Role of astrocytic mu-opioid receptors of the ventrolateral periaqueductal gray in modulating anxiety-like responses

Role of astrocytic mu-opioid receptors of the ventrolateral periaqueductal gray in modulating anxiety-like responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of astrocytic mu-opioid receptors of the ventrolateral periaqueductal gray in modulating anxiety-like responses Yinan Du, Aozhuo Zhang, Zhiwei Li, Yukui Zhao, Shuyi Liu, Chunling Wei, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6262877/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Behavioral and Brain Functions → Version 1 posted 11 You are reading this latest preprint version Abstract Background Mu-opioid receptors (MORs) are critical regulators mediating the modulation of several behavioral reactions, including analgesia, addiction, and sedation. Recent studies have reported that MORs are closely associated with mood disorders or anxiety behaviors; however, the underlying neural mechanisms remain unclear. The periaqueductal gray (PAG), a key brain area, participates in the modulation of aversive emotional behaviors. MORs show a high expression in the ventrolateral PAG (vlPAG) region. This study explored the preliminary role of MORs expressed in the vlPAG in modulating emotional behaviors. Results Bilateral administration of DAMGO, an MOR-specific agonist, into the vlPAG of male mice elicited anxiety-like behaviors in elevated plus maze tests. This phenotype was reversed by conditional knockdown of astrocytic MORs. In contrast, glutamatergic or GABAergic MORs were not involved in vlPAG MOR-dependent anxiety-like behaviors. By using in vitro calcium imaging of vlPAG astrocytes and chemical genetic technologies, we found that vlPAG astrocytic MORs can promote astrocytic calcium signaling, which can efficiently induce anxiety-like behaviors. Accordingly, the interference of astrocytic calcium signaling by viral infection reversed vlPAG-dependent anxiety-like behaviors. Conclusion Our findings demonstrated that vlPAG astrocytic, but not glutamatergic or GABAergic, MORs are involved in modulating emotional reactions, and these effects are accomplished by MOR-elicited astrocytic calcium signaling mechanisms. The present study provides a theoretical basis for treating emotional dysfunctions during MOR-targeted management. Anxiety mu-opioid receptors periaqueductal gray astrocytes calcium signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mu-opioid receptor (MOR), a member of the endogenous opioid system, is a major regulator of pain sensation and reaction in the central nervous system (CNS) [ 1 , 2 ]. According to several studies, a strong correlation exists between MOR activity levels and anxiety behavior [ 3 – 5 ]. A high prevalence of mood disorders and anxiety behavior is observed in individuals habitually using MOR-targeted analgesics [ 4 , 5 ]. Thus, the role of MORs in influencing anxiety or anxiety-like behaviors requires greater attention. MORs are widely expressed in several brain areas that control emotional behaviors, such as the amygdala, hippocampus, prefrontal cortex, bed nucleus of the stria terminalis, and periaqueductal gray (PAG) [ 6 , 7 ]. Among these areas, the PAG is a brainstem region with predominant involvement in modulating aversive emotional or defensive behaviors [ 8 – 10 ]. It receives afferent connections from the prefrontal cortex, central amygdala, hypothalamus, and habenula nucleus; it integrates neurotransmission from these brain areas and exports an emotional primary process [ 8 , 10 ]. The PAG contains four longitudinal columns: the dorsomedial PAG (dmPAG), dorsolateral PAG (dlPAG), lateral PAG (lPAG), and ventrolateral PAG (vlPAG) [ 8 – 10 ]. The vlPAG has a close association with negative emotional responses [ 11 , 12 ]. Altered excitability of vlPAG neurons has been observed in emotional-related disorders, and chemogenetic activation of vlPAG glutamatergic neurons induces anxiety-like behavior in the light/dark test and open field test in rodents [ 11 ]. These findings indicate the functional role of the vlPAG in modulating negative emotional behavior. Both morphological and biochemical investigations have reported the dense distribution of MORs in the vlPAG [ 2 , 7 ]. Additionally, pharmacological or genetic intervention targeted toward the functions of vlPAG MORs can fluctuate the neural excitability of the PAG [ 7 , 13 ]. These findings suggest a potential role of PAG MORs in modulating emotional behavior. Moreover, by using combined in situ hybridization and immunofluorescence technology, several cell types, including glutamatergic (MOR Glut ), gamma-aminobutyric acid (GABA) ergic (MOR GABA ), and astrocytic (MOR Astro ) neurocytes, were found to express MORs in the vlPAG [ 7 , 14 ]; this suggests that the multiple interactions between MORs on these different neuronal types might be involved in emotional behavior control. Here, we performed bilateral micro-administration of the MOR-specific agonist DAMGO in male mice and observed how local activation of vlPAG MORs affected anxiety-like behavior through the elevated plus maze (EPM) test. By developing three male murine models in which glutamatergic, GABAergic, or astrocytic MORs were conditionally knocked down, we detected the role of divergent neuronal types of vlPAG MORs on the modulation of anxiety-like behaviors. Furthermore, by using in vitro calcium imaging and chemogenetic and virus-interfering technologies, we elucidated the preliminary mechanisms of these effects. DAMGO induced significant MOR-dependent anxiety-like effects in the vlPAG. Interestingly, these effects were induced by MOR Astro but not by MOR Glut or MOR GABA . Moreover, the induction and enhancement of calcium signaling could be critical mechanisms for inducing vlPAG MOR Astro -dependent anxiety-like behavior. Our study highlights a new MOR Astro elicited anxiety-like behavior paradigm in the PAG. These observations offer a theoretical premise for treating emotional dysfunctions during MOR-targeted management. Materials and methods Animals The study involved 78 C57BL/6J male mice, 42 MOR mutant male mice, and 42 littermate male controls (8–12 weeks old). All mice were supplied by the Experimental Animal Center of Shaanxi Normal University, MOE Key Laboratory of Modern Teaching Technology. The mice were housed in individually ventilated cages in groups (5–6 animals per group) and maintained under the following conditions: temperature: 21°C ± 2°C; relative humidity: 50% ± 5%; sufficient food and water; and 12-h light/dark cycle. Before the behavioral experiments, the animals were acclimated to the environment and apparatus. The use count of mice in each experiment is detailed in the Results section. The mutant mouse lines were generated as reported previously [ 7 , 15 ]. Briefly, mice specifically lacking MORs in MOR Astro , MOR GABA , or MOR Glut were generated by crossing Oprm1 -floxed mice to GFAP-CreERT2 , GAD2 - iCreERT2 , or vGlut2-iCreERT2 mice, respectively. The adult Oprm1 loxP/loxP : GFAP-CreERT2 (MOR Astro −/−), Oprm1 loxP/loxP : Gad2-iCreERT2 (MOR GABA −/−), or Oprm1 loxP/loxP : vGlut2-iCreERT2 (MOR Glut −/−) mice were intraperitoneally administered for 7 consecutive days with tamoxifen (2 mg/day; Sigma-Aldrich) for inducing MOR knockdown and subsequently utilized for the experiments at 2 weeks after the final injection. The littermates of these three types of mice ( Oprm1-flox +/+: CreERT2 −/−) receiving identical tamoxifen treatment were considered controls (MOR Astro +/+, MOR GABA +/+, or MOR Glut +/+). Surgery and intra-vlPAG injections After inducing anesthesia with 4% isoflurane inhalant, mice were placed in a brain stereotaxic apparatus (RWD, China) for the surgical insertion of guide cannulas. The nasopharynx of each mouse was continuously treated with 1% isoflurane inhalant for maintaining anesthesia during surgery. After the skull was exposed, the guide cannulas (double-barreled, 0.8 mm apart; length: 4 mm, internal diameter: 0.34 mm, external diameter: 0.48 mm; RWD) were inserted into the bilateral of the vlPAG (site: anteroposterior: −4.80 mm, mediolateral: ± 0.40 mm, dorsoventral: −2.85 mm) according to the mouse brain atlas. The upper-half parts of the guide cannulas were secured to the skull through the cannulas and pre-covered with protective caps before intravenous PEG administration to prevent clogging. A diclofenac sodium gel was smeared near the wounds to achieve postoperative pain relief. After surgery, the mice were transferred to a newly ventilated cage with a clean and pathogen-free environment. All animals underwent recovery for at least 7 days before intra-vlPAG injection. The intra-vlPAG injections were processed as described previously. Briefly, the drugs for injection were aspirated into 0.5 µL needle-tipped micro-syringes (the process was regulated by a micro-infusion pump; RWD). The micro-syringes were then