated Ca2+ response was not blocked by TTX, implying that this response is not a secondary response caused by neuronal activation. Our study proposes an age-specific molecular target of DA-induced astrocytic Ca2+ signaling: MAO-B in young mice and DAR in adult mice.
Dopamine (DA) is a neurotransmitter that functions in the brain to regulate neural processes, including the reward system, motor control, cognition, and memory . The main source of DA in the brain is the DAergic neurons residing in the ventral tegmental area, and their primary target includes the striatum, cortical areas, and recently hippocampus [2, 3]. Among these brain regions, the medial prefrontal cortex (mPFC) is regarded as a center for cognitive control, which integrates sensory information and associates many complex cognitive processes [4, 5]. In terms of the DA signaling pathway, in neurons, it has been well studied that DA activates dopamine receptors (DARs), and their activation upregulates or downregulates intracellular cyclic AMP (cAMP) levels depending on the receptor types. However, the signaling pathway and physiological roles of DA in astrocytes are still under active investigation.
Astrocytes are the most abundant cell types in the brain and play a vital role in both physiological [6,7,8] and pathological [9,10,11,12,13] conditions. The astrocytic Ca2+ signaling is critically involved in various gliotransmitter releases such as glutamate , GABA , ATP , and d-serine , which are critical for glia-neuron communication. While there are many signaling pathways that trigger intracellular Ca2+ influx in astrocytes, a Gq-coupled G-protein coupled receptor (GPCR) is the predominant form of Ca2+ elevations in astrocytes via IP3R2 in the endoplasmic reticulum (ER) . There are a plethora of neurotransmitters or neuromodulators that can trigger astrocytic Ca2+ (i.e. ATP, glutamate, norepinephrine, and acetylcholine) via Gq-GPCR pathways . On the other hand, there is a noncanonical GPCR pathway that can evoke Ca2+ signaling by oligomerization of Gi-coupled GABAB receptors .
In contrast to neurons, a recent study reported that DA does not modulate cAMP level of astrocytes . Instead, astrocytes have been shown to elevate their intracellular Ca2+ in response to DA in vitro , ex vivo [16, 23], and in vivo  in various brain regions. The suggested pathways of DA-mediate astrocytic Ca2+ signaling are (1) monoamine oxidase B (MAO-B) pathway  and (2) DAR pathway [16, 21, 23]. In the MAO-B pathway, ROS generated during DA metabolism causes lipid peroxidation and phospholipase C (PLC) activation, which induces the IP3R2-mediated Ca2+ release from the endoplasmic reticulum (ER). On the other hand, we have previously demonstrated that MAO-B in adult striatum is responsible for GABA synthesis, but not DA degradation . Meanwhile, DAR-mediated Ca2+ signaling in astrocytes is not well characterized but expected to share the IP3R2-mediated Ca2+ release from ER. Previous reports suggest that in nucleus accumbens, astrocytic D1R  or neuronal D2R  can induce intracellular Ca2+ response. Another possibility is D1R-D2R heteromerization observed in artificial in vitro systems [26, 27]. However, which molecular target is utilized under what physiological condition still remains elusive.
In this study, we investigate the age-dependent change of DA-induced Ca2+ signaling in mouse mPFC astrocytes. Using genetically encoded calcium indicator (GECI), GCaMP6f, we have found that DA-induced astrocytic Ca2+ signaling switches from the MAO-B pathway (5–6 weeks) to the DAR pathway (8–12 weeks). Our findings show the age-dependent switching of DA-induced astrocytic Ca2+ signaling during development and suggest an age-specific target to modulate astrocytic Ca2+ signaling.
Materials and methods
Young and adult C57BL/6J mice were provided by IBS Research Solution Center. MAO-B KO mice (014133, Jackson Laboratory) were maintained on a heterozygote. MAO-B WT and MAO-B KO mice were obtained from mating cages of heterozygous males and females. The animals were kept on a 12 h light–dark cycle with controlled temperature and humidity and had free access to food and water. Mice were kept in group-housed (4 to 5 mice together). Experimenters and animal managers took care of mice following National Institutes of Health (NIH) guidelines. Every animal experimental procedure was approved by the Institutional Animal Care and Use Committee of the Institute for Basic Science (IBS, Daejeon, Korea).
The AAV containing GfaABC1D-GCaMP6f (titer = 4.09 × 1012) was packaged by the IBS virus facility (Daejeon, Korea). Before injection, the virus was diluted by 3 times with saline.
For ex vivo astrocyte Ca2+ imaging, KDS2010 (Kindly provided by Dr. Ki Duk Park, KIST), haloperidol (H1512, Sigma-Aldrich), SCH-23390 (0925, Tocris), and TTX (T-550, Alomone labs) were bath applied for 15 min. The concentration of each chemical is indicated in each experiment.