connected with the injecting cannulas (double-barreled, 0.8 mm apart; length: 4 mm, internal diameter: 0.14 mm, external diameter: 0.30 mm; RWD) by using plastic hoses. The injecting cannulas were then inserted into guide cannulas. Subsequently, the drugs were intra-vlPAG injected through micro-infusion pump control (0.1 µL each side, 0.04 µL/min, duration: 3 min). The following drugs were used in the intra-vlPAG injections: DAMGO (100 µM, dissolved in normal saline, concentration based on a previous study [ 16 ]; Tocris, UK) and CTAP (1 mM, dissolved in normal saline; concentration based on a previous study [ 16 ]; Tocris). To minimize the effects of stress elicited by the injection procedures during behavioral tests, mice were subjected to daily simulated intracerebral injections for 3 consecutive days before behavioral tests. The injection sites of each mouse were morphologically examined after the behavioral test. Data of mice with injection sites different from vlPAG were rejected. EPM test The EPM testing device comprised two mutually orthogonal open arms and two closed arms (length × width: 28 × 5.8 cm) intersected at a central square (length × width: 5.8 × 5.8 cm). The two close arms were placed opposite to each other and were surrounded by 15.5-cm-high walls. The height of the maze was 55 cm above the ground. The image-capturing camera was placed directly above the central square of the EPM. During the test, the mouse was placed in the central square, and its head faced one of the open arms. Each mouse was allowed 5 min of free exploration. The total distance traveled, percentage of time spent in the open arms, and percentage of open arm entries were determined using Mouse EthoVision XT (Noldus, Holland). In each mouse, the percentage of open arm entries was estimated as follows: number of open arm entries/number of total arm entries × 100%; the percentage of time spent in the open arms was estimated as follows: time spent in open arms/time spent in all arms × 100%. After each test, the testing device was cleaned and disinfected with 75% ethanol. Recombinant adeno-associated virus (rAAV) injection The surgery and rAAV injection were performed as described previously. Briefly, after inducing anesthesia with 4% isoflurane inhalant, the mice were placed in the brain stereotaxic apparatus. After exposing the skull, the corresponding rAAVs were injected into the vlPAG at the rate of 30 nL/min. Following this injection, the head skin of each mouse was carefully sutured. The postoperative treatment was identical to that of embedding of the guide cannula. The following rAAVs were used: rAAV2/5-GfaABC1D-cyto-GCaMP6f-SV40 pA (100 nL/injection, 1.20 × 10 13 vg/mL; Brain VTA, China) was used for fluorescent visualization of calcium activity of astrocytes in the vlPAG; rAAV2/5-GfaABC1D-hM3D(Gq)-mCherry-SV40 pA (170 nL/injection, 5.93 × 10 12 vg/mL; Brain VTA) was used to homogenetically activate the calcium signaling of astrocytes in the vlPAG; rAAV2/5-GfaABC1D-hPMCA2w/b-mCherry-SV40 pA (170 nL/injection, 5.27 × 10 12 vg/mL; Brain VTA) was used to specifically intercept calcium signaling of astrocytes in the vlPAG; and rAAV2/5-GfaABC1D-mCherry-SV40 pA (170 nL/injection, 5.27 × 10 12 vg/mL; Brain VTA) was used as a control for the above rAAVs. All animals underwent recovery for at least 21 days for ensuring complete expression of rAAVs. In vitro calcium imaging of vlPAG astrocytes The calcium activity of vlPAG astrocytes was monitored and analyzed by in vitro calcium imaging as described previously. Under 4% isoflurane-induced anesthesia, mice injected with rAAV2/5-GfaABC1D-cyto-GCaMP6f-SV40 pA were rapidly decapitated, and their brains were removed and rapidly transferred into oxygenated modified artificial cerebrospinal fluid (ACSF, including [in mM]: NaCl, 125; KCl, 2.5; NaH 2 PO 4 , 1.25; MgCl 2 , 2; CaCl 2 , 2; glucose, 25; and NaHCO 3 , 25; pH 7.4). By using a vibrating slicer (1000 plus; Vibratome Company, St. Louis, MO, USA), 300-µm-thick coronal slices containing vlPAG were cut. The vlPAG astrocytes were imaged using continuous fluorescence excitation with a 488-nm light source (Leica, Germany). The imaging sessions were conducted at the rate of 1 frame/s. Brain slice processing and astrocyte imaging were performed under oxygenated ACSF incubation. The bath solution for imaging contained the following materials individually or mixed: DAMGO (1 µM), CTAP (10 µM), and/or tetrodotoxin (TTX, 1 µM; 554412, Sigma-Aldrich, USA). TTX and CTAP were added to the bath 10 min before the start of recording. Imaging data were acquired and analyzed using ImageJ software. Morphological assessment of mutant mouse lines To confirm the knockdown efficiency of MOR Astro −/−, MOR GABA −/−, and MOR Glut −/− mice, fluorescence in situ hybridization (FISH) with RNAscope and immunofluorescence were used as described previously [ 7 , 15 ]. Briefly, mice were deeply anesthetized with 4% isoflurane inhalant and subjected to cardiovascular perfusion with 0.9% saline for 5 min. The brains were rapidly removed and embedded in an optimal cutting temperature (OCT) compound (SAKURA Tissue-Tek, Japan) at − 22°C. A CM1950 freezing microtome (Leica, Germany) was utilized for cutting fresh frozen sections (16 µm) containing the vlPAG region (coronal plane). After fixation with 4% paraformaldehyde (PFA) for 30 min at 4°C, the sections were dehydrated using three grades of ethanol (50%, 75%, and 100%) for 5 min each at 25°C. For fluorescence staining of MOR Glut −/−, MOR GABA −/− mice and their littermates, FISH alone was used. The sections were pretreated with hydrogen dioxide and protease IV for 10 and 15 min, respectively. The sections were incubated with probes for vglut2 (416631-C1, ACD, USA) conjugated to Atto 520, Oprm1 (544731-C2, ACD) conjugated to Atto 570, and GAT (424548-C3, ACD) conjugated to Atto 650. In situ hybridization was performed in a HybEZTM oven (ACD) by using an RNAscope Multiplex Fluorescent Reagent Kit (ACD) in accordance with the manufacturer’s protocol. Finally, the sections were mounted in a DAPI-containing anti-fade mounting medium. Both FISH with RNAscope and immunofluorescence were conducted for fluorescence staining of MOR Astro −/− mice and littermates. Fluorescence labeling of Oprm1 was conducted following the protocol for FISH. The fluorescence labeling of astrocytes was performed by sequentially incubating the sections with rabbit anti-GFAP primary antibodies (1:500, 16825-1-AP, Proteintech, China) at 4°C (12 h) and dylight488-conjugated goat anti-rabbit antibodies (1:500, A23240, Abbkine, China) at room temperature (2 h). Subsequently, the sections were mounted with a DAPI-containing anti-fade mounting medium when both Oprm1 and astrocytes were labeled. A fluorescence microscope (Zeiss, Germany) was used to acquire confocal images of all sections, and cells showing positive labeling were enumerated. Morphological examination for confirmation of the specific expression of rAAVs To confirm the specific expression of rAAVs coupled with the GfaABC1D promoter in vlPAG astrocytes, immunofluorescence was performed as reported previously [ 17 ]. Briefly, mice injected with rAAVs were anesthetized by 4% isoflurane inhalant, before transcardial perfusion with 0.9% sodium chloride solution (Kelun, China) and 4% PFA fixative (BL539A, Biosharp, China). The brains were removed, post-fixed with 4% PFA fixative for 24 h, and then immersed in 30% sucrose phosphate-buffered saline (PBS) solution at 4°C (48 h). Subsequently, the brains were embedded in an OCT compound at -22°C. Fresh frozen sections (16 µm) containing the vlPAG region (coronal plane) were cut. For immunofluorescence assay, the sections were incubated for 60 min with PBS supplemented with 10% non-immune donkey serum (T8200, SolarBio, China) and 0.5% Triton X-100 (BL939A, Biosharp, China). The sections were sequentially incubated with rabbit anti-GFAP antibodies (1:300, 16825-1-AP, Proteintech) at 4°C (12 h) and dylight680-conjugated goat anti-rabbit antibodies (1:500, A23720, Abbkine) at room temperature (2 h). After labeling, the sections were confocally imaged using a fluorescence microscope to acquire confocal images of the sections, and cells showing positive labeling were enumerated. Statistical analysis Data are expressed in their original form or as mean ± SEM. GraphPad Prism 9.0 was utilized for data analysis. Comparison of two groups was achieved through unpaired or paired Student’s t test, and comparison of multiple groups was performed with one-way analysis of variance (ANOVA) and Sidak’s multiple comparison test. Statistical significance was considered at p < 0.05. Results vlPAG MOR activation triggered anxiety-like behavior MORs play a role in modulating emotional responses; however, the specific brain mechanisms underlying this effect remain unclear. To determine how MORs expressed on the vlPAG modulate anxiety-like behavior, we conducted bilateral insertion of guide cannulas into the vlPAG of mice (Figs. 1 A, B) to specifically activate vlPAG MORs through pharmacological administration. The cannula position was confirmed morphologically (Fig. 1 B). Next, we determined the effect of intravenous DAMGO, saline, or DAMGO + CTAP on anxiety-like behaviors. We found that the total distance traveled by mice in the EPM tests was comparable between the three mice groups, suggesting that locomotor activity was not affected by DAMGO (Figs. 1 C, D). However, intra-vlPAG DAMGO induced a significant anxiogenic performance, as shown by the lower percentage of open arm entries and time spent in the open arms in control mice than in those with intra-vlPAG saline administration (Figs. 1 C, E, F). These effects were reversed by CTAP (Figs. 1 C, E, F), thus indicating the functional role of vlPAG MORs in modulating anxiety responses. MOR Astro , but not MOR GABA or MOR Glut , is involved in vlPAG-MOR-induced anxiety-like behavior In the vlPAG, MORs exhibit a high expression level in different cell types such as astrocytes, GABAergic neurons, and glutaminergic neurons [ 7 , 14 ]. To assess the distinct effects of vlPAG MOR Astro , MOR GABA , and MOR Glut on the vlPAG-MOR elicited anxiety-like behavior, three mouse lines specifically lacking MOR Astro −/−, MOR GABA −/−, and MOR Glut −/−, respectively, and their controls (MOR Astro +/+, MOR GABA +/+, or MOR Glut +/+, respectively) were generated according to previously reported methods. To further confirm the validity of MOR deletions, the absence of astrocytic MORs of MOR Astro −/− mice (Figs. 2 A, B), GABAergic MORs of MOR GABA −/− mice (Figs. 3 A, B), and glutamatergic MORs of MOR Glut −/− mice (Figs. 4 A, B) were morphologically detected by combined FISH with RNAscope and immunofluorescence technology. We inserted guide cannulas into the bilateral vlPAG of these mice and tested anxiety-like behavior through the EPM test after intra-vlPAG DAMGO or saline administration. During assessment of the effects of vlPAG MOR Astro on the vlPAG-MOR-elicited anxiety-like behavior, EPM tests showed that the total distance traveled by mice was slightly different among the four mice groups, suggesting that genetic absence of MOR Astro did not affect locomotor activity (Fig. 2 C). MOR Astro −/− and MOR Astro +/+ mice with intra-vlPAG saline administration (Figs. 2 D, E) showed no significant differences in the percentage of open arm entries and time spent in the open arms, indicating that the absence of MOR Astro did not alter the basal emotional responses of mice. However, the anxiogenic effects induced by vlPAG MORs were significantly reversed by MOR Astro −/−, as shown by the reduced percentage of open arm entries and time spent in the open arms of MOR Astro +/+ mice with intra-vlPAG DAMGO administration compared to that of MOR Astro +/+ mice with intra-vlPAG saline administration; in contrast, no significant differences were noted in the percentage of open arm entries and time spent in the open arms between MOR Astro −/− mice with intra-vlPAG DAMGO administration and MOR Astro −/− mice with intra-vlPAG saline administration (Figs. 2 D, E). A remarkable difference was found in the percentage of open arm entries and time spent in the open arms between MOR Astro +/+ mice administered with intra-vlPAG DAMGO administration and MOR Astro −/− mice with intra-vlPAG DAMGO administration (Figs. 2 D, E). Thus, MORs Astro have a crucial role in vlPAG-MOR-induced anxiety-like behavior. We then assessed the effects of vlPAG MOR GABA on vlPAG-MOR-elicited anxiety-like behavior and found that the genetic absence of MOR GABA did not affect locomotor activity; this was confirmed by the observation that the total distance traveled by mice was slightly different among the four mice groups (Fig. 3 C). The percentage of open arm entries and time spent in the open arms displayed no significant differences between MOR GABA −/− and MOR GABA +/+ mice with intra-vlPAG saline administration (Figs. 3 D, E), indicating that the absence of MOR GABA did not alter the basal emotional responses of mice. Both MOR GABA −/− and MOR GABA +/+ mice were anxiogenic after intra-vlPAG DAMGO administration, and no significant difference was noted between MOR GABA +/+ mice with intra-vlPAG DAMGO administration and MOR GABA −/− mice with intra-vlPAG DAMGO administration (Figs. 3 D, E). Thus, MOR GABA hardly participates in vlPAG-MOR-induced anxiety-like behavior. Similar to MOR GABA , the assessment of how vlPAG MOR GABA affects vlPAG-MOR-elicited anxiety-like behavior revealed no influence of the genetic absence of MOR Glut on locomotor activity (Fig. 4 C). No significant variations were noted in the percentage of open arm entries and time spent in the open arms between MOR Glut −/− and MOR Glut +/+ mice following intra-vlPAG saline administration (Figs. 4 D, E), thus indicating that the absence of MOR Glut did not alter the basal emotional responses of mice. Both MOR Glut −/− and MOR Glut +/+ mice were anxiogenic after intra-vlPAG DAMGO administration, and there was no significant difference between MOR Glut +/+ mice with intra-vlPAG DAMGO administration and MOR Glu −/− mice with intra-vlPAG DAMGO administration (Figs. 4 D, E). Thus, MOR Glut barely participates in vlPAG-MOR-induced anxiety-like behavior. Taken together, our results indicate that MOR Astro , but not MOR GABA or MOR Glut , is involved in vlPAG-MOR-induced anxiety-like behavior. vlPAG MOR Astro induces anxiety-like behavior through astrocytic calcium signaling Next, to investigate the cellular mechanisms underlying MOR Astro -dependent anxiety-like behavior, we focused on the role of vlPAG MOR Astro activation and its effects on intracellular calcium signaling in astrocytes. The activation of MOR Astro can trigger calcium signaling in the CNS [ 18 ]. As described previously [ 19 , 20 ], GCamp6f was expressed in PAG astrocytes by using a viral vector with the GfaABC1D promoter for calcium imaging (Fig. 5 A). Immunohistochemical assay confirmed the predominant expression of GCamp6f in astrocytes (Figs. 5 B, C). The treatment of vlPAG slices with the specific MOR agonist DAMGO (1 µM) significantly increased calcium levels in vlPAG astrocytes (Figs. 5 D, F); this was not observed in the control group perfused with DAMGO-free ACSF (Fig. 5 E). The increase in calcium levels was inhibited by CTAP (10 µM) (Fig. 5 G) but not by TTX (1 µM) (Fig. 5 H), suggesting that this increase was specifically mediated by MOR Astro activation. To further investigate the involvement of astrocytic calcium signaling in the vlPAG in modulating anxiogenic performance, we examined whether direct activation of PAG astrocytes alone is sufficient to produce anxiety-like behavior. Designer receptors exclusively activated by designer drugs (DREADDs) coupled with the GfaABC1D promotor virus were used, which efficiently activated the calcium signals of astrocytes, as described previously [ 21 , 22 ]. We injected rAAV5-GfaABC1D-hM3Dq-mCherry-WPRE-pA into the vlPAG targeting astrocytes (Astro hM3Dq ), with rAAV5-GfaABC1D-mCherry-WPRE-pA (Astro control ) as the control (Fig. 6 A). Immunofluorescence results showed that the mCherry report proteins and GFAP were mostly co-labeled (Figs. 6 B, C), indicating that hM3Dq rAAVs were specifically expressed in PAG astrocytes. The intraperitoneal administration of 1 mg/kg CNO remarkably caused anxiety-like behavior in Astro hM3Dq mice; this was confirmed by the lower percentage of open arm entries and time spent in the open arms as compared to those of Astro control mice with CNO administration or Astro hM3Dq mice with saline administration (Figs. 6 E, F). The total distance traveled by mice was comparable between these three mice groups, suggesting no influence of Astro hM3D q on locomotor activity (Fig. 6 D). Thus, the direct activation of vlPAG astrocytic calcium signaling elicits anxiety-like behavior. Next, we examined whether MOR Astro -triggered calcium signaling is the cellular mechanism underlying the vlPAG MOR Astro -dependent anxiety-like behavior. For this purpose, we bilaterally delivered rAAV5-GfaABC1D-hPMCA2w/b-mCherry-WPRE-SV40 into the PAG (Astro hPMCA2w/b ) (Fig. 7 A), which specifically extruded cytoplasmic Ca 2+ and reduced Ca 2+ oscillations in PAG astrocytes, as described previously [ 23 – 25 ]. The rAAV5-GfaABC1D-mCherry-WPRE-SV40 virus was also injected into the PAG to generate control mice (Astro control ). The results of immunofluorescence assay showed that the mCherry report proteins and GFAP were mostly co-labeled (Figs. 7 B, C), indicating that hPMCA2w/b rAAVs were specifically expressed in PAG astrocytes. We inserted guide cannulas into the bilateral vlPAG of these mice and tested their anxiety-like behavior through EPM tests following intra-vlPAG DAMGO or saline administration. Astro hPMCA2w/b and Astro control mice with intra-vlPAG saline administration showed no apparent differences