Mice were deeply anesthetized with isoflurane (3% for induction and 1.5% during surgery) and placed on a stereotaxic device (68537, RWD). After an incision of the scalp, the skull was cleaned and matched the dorsoventral level of bregma and lambda for accurate targeting of a brain region. The holes were drilled bilaterally in the skull targeting the mPFC using the following coordinates: AP, 1.8 mm; ML, ± 0.4 mm; DV, − 2.3 (young) or − 2.5 (adult) mm from the bregma. A glass capillary (53508–375, VWR) was pulled by a vertical puller (PC-100, Narishige) and was connected to a microinfusion pump (Legato 130, KD Scientific) through polyethylene tubings (PE30 and PE90, BD intramedic). The AAV-GfaABC1D-GCaMP6f virus was loaded into the glass capillary and slowly infused into mPFC at a rate of 0.1 μL/min with 0.5 μL of total volume in each hemisphere. The injection needle was left in place for an additional 10 min before the withdrawal. For young mice, the virus was injected at postnatal day 25. After at least 2 weeks of recovery, the mice were sacrificed and used for imaging experiments.
Acute brain slice preparation
Mice were deeply anesthetized with 3% isoflurane and decapitated. The brain was quickly removed from the skull and submerged in cold sucrose-based dissection buffer (in mM): 212.5 sucrose, 26 NaHCO3, 10 d-glucose, 5 MgCl2, 3 KCl, 1.25 NaH2PO4 and 0.1 CaCl2; pH 7.4, saturated with 95% O2 and 5% CO2. To obtain mPFC brain slices, the brain was attached to the chamber of a vibratome (D.S.K LinearSlicer pro 7, Dosaka EM Co. Ltd) and 300-μm-thick coronal slices were collected. Slices were then transferred to an incubation chamber filled with artificial cerebrospinal fluid (aCSF) (in mM): 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.5 CaCl2, 1.5 MgCl2 and 10 glucose; pH 7.4, saturated with 95% O2 and 5% CO2, at room temperature. All imaging experiments were conducted after 1 h of stabilization.
Ex vivo astrocyte Ca2+ imaging
After stabilization, obtained brain slices were moved to a recording chamber where aCSF continuously flowed over slices at around 1 mL/min. To perform Ca2+ imaging, a blue light (pE340fura, CoolLED) was applied to brain slices under a fluorescent upright microscope (Zeiss Examiner.D1, Zeiss) with a 63 × water objective (Zeiss). Drugs were treated by bath application which is controlled by a valve controller (VC-6 six channel valve controller, Warner instrument). To measure Ca2+ response, the peak of GCaMP6f signals after drug treatment was normalized by its baseline (ΔF/F0 ratio). Analyzed ROIs were chosen based on the response from the 1st (or before drug) DA treatment. Image acquisition and ROI analysis were performed using Imaging Workbench (INDEC Biosystems) and ImageJ (NIH).
All data are given as mean ± SEM. Statistical comparisons were performed using an unpaired Student’s t-test or a one-way ANOVA test using Prism 9 (GraphPad). Statistical significance is indicated as follows: ***p < 0.001; ****p < 0.0001; ns, not significant.
A recent study suggested that mPFC astrocytes were shown to elevate intracellular Ca2+ in response to DA . To recapitulate the previous finding, we injected the AAV-GfaABC1D-GCaMP6f virus into the mPFC region in young (5–6 weeks) and adult (> 8 weeks) mice and conducted ex vivo astrocyte Ca2+ imaging (Fig. 1A and B). We found that DA application to the mPFC astrocytes faithfully elevated the Ca2+ responses in both young (61.01 ± 2.620) and adult mice (66.89 ± 2.852), which are consistent with previous findings (Fig. 1C and D). To elucidate the molecular mechanism of DA-induced Ca2+ signaling pathway in mPFC astrocytes, we pharmacologically inhibited two potential pathways: the MAO-B-mediated pathway and the DAR-mediated pathway (Fig. 1A). We found that KDS2010, a reversible MAO-B inhibitor, significantly decreased DA-induced Ca2+ response in young (35.47 ± 2.210), whereas KDS2010 significantly increased in adult mPFC astrocytes (82.51 ± 1.954, Fig. 1E and F). To test whether DA-induced Ca2+ response in the adult is mediated by DARs, we applied D1R antagonist (SCH-23390) and D2R antagonist (haloperidol) simultaneously. In contrast to the result from MAO-B inhibitor condition, DAR antagonists significantly reduced DA-induced Ca2+ response in an adult (27.92 ± 2.591) but not in young mPFC astrocytes (59.79 ± 3.475, Fig. 1G and H). Taken together, these results highlight the age-dependent switching of DA-induced Ca2+ signaling pathway in mPFC astrocytes.