in the percentage of open arm entries and time spent in the open arms (Figs. 7 E, F); this finding indicated that Astro hPMCA2w/b did not alter the basal emotional responses of mice. However, the anxiogenic performance elicited by vlPAG MORs were significantly reversed by Astro hPMCA2w/b , as shown by the decreased percentage of open arm entries and time spent in the open arms of Astro control mice with intra-vlPAG DAMGO administration as compared to those of Astro control mice with intra-vlPAG saline administration. In contrast, Astro hPMCA2w/b mice with intra-vlPAG DAMGO administration and Astro hPMCA2w/b mice with intra-vlPAG saline administration showed no significant differences in the two abovementioned parameters (Figs. 7 E, F). The percentage of open arm entries as well as the time spent in the open arms displayed significant differences between Astro control mice with intra-vlPAG DAMGO administration and Astro hPMCA2w/b mice with intra-vlPAG DAMGO administration (Figs. 7 E, F). These four mice groups showed comparable total distance traveled, suggesting that the locomotor activity was not influenced by Astro hPMCA2w/b (Fig. 6 D). Thus, astrocytic calcium signaling is the cellular mechanism underlying vlPAG MOR Astro -dependent anxiety-like behavior. In summary, the obtained results indicate that vlPAG MOR Astro elicits anxiety-like behavior through astrocytic calcium signaling. Discussion Here, we examined the role of PAG MORs in modulating anxiety-like behavior. We observed that the local activation of PAG MORs through microinjection of DAMGO in the PAG elicited an apparent MOR-dependent anxiety-like behavior in the EPM tests. Surprisingly, neither MOR Glut nor MOR GABA was involved in modulating this form of MOR-dependent anxiety-like behavior. In contrast, in the EPM test, conditional knockdown of MOR Astro reversed the DAMGO-induced reduction in the percentage of open arm entries and time spent in the open arms. By using calcium imaging and chemogenetic technologies, we further demonstrated the critical role of MOR Astro in inducing astrocytic calcium signaling during the modulation of MOR Astro -dependent anxiety-like behavior. Our study demonstrates a novel MOR Astro -dependent emotional response in the PAG, which reveals a possible solution for treating emotional dysfunctions during MOR-targeted management in clinical settings. The functional role of MORs in analgesia has been widely studied, with corresponding theories implemented in the area of clinical pain management [ 1 , 2 ]. However, several clinical studies have cautioned about the increased risks of emotional dysfunction among patients who use opioid drugs during pain management [ 3 – 5 ]. In addition to pain modulation, MORs are extensively involved in emotional responses [ 26 – 28 ]. In clinical settings, patients with major depressive disorder display lower MOR availability [ 3 ]. Indeed, according to previous studies, blocking the effect of MORs by oral administration of naltrexone increased the remarkable panic provocation of the volunteers under 35% CO 2 inhalation stress [ 27 ]. However, anxiety and depression disorders are also enhanced in individuals with long-term opioid-related drug use [ 3 – 5 ]. These conflicting reports suggest that the role of MORs in modulating emotional responses may be bidirectional. MORs are extensively expressed in several emotion-related brain areas that control different aspects of the transmission of emotive information. The modulation of emotional behavior by MORs in different brain regions exhibit region-specificity. For example, MORs expressed on the basolateral amygdala and dorsal raphe nucleus induce anxiolytic effects upon their activation [ 29 , 30 ], whereas MORs expressed on the central amygdala and lateral septum elicit anxiety-like behavior upon their activation in rodents [ 31 , 32 ]. Thus, the precise mechanism by which different local brain regions of MORs modulate emotional responses is advantageous for risk aversion to emotional dysfunctions during MOR-targeted pain management. The PAG is a critical brainstem region that controls the activation of the descending pain inhibitory pathway. MORs are densely expressed in the PAG [ 7 ]. As shown in rodent studies, both endogenous and exogenous activation of these MORs induce remarkable analgesia [ 7 , 33 ]. The PAG also efficiently participates in the modulation of emotional responses such as aversive and defensive behaviors [ 8 – 10 ]. Homogenetically activating PAG vglut2-positive excitatory neurons elicits anxiety-like behaviors in open field and light/dark tests [ 11 ]. Here, we found that activated PAG MORs caused a decrease in the percentage of open arm entries and time spent in the open arms by mice; moreover, these effects were reversed by pharmacological blockage of PAG MORs. Thus, PAG MORs have a functional role in modulating anxiety-related behavior, which may explain the elevated emotional disorders of patients using opioid-related drugs. The most conspicuous results of our work were that the anxiety-like behaviors elicited by the activation of PAG MORs were primarily contributed by PAG MOR Astro , whereas MOR Glut and MOR GABA only had a negligible effect on anxiety-related responses. Several morphological studies have confirmed that MORs are distributed in various types of neurocytes [ 7 , 14 ]. However, their functional modulatory role in the CNS is not fully understood. By developing three lines of MOR-knockdown mice, including MOR Glut −/−, MOR GABA −/−, and MOR Astro −/− [ 7 , 15 ], we detected the functional role of divergent cell types of PAG MORs on the modulation of anxiety-like behaviors. We found that anxiety-like behaviors induced by the local activation of vlPAG MORs were reversed by the conditional knockdown of MOR Astro . In contrast, anxiety-like behaviors were marginally modulated in MOR Glut −/− or MOR GABA −/− mice. These results are exhilarating because of confirmation of the functional role of PAG MOR GABA in pain modulation. As an inhibitory G protein-coupled receptor (GiPCR), the activation of MOR GABA could decrease GABAergic neuron excitability and subsequently weaken the tonic inhibition from GABAergic neurons to PAG-rostral ventromedial medulla (RVM) excitatory projections to elicit analgesic effects [ 7 , 34 ]. Our results demonstrated the previously unappreciated role of MOR GABA in modulating anxiety-related responses, with MOR Astro displaying critical effects in these responses. Thus, theoretically, the targeted interception of the activation of MOR Astro would reduce the aversive emotional responses without affecting the analgesic effects during PAG MOR activation. This would provide a potential possibility to address the elevated emotional disorders of patients using opioid-related drugs targeting astrocytes of the PAG. We also found that MOR Glut slightly influenced the modulation of anxiety-like behaviors. This finding aligned with the observation that the chemogenetic activation of GiPCR signaling in vGlut2-positive neurons of the PAG minimally contributed to emotional responses [ 11 ]. Beyond the modulation of pain and emotion, the PAG is associated with several other brain-related functions, including respiratory, cardiovascular, and sleep and wakefulness regulation [ 35 , 36 ]. Thus, MOR expression on PAG glutamatergic neurons might have a role in modulating autonomic nervous system function or sleep-wake reactions. By using in vitro calcium imaging combined with chemical genetics [ 19 , 20 ], we further demonstrated that the PAG MOR Astro elicits anxiety-like behavior through an astrocytic calcium signaling mechanism. As confirmed previously, astrocytic GiPCR activation elevates calcium ion levels in astrocytes [ 18 ]. In the nucleus accumbens, the activation of MOR Astro by DAMGO also elicited calcium oscillations in astrocytes [ 18 ]. In agreement with these studies, we found that the activation of PAG MORs induced significant enhancement of calcium levels in astrocytes, and this phenomenon was not blocked by TTX, indicating that this increase was specifically mediated by MOR Astro activation. The chemical activation of calcium signaling in PAG astrocytes further confirmed that calcium signaling in PAG astrocytes efficiently induces anxiogenic behavior. In contrast, anxiety-like behaviors elicited by the activation of PAG MORs were reversed by specific interception of astrocytic calcium signaling. As reported earlier, astrocytic calcium signaling activation can promote the release of gliotransmitters and subsequently modulate neural excitability through the control of synaptic transmission [ 37 – 40 ]. Thus, the activation of MOR Astro seemed to modulate synaptic transmission through astrocytic calcium signaling, thereby controlling the release of gliotransmitters in the PAG to elicit anxiogenic effects. However, further research is required to support our hypothesis. Although our study has certain strengths, there are also some limitations. First, male mice alone were used because female mice show nominal differences in the MOR-dependent modulation of emotional responses compared to male mice. Additional investigations with female mice are required for improved understanding of the role of PAG MORs in the modulation of emotional responses. Second, we confirmed only the action of the MOR Astro -induced astrocytic calcium signaling mechanism on the modulation of vlPAG MOR-induced anxiety-like behavior. However, the role of astrocytes in modulating brain behaviors is always accomplished by regulating neural activity through control of synaptic transmission or intrinsic excitability of neurons. Further electrophysiological studies should focus on explaining the astrocytic control of the neural mechanisms of PAG MOR-induced anxiogenic effects. Declarations Ethical Approval All animal experiments were carried out by following the guidelines of the Chinese Council on Animal Care. The study protocol was approved by the Animal Protection Committee of Shaanxi Normal University and the Animal Care Committee of The First Affiliated Hospital of Xi'an Medical University. Competing interests The authors declare that they have no conflict of interest. Author contributions Yinan Du and Zhiqiang Liu designed experiments, conceived the project and prepared the manuscript. Yinan Du performed behavior test, relevant surgery and in vitro calcium imaging. Aozhuo Zhang, Zhiwei Li and Yukui Zhao performed morphological test and statistical analysis. Other authors helped to write and refine the manuscript. Fundings This work was supported by the National Natural Science Foundation of China (NSFC) (grants 91949105, 82071516) and the Science and Technology Innovation Team Project of Xi'an Medical University (grants 2021TD14). References Cuitavi J, Torres-Pérez JV, Lorente JD, Campos-Jurado Y, Andrés-Herrera P, Polache A, et al. Crosstalk between mu-opioid receptors and neuroinflammation: Consequences for drug addiction and pain. Neurosci Biobehav Rev. 2023;145:105011. Corder G, Castro DC, Bruchas MR, Scherrer G. Endogenous and exogenous opioids in pain. 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Supplementary Files Figure2Aoriginaldata.rar Figure3Aoriginaldata.rar Figure4Aoriginaldata.rar Figure5originaldata.rar Figure6originaldata.rar Figure7originaldata.rar Cite Share Download PDF Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Behavioral and Brain Functions → Version 1 posted Editorial decision: Revision requested 26 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 02 Apr, 2025 Editor assigned by journal 21 Mar, 2025 Submission checks completed at journal 21 Mar, 2025 First submitted to journal 19 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6262877","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444151283,"identity":"95b79310-1fd5-4e9e-b8be-fa9e19c326d7","order_by":0,"name":"Yinan 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15:08:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6262877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6262877/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12993-025-00291-0","type":"published","date":"2025-07-23T15:58:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81932688,"identity":"e1cdc51d-815e-489e-97de-0d3c9006ad4a","added_by":"auto","created_at":"2025-05-05 05:37:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":461142,"visible":true,"origin":"","legend":"\u003cp\u003eActivation of ventrolateral periaqueductal gray (vlPAG) mu-opioid receptors (MORs) elicited anxiety-like behavior.\u003cstrong\u003e A.\u003c/strong\u003e Schematic showing the implantation and morphological confirmation of the guide cannulas in the vlPAG. Scale bar: 400 μm. \u003cstrong\u003eB\u003c/strong\u003e. Experimental schedule. \u003cstrong\u003eC.\u003c/strong\u003e Typical representative activity tracking in EPM tests. \u003cstrong\u003eD–F\u003c/strong\u003e. Summary plots of the total distance (\u003cstrong\u003eD\u003c/strong\u003e), percentage of open arm entries (\u003cstrong\u003eE\u003c/strong\u003e), and percentage of time spent in the open arms (\u003cstrong\u003eF\u003c/strong\u003e) during EPM tests. Saline-treated mice: n = 8; DAMGO-treated mice: n = 8; DAMGO+CTAP-treated mice: n = 8. Total distance: ordinary one-way ANOVA measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,21)\u003c/sub\u003e = 0.9410, \u003cem\u003ep\u003c/em\u003e = 0.4061. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,21)\u003c/sub\u003e = 12.85, \u003cem\u003ep\u003c/em\u003e = 0.0002 (**), saline vs. DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0005 (**); saline vs. DAMGO+CTAP, \u003cem\u003ep\u003c/em\u003e = 0.9358; DAMGO vs. DAMGO+CTAP, \u003cem\u003ep\u003c/em\u003e = 0.0011 (**). Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,21)\u003c/sub\u003e = 12.53, \u003cem\u003ep\u003c/em\u003e = 0.0003 (**), saline vs. DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0002 (**); saline vs. DAMGO+CTAP, \u003cem\u003ep\u003c/em\u003e = 0.3128; DAMGO vs. DAMGO+CTAP, \u003cem\u003ep\u003c/em\u003e = 0.0075 (**).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/0439ca7eb2b851f9ac619036.png"},{"id":81932690,"identity":"75c4533a-c6e7-4026-b8a5-cb3979ae9190","added_by":"auto","created_at":"2025-05-05 05:37:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1042172,"visible":true,"origin":"","legend":"\u003cp\u003eMORs\u003csub\u003eAstro\u003c/sub\u003e are involved in vlPAG-MOR-elicited anxiety-like behavior. \u003cstrong\u003eA\u003c/strong\u003e. Schematic of \u003cem\u003ein situ\u003c/em\u003e hybridization for \u003cem\u003eOprm1\u003c/em\u003e mRNA and immunofluorescence for the GFAP protein in the vlPAG areas in MOR\u003csub\u003eAstro\u003c/sub\u003e−/− and MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ mice. The nucleus is stained in blue (DAPI), GFAP is stained in green, and \u003cem\u003eOprm1\u003c/em\u003e mRNA is stained in red. Scale bar: 10 μm. The white arrowheads indicate cells double-labeled with \u003cem\u003eOprm1\u003c/em\u003e mRNA and GFAP; the purple arrowheads represent \u003cem\u003eOprm1\u003c/em\u003e mRNA localization in GFAP-negative cells; and the yellow arrowheads represent GFAP-positive cells without \u003cem\u003eOprm1\u003c/em\u003e mRNA. \u003cstrong\u003eB.\u003c/strong\u003e Quantitative analysis of the percentage of double-positive cells (\u003cem\u003eOprm1\u003c/em\u003e and GFAP) against GFAP-positive cells. \u003cem\u003et\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e = 19.96, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, unpaired Student’s \u003cem\u003et \u003c/em\u003etest, n = 3 mice per group. \u003cstrong\u003eC–E\u003c/strong\u003e. Summary plots of the total distance (\u003cstrong\u003eC\u003c/strong\u003e), percentage of open arm entries (\u003cstrong\u003eD\u003c/strong\u003e), and percentage of time spent in the open arms (\u003cstrong\u003eE\u003c/strong\u003e) during EPM tests. MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +saline-treated mice: n = 7; MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +saline-treated mice: n = 7; MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +DAMGO-treated mice: n = 7; MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 2.177, \u003cem\u003ep\u003c/em\u003e = 0.1169. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 6.856, \u003cem\u003ep\u003c/em\u003e = 0.0017 (**), MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +saline vs. MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0027 (**); MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +saline vs. MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.9988; MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +DAMGO vs. MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0221 (*). Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 10.69, \u003cem\u003ep\u003c/em\u003e = 0.0001 (**), MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +saline vs. MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0028 (**); MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +saline vs. MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.9999; MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ +DAMGO vs. MOR\u003csub\u003eAstro\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0003 (**).