To investigate which type of DA receptors evokes Ca2+ response in adult mPFC astrocytes, we separately applied the antagonists of D1R (SCH-23390) and D2R (haloperidol). DA-induced Ca2+ response in adult mPFC astrocytes was almost completely blocked by both SCH-23390 (10.20 ± 2.394) and haloperidol (9.185 ± 2.412) compared to control (69.41 ± 5.006; Fig. 2A and B). To examine the involvement of neuronal activity, we applied tetrodotoxin (TTX), a neuronal activity blocker, and found no significant difference compared to control (76.47 ± 3.747; Fig. 2A and B), suggesting that astrocytic Ca2+ response is not a secondary effect of neuronal activity. In addition to pharmacological inhibition of MAO-B, we utilized MAO-B knockout (KO) mice. DA-induced astrocytic Ca2+ responses were not significantly different between MAO-B WT (79.07 ± 10.52) and MAO-B KO (80.94 ± 8.578), suggesting that MAO-B is not a mediator of DA-induced Ca2+ signaling in adult astrocytes (Fig. 2C and D). Taken together, our results strongly suggest that DA-induced astrocytic Ca2+ signaling in adult mice is mediated by D1R, D2R, or both, but not by MAO-B.
In this study, we have investigated the astrocytic Ca2+ signaling in response to DA in young and adult periods. Our results indicate that MAO-B-mediated Ca2+ signaling is the predominant pathway during the young (5–6 weeks) period, whereas DAR-mediated Ca2+ signaling is the major form during the adult (8–12 weeks) period. It is intriguing that DA response is age-dependently switched from MAO-B to DAR pathway within 2 weeks of the juvenile period.
One possible explanation for this switching phenomenon might be due to a change of astrocytic role in response to DA. There is a previous report that astrocyte is critical for DA homeostasis in the developing prefrontal cortex . Based on this report, in mPFC of young mice, DA is actively taken up by astrocytes through vesicular monoamine transporter 2 (VMAT2) and is degraded by MAO-B. During this process, hydrogen peroxide (H2O2) is produced and it might evoke ER Ca2+ release . This Ca2+ release might be crucial for the expression of synapse-regulating genes and synaptic development . On the other hand, rather than homeostatic control of DA, DA-induced Ca2+ signaling in adult mPFC astrocytes possibly induces a release of various gliotransmitter such as glutamate, D-serine, GABA, and ATP. It would be interesting to investigate which gliotransmitter is released in future studies.
In this study, we found that DA-induced astrocytic Ca2+ response is majorly through MAO-B in mPFC of young mice. However, in adult mice, MAO-B-dependent Ca2+ elevation in response to DA disappeared. This is an interesting phenomenon because MAO-B expression level is known to increase along with aging in most structures of rodent brains . The previous study suggested that astroglial VMAT2 is essential for DA uptake and degradation in the developing prefrontal cortex . However, there has been no report about the expression level of astrocytic VMAT2 in adulthood, which should be investigated in the future. Additionally, we reported that MAO-B is not responsible for DA degradation in the adult striatum . Based on these results, we expect that in adult mPFC astrocytes, the level of dopamine transporters is low, which leads to minimal DA uptake in MAO-B-expressing astrocytes. Therefore, there is no link between DA-induced Ca2+ response and MAO-B in adulthood. Then what could be the role of MAO-B in the adult mPFC? As we have recently demonstrated, MAO-B is responsible for synthesizing GABA in adult striatal astrocytes, rather than DA degradation . Based on this report, it would be possible that MAO-B in adult mPFC synthesizes GABA to modulate nearby neuronal activities. Also, this age-dependent astrocytic role of MAO-B (DA degradation in young mice and GABA synthesis in adult mice) might occur in other brain regions such as nucleus accumbens, striatum, hippocampus, and other cortical areas. These exciting possibilities await future investigation.
Availability of data and materials
Medial prefrontal cortex
Monoamine oxidase B
Dopamine receptor D1
Dopamine receptor D2
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Special thanks to Dr. Ki Duk Park for synthesizing and providing KDS2010.
This work is supported by the Institute for Basic Science (IBS), Center for Cognition and Sociality (IBS-R001-D2) funded by the Ministry of Science to C.J.L.
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This study was approved by the Institutional Animal Care and Use Committee of the Institute for Basic Science (IBS, Daejeon, Korea).
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Kim, S., Kwon, J., Park, M.G. et al. Dopamine-induced astrocytic Ca2+ signaling in mPFC is mediated by MAO-B in young mice, but by dopamine receptors in adult mice. Mol Brain 15, 90 (2022). https://doi.org/10.1186/s13041-022-00977-w
- Ca2+ response
- Dopamine receptors