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/18f0a48c6e317a99fae6ef3b.png"},{"id":81932689,"identity":"3873c848-7d5a-49d7-8fe1-de2488d7fc59","added_by":"auto","created_at":"2025-05-05 05:37:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":831683,"visible":true,"origin":"","legend":"\u003cp\u003eMORs\u003csub\u003eGABA\u003c/sub\u003e are hardly involved in vlPAG-MOR-elicited anxiety-like behavior. \u003cstrong\u003eA\u003c/strong\u003e. Schematic of \u003cem\u003ein situ\u003c/em\u003e hybridization for \u003cem\u003eOprm1\u003c/em\u003e mRNA and\u003cem\u003e GAT\u003c/em\u003e mRNA in the vlPAG areas in MOR\u003csub\u003eGABA\u003c/sub\u003e−/− and MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ mice. The nucleus is stained in blue (DAPI), \u003cem\u003eGAT\u003c/em\u003e is stained in green, and \u003cem\u003eOprm1\u003c/em\u003e mRNA is stained in red. Scale bar: 10 μm. The white arrowheads indicate cells double-labeled with \u003cem\u003eOprm1\u003c/em\u003e mRNA and \u003cem\u003eGAT\u003c/em\u003e mRNA; the purple arrowheads represent \u003cem\u003eOprm1\u003c/em\u003e mRNA localization in \u003cem\u003eGAT\u003c/em\u003e-negative cells; and the yellow arrowheads represent \u003cem\u003eGAT\u003c/em\u003e-positive cells without \u003cem\u003eOprm1\u003c/em\u003e mRNA. \u003cstrong\u003eB.\u003c/strong\u003e Quantitative analysis of the percentage of double-positive cells (\u003cem\u003eOprm1\u003c/em\u003e and \u003cem\u003eGAT\u003c/em\u003e) against \u003cem\u003eGAT\u003c/em\u003e-positive cells. \u003cem\u003et\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e = 8.194, \u003cem\u003ep\u003c/em\u003e = 0.0012, unpaired Student’s \u003cem\u003et \u003c/em\u003etest, n = 3 mice per group. \u003cstrong\u003eC–E\u003c/strong\u003e. Summary plots of total distance (\u003cstrong\u003eC\u003c/strong\u003e), percentage of open arm entries (\u003cstrong\u003eD\u003c/strong\u003e), and percentage of time spent in the open arms (\u003cstrong\u003eE\u003c/strong\u003e) during EPM tests. MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +saline-treated mice: n = 7; MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +saline-treated mice: n = 7; MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +DAMGO-treated mice: n = 7; MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 1.399, \u003cem\u003ep\u003c/em\u003e = 0.2675. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 9.688, \u003cem\u003ep\u003c/em\u003e = 0.0002 (**), MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +saline vs. MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0012 (**); MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +saline vs. MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0345(*); MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +DAMGO vs. MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.9999. Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 23.57, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**), MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +saline vs. MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0001 (**); MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +saline vs. MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**); MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ +DAMGO vs. MOR\u003csub\u003eGABA\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.8229.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/1f4a8ed8c5ae048b809f1879.png"},{"id":81936730,"identity":"2a5bbf75-06f9-457e-ba59-c8ac39e5e445","added_by":"auto","created_at":"2025-05-05 06:13:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1093558,"visible":true,"origin":"","legend":"\u003cp\u003eMORs\u003csub\u003eGlut\u003c/sub\u003e are barely involved in vlPAG-MOR-elicited anxiety-like behavior. \u003cstrong\u003eA\u003c/strong\u003e. Schematic of \u003cem\u003ein situ\u003c/em\u003e hybridization for \u003cem\u003eOprm1\u003c/em\u003e mRNA and\u003cem\u003e Glut\u003c/em\u003e mRNA in the vlPAG areas in MOR\u003csub\u003eGlut\u003c/sub\u003e−/− and MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ mice. The nucleus is stained in blue (DAPI), \u003cem\u003eGlut\u003c/em\u003e is stained in green, and \u003cem\u003eOprm1\u003c/em\u003e mRNA is stained in red. Scale bar: 10 μm. The white arrowheads indicate cells double-labeled with \u003cem\u003eOprm1\u003c/em\u003e mRNA and \u003cem\u003eGlut\u003c/em\u003e mRNA; the purple arrowheads represent \u003cem\u003eOprm1\u003c/em\u003e mRNA localization in \u003cem\u003eGlut\u003c/em\u003e-negative cells; and the yellow arrowheads represent \u003cem\u003eGlut\u003c/em\u003e-positive cells without \u003cem\u003eOprm1\u003c/em\u003e mRNA. \u003cstrong\u003eB.\u003c/strong\u003e Quantitative analysis of the percentage of double-positive cells (\u003cem\u003eOprm1\u003c/em\u003e and \u003cem\u003eGlut\u003c/em\u003e) against \u003cem\u003eGlut\u003c/em\u003e-positive cells. \u003cem\u003et\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e = 14.46, \u003cem\u003ep\u003c/em\u003e = 0.0001, unpaired Student’s \u003cem\u003et \u003c/em\u003etest, n = 3 mice per group. \u003cstrong\u003eC–E\u003c/strong\u003e. Summary plots of the total distance (\u003cstrong\u003eC\u003c/strong\u003e), percentage of open arm entries (\u003cstrong\u003eD\u003c/strong\u003e), and percentage of time spent in the open arms (\u003cstrong\u003eE\u003c/strong\u003e) during EPM tests. MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +saline-treated mice: n = 7; MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +saline-treated mice: n = 7; MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +DAMGO-treated mice: n = 7; MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 1.985, \u003cem\u003ep\u003c/em\u003e = 0.1432. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 12.77, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**), MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +saline vs. MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0001 (**); MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +saline vs. MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0355(*); MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +DAMGO vs. MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.2730. Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple-comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 24.90, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**), MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +saline vs. MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0001 (**); MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +saline vs. MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**); MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ +DAMGO vs. MOR\u003csub\u003eGlut\u003c/sub\u003e−/− +DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.9475.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/7e95fded6b0e0b73deda1a82.png"},{"id":81936012,"identity":"d311ce67-c13a-41ff-a2b3-63904720890f","added_by":"auto","created_at":"2025-05-05 06:01:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":719544,"visible":true,"origin":"","legend":"\u003cp\u003eActivation of PAG MOR\u003csub\u003eAstro\u003c/sub\u003e triggers astrocytic calcium signaling. \u003cstrong\u003eA\u003c/strong\u003e. Schematic of the rAAVs monitored to specifically exhibit astrocytic calcium levels in the vlPAG. \u003cstrong\u003eB\u003c/strong\u003e. Double-immunofluorescence staining of GCamp (green) with GFAP (red). Scale bar: 10 μm. The white arrows represent the co-labeled cells. \u003cstrong\u003eC.\u003c/strong\u003e Statistics of the co-labeling rate of GCamp coupled with GFAP (n = 3 mice). \u003cstrong\u003eD\u003c/strong\u003e. Typical change and representative traces of Ca\u003csup\u003e2+\u003c/sup\u003esignals of a single PAG astrocyte during DAMGO (1 μM) perfusion (arrow indicates DAMGO application). \u003cstrong\u003eE–H\u003c/strong\u003e. Summary bar graph of the change in the △F/F of the regions of interest during treatment with ACSF (\u003cstrong\u003eE\u003c/strong\u003e) (\u003cem\u003et\u003c/em\u003e\u003csub\u003e7\u003c/sub\u003e = 0.8204, \u003cem\u003ep\u003c/em\u003e = 0.4353, paired Student’s \u003cem\u003et \u003c/em\u003etest, n = 8 cells from n = 2 mice), 1 μM DAMGO (\u003cstrong\u003eF\u003c/strong\u003e) (\u003cem\u003et\u003c/em\u003e\u003csub\u003e7\u003c/sub\u003e = 2.565, \u003cem\u003ep\u003c/em\u003e = 0.0373 (*), paired Student’s \u003cem\u003et \u003c/em\u003etest, n = 8 cells from n = 2 mice), 1 μM DAMGO + 10 μM CTAP (\u003cstrong\u003eG\u003c/strong\u003e) (\u003cem\u003et\u003c/em\u003e\u003csub\u003e7\u003c/sub\u003e = 0.9734, \u003cem\u003ep\u003c/em\u003e = 0.3628, paired Student’s \u003cem\u003et \u003c/em\u003etest, n = 8 cells from n = 2 mice), and 1 μM DAMGO + 1 μM TTX (\u003cstrong\u003eH\u003c/strong\u003e) (\u003cem\u003et\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e = 3.297, \u003cem\u003ep\u003c/em\u003e = 0.0165 (*), paired Student’s \u003cem\u003et \u003c/em\u003etest, n = 7 cells from n = 2 mice) perfusion.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/9113e61b19d6f6cb74e15c95.png"},{"id":81932712,"identity":"0ac92e7e-ed08-4892-b08f-6bcc445a9389","added_by":"auto","created_at":"2025-05-05 05:37:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":436353,"visible":true,"origin":"","legend":"\u003cp\u003eDirect activation of vlPAG astrocytic calcium signaling elicits anxiety-like behavior. \u003cstrong\u003eA.\u003c/strong\u003e Schematic and fluorescent signal image of the rAAVs engineered to specifically activate vlPAG astrocytic calcium signaling. Aq: Aqueduct. Scale bar: 100 μm. \u003cstrong\u003eB.\u003c/strong\u003e Double-immunofluorescence staining of hM3Dq (red) with GFAP (green). Scale bar: 10 μm. The white arrows represent the co-labeled cells. \u003cstrong\u003eC.\u003c/strong\u003e Statistics of the co-labeling rate of hM3Dq coupled with GFAP (n = 3 mice). \u003cstrong\u003eD–F.\u003c/strong\u003e Summary plots of the total distance (\u003cstrong\u003eD\u003c/strong\u003e), percentage of open arm entries (\u003cstrong\u003eE\u003c/strong\u003e), and percentage of time spent in the open arms (\u003cstrong\u003eF\u003c/strong\u003e) during EPM tests. Astro\u003csub\u003econtrol\u003c/sub\u003e+CNO-treated mice: n = 6; Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+CNO-treated mice: n = 6; Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+saline-treated mice: n = 6. Total distance: ordinary one-way ANOVA measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,15)\u003c/sub\u003e = 0.4609, \u003cem\u003ep\u003c/em\u003e = 0.6393. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,15)\u003c/sub\u003e = 12.04, \u003cem\u003ep\u003c/em\u003e = 0.0008 (**), Astro\u003csub\u003econtrol\u003c/sub\u003e+CNO vs. Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+CNO, \u003cem\u003ep\u003c/em\u003e = 0.0008 (**); Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+CNO vs. Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+saline, \u003cem\u003ep\u003c/em\u003e = 0.0084 (**); Astro\u003csub\u003econtrol\u003c/sub\u003e+CNO vs. Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+saline, \u003cem\u003ep\u003c/em\u003e = 0.6171. Percentage of time spent in open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,15)\u003c/sub\u003e = 11.91, \u003cem\u003ep\u003c/em\u003e = 0.0013 (**), Astro\u003csub\u003econtrol\u003c/sub\u003e+CNO vs. Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+CNO, \u003cem\u003ep\u003c/em\u003e = 0.0013 (**); Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+CNO vs. Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+saline, \u003cem\u003ep\u003c/em\u003e = 0.0040 (**); Astro\u003csub\u003econtrol\u003c/sub\u003e+CNO vs. Astro\u003csub\u003ehM3Dq\u003c/sub\u003e+saline, \u003cem\u003ep\u003c/em\u003e = 0.9323.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/e12083d00d771a99af7dfb24.png"},{"id":81936208,"identity":"ba70c3d4-e608-4112-9fb8-65cc8c32c85f","added_by":"auto","created_at":"2025-05-05 06:05:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":462015,"visible":true,"origin":"","legend":"\u003cp\u003eAstrocytic calcium signaling is the cellular mechanism underlying vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent anxiety-like behavior. \u003cstrong\u003eA.\u003c/strong\u003e Schematic and fluorescent signal image of the rAAVs engineered to specifically extrude cytoplasmic calcium and reduce calcium oscillations in vlPAG astrocytes. Aq: Aqueduct. \u003cstrong\u003eB.\u003c/strong\u003e Double-immunofluorescence staining of hPMCA2w/b (red) with GFAP (green). Scale bar: 10 μm. \u003cstrong\u003eC.\u003c/strong\u003e Statistics of the co-labeling rate of hPMCA2w/b coupled with GFAP (n = 3 mice). \u003cstrong\u003eD–F\u003c/strong\u003e. Summary plots of the total distance (\u003cstrong\u003eD\u003c/strong\u003e), percentage of open arm entries (\u003cstrong\u003eE\u003c/strong\u003e), and percentage of time spent in the open arms (\u003cstrong\u003eF\u003c/strong\u003e) during EPM tests. Astro\u003csub\u003econtrol\u003c/sub\u003e+saline-treated mice: n = 7; Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+saline-treated mice: n = 7; Astro\u003csub\u003econtrol\u003c/sub\u003e+DAMGO-treated mice: n = 7; Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+DAMGO-treated mice: n = 7. Total distance: ordinary one-way ANOVA measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 1.007, \u003cem\u003ep\u003c/em\u003e = 0.4068. Percentage of open arm entries: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 13.21, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**), Astro\u003csub\u003econtrol\u003c/sub\u003e+saline vs. Astro\u003csub\u003econtrol\u003c/sub\u003e+DAMGO, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**); Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+saline vs. Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.9999; Astro\u003csub\u003econtrol\u003c/sub\u003e+DAMGO vs. Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0005 (**). Percentage of time spent in the open arms: ordinary one-way ANOVA followed by Sidak’s multiple comparison test measures, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,24)\u003c/sub\u003e = 13.35, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 (**), Astro\u003csub\u003econtrol\u003c/sub\u003e+saline vs. Astro\u003csub\u003econtrol\u003c/sub\u003e+DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0013 (**); Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+saline vs. Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.6004; Astro\u003csub\u003econtrol\u003c/sub\u003e+DAMGO vs. Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e+DAMGO, \u003cem\u003ep\u003c/em\u003e = 0.0009 (**).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/ec46258f988bd385fe4d2694.png"},{"id":87756937,"identity":"aa96879d-5804-4fec-b08f-c16b112aeb3b","added_by":"auto","created_at":"2025-07-28 16:10:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5699943,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/aa6128d1-6b28-4748-ae00-3fd112f8269b.pdf"},{"id":81932693,"identity":"1a6f0399-6854-40c6-a24e-e32029b9687c","added_by":"auto","created_at":"2025-05-05 05:37:15","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":794266,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2Aoriginaldata.rar","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/9b84e1f9ea6196ff10867b72.rar"},{"id":81934916,"identity":"2aeec0df-9fb3-4105-8d7d-7c936a23df5d","added_by":"auto","created_at":"2025-05-05 05:59:45","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":703907,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3Aoriginaldata.rar","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/543e9fa98aa2ba4e8c4680e0.rar"},{"id":81936220,"identity":"37ab6c6a-c5a6-43e7-8b16-e4287b932d7b","added_by":"auto","created_at":"2025-05-05 06:06:05","extension":"rar","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":886030,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4Aoriginaldata.rar","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/f861f5d52a16eab319a0c669.rar"},{"id":81936207,"identity":"5fd8e956-da9a-47cd-934a-9bd821b0f1cf","added_by":"auto","created_at":"2025-05-05 06:05:51","extension":"rar","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":323825,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5originaldata.rar","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/c79e31ad957f7d9f5cc4a572.rar"},{"id":81932698,"identity":"b049a304-5cd1-440d-a425-56f42452d6e9","added_by":"auto","created_at":"2025-05-05 05:37:15","extension":"rar","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":704859,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6originaldata.rar","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/ac8bd34f8b68ba75eeacf53e.rar"},{"id":81936271,"identity":"0797fd18-e75d-4adf-8753-7a7b69c6404e","added_by":"auto","created_at":"2025-05-05 06:07:53","extension":"rar","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1036788,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7originaldata.rar","url":"https://assets-eu.researchsquare.com/files/rs-6262877/v1/159ccc486859f7f549b3b6cc.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of astrocytic mu-opioid receptors of the ventrolateral periaqueductal gray in modulating anxiety-like responses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMu-opioid receptor (MOR), a member of the endogenous opioid system, is a major regulator of pain sensation and reaction in the central nervous system (CNS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to several studies, a strong correlation exists between MOR activity levels and anxiety behavior [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A high prevalence of mood disorders and anxiety behavior is observed in individuals habitually using MOR-targeted analgesics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, the role of MORs in influencing anxiety or anxiety-like behaviors requires greater attention.\u003c/p\u003e \u003cp\u003eMORs are widely expressed in several brain areas that control emotional behaviors, such as the amygdala, hippocampus, prefrontal cortex, bed nucleus of the stria terminalis, and periaqueductal gray (PAG) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among these areas, the PAG is a brainstem region with predominant involvement in modulating aversive emotional or defensive behaviors [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It receives afferent connections from the prefrontal cortex, central amygdala, hypothalamus, and habenula nucleus; it integrates neurotransmission from these brain areas and exports an emotional primary process [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The PAG contains four longitudinal columns: the dorsomedial PAG (dmPAG), dorsolateral PAG (dlPAG), lateral PAG (lPAG), and ventrolateral PAG (vlPAG) [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The vlPAG has a close association with negative emotional responses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Altered excitability of vlPAG neurons has been observed in emotional-related disorders, and chemogenetic activation of vlPAG glutamatergic neurons induces anxiety-like behavior in the light/dark test and open field test in rodents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings indicate the functional role of the vlPAG in modulating negative emotional behavior. Both morphological and biochemical investigations have reported the dense distribution of MORs in the vlPAG [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, pharmacological or genetic intervention targeted toward the functions of vlPAG MORs can fluctuate the neural excitability of the PAG [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These findings suggest a potential role of PAG MORs in modulating emotional behavior. Moreover, by using combined \u003cem\u003ein situ\u003c/em\u003e hybridization and immunofluorescence technology, several cell types, including glutamatergic (MOR\u003csub\u003eGlut\u003c/sub\u003e), gamma-aminobutyric acid (GABA) ergic (MOR\u003csub\u003eGABA\u003c/sub\u003e), and astrocytic (MOR\u003csub\u003eAstro\u003c/sub\u003e) neurocytes, were found to express MORs in the vlPAG [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; this suggests that the multiple interactions between MORs on these different neuronal types might be involved in emotional behavior control.\u003c/p\u003e \u003cp\u003eHere, we performed bilateral micro-administration of the MOR-specific agonist DAMGO in male mice and observed how local activation of vlPAG MORs affected anxiety-like behavior through the elevated plus maze (EPM) test. By developing three male murine models in which glutamatergic, GABAergic, or astrocytic MORs were conditionally knocked down, we detected the role of divergent neuronal types of vlPAG MORs on the modulation of anxiety-like behaviors. Furthermore, by using \u003cem\u003ein vitro\u003c/em\u003e calcium imaging and chemogenetic and virus-interfering technologies, we elucidated the preliminary mechanisms of these effects. DAMGO induced significant MOR-dependent anxiety-like effects in the vlPAG. Interestingly, these effects were induced by MOR\u003csub\u003eAstro\u003c/sub\u003e but not by MOR\u003csub\u003eGlut\u003c/sub\u003e or MOR\u003csub\u003eGABA\u003c/sub\u003e. Moreover, the induction and enhancement of calcium signaling could be critical mechanisms for inducing vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent anxiety-like behavior. Our study highlights a new MOR\u003csub\u003eAstro\u003c/sub\u003e elicited anxiety-like behavior paradigm in the PAG. These observations offer a theoretical premise for treating emotional dysfunctions during MOR-targeted management.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe study involved 78 C57BL/6J male mice, 42 MOR mutant male mice, and 42 littermate male controls (8\u0026ndash;12 weeks old). All mice were supplied by the Experimental Animal Center of Shaanxi Normal University, MOE Key Laboratory of Modern Teaching Technology. The mice were housed in individually ventilated cages in groups (5\u0026ndash;6 animals per group) and maintained under the following conditions: temperature: 21\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; relative humidity: 50% \u0026plusmn; 5%; sufficient food and water; and 12-h light/dark cycle. Before the behavioral experiments, the animals were acclimated to the environment and apparatus. The use count of mice in each experiment is detailed in the Results section.\u003c/p\u003e \u003cp\u003eThe mutant mouse lines were generated as reported previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, mice specifically lacking MORs in MOR\u003csub\u003eAstro\u003c/sub\u003e, MOR\u003csub\u003eGABA\u003c/sub\u003e, or MOR\u003csub\u003eGlut\u003c/sub\u003e were generated by crossing \u003cem\u003eOprm1\u003c/em\u003e-floxed mice to \u003cem\u003eGFAP-CreERT2\u003c/em\u003e, \u003cem\u003eGAD2\u003c/em\u003e-\u003cem\u003eiCreERT2\u003c/em\u003e, or \u003cem\u003evGlut2-iCreERT2\u003c/em\u003e mice, respectively. The adult \u003cem\u003eOprm1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxP/loxP\u003c/em\u003e\u003c/sup\u003e:\u003cem\u003eGFAP-CreERT2\u003c/em\u003e (MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus;), \u003cem\u003eOprm1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxP/loxP\u003c/em\u003e\u003c/sup\u003e:\u003cem\u003eGad2-iCreERT2\u003c/em\u003e (MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus;), or \u003cem\u003eOprm1\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxP/loxP\u003c/em\u003e\u003c/sup\u003e:\u003cem\u003evGlut2-iCreERT2\u003c/em\u003e (MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus;) mice were intraperitoneally administered for 7 consecutive days with tamoxifen (2 mg/day; Sigma-Aldrich) for inducing MOR knockdown and subsequently utilized for the experiments at 2 weeks after the final injection. The littermates of these three types of mice (\u003cem\u003eOprm1-flox\u003c/em\u003e+/+:\u003cem\u003eCreERT2\u003c/em\u003e\u0026minus;/\u0026minus;) receiving identical tamoxifen treatment were considered controls (MOR\u003csub\u003eAstro\u003c/sub\u003e+/+, MOR\u003csub\u003eGABA\u003c/sub\u003e+/+, or MOR\u003csub\u003eGlut\u003c/sub\u003e+/+).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurgery and intra-vlPAG injections\u003c/h3\u003e\n\u003cp\u003eAfter inducing anesthesia with 4% isoflurane inhalant, mice were placed in a brain stereotaxic apparatus (RWD, China) for the surgical insertion of guide cannulas. The nasopharynx of each mouse was continuously treated with 1% isoflurane inhalant for maintaining anesthesia during surgery. After the skull was exposed, the guide cannulas (double-barreled, 0.8 mm apart; length: 4 mm, internal diameter: 0.34 mm, external diameter: 0.48 mm; RWD) were inserted into the bilateral of the vlPAG (site: anteroposterior: \u0026minus;4.80 mm, mediolateral: \u0026plusmn; 0.40 mm, dorsoventral: \u0026minus;2.85 mm) according to the mouse brain atlas. The upper-half parts of the guide cannulas were secured to the skull through the cannulas and pre-covered with protective caps before intravenous PEG administration to prevent clogging. A diclofenac sodium gel was smeared near the wounds to achieve postoperative pain relief. After surgery, the mice were transferred to a newly ventilated cage with a clean and pathogen-free environment. All animals underwent recovery for at least 7 days before intra-vlPAG injection.\u003c/p\u003e \u003cp\u003eThe intra-vlPAG injections were processed as described previously. Briefly, the drugs for injection were aspirated into 0.5 \u0026micro;L needle-tipped micro-syringes (the process was regulated by a micro-infusion pump; RWD). The micro-syringes were then connected with the injecting cannulas (double-barreled, 0.8 mm apart; length: 4 mm, internal diameter: 0.14 mm, external diameter: 0.30 mm; RWD) by using plastic hoses. The injecting cannulas were then inserted into guide cannulas. Subsequently, the drugs were intra-vlPAG injected through micro-infusion pump control (0.1 \u0026micro;L each side, 0.04 \u0026micro;L/min, duration: 3 min). The following drugs were used in the intra-vlPAG injections: DAMGO (100 \u0026micro;M, dissolved in normal saline, concentration based on a previous study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; Tocris, UK) and CTAP (1 mM, dissolved in normal saline; concentration based on a previous study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; Tocris). To minimize the effects of stress elicited by the injection procedures during behavioral tests, mice were subjected to daily simulated intracerebral injections for 3 consecutive days before behavioral tests. The injection sites of each mouse were morphologically examined after the behavioral test. Data of mice with injection sites different from vlPAG were rejected.\u003c/p\u003e\n\u003ch3\u003eEPM test\u003c/h3\u003e\n\u003cp\u003eThe EPM testing device comprised two mutually orthogonal open arms and two closed arms (length \u0026times; width: 28 \u0026times; 5.8 cm) intersected at a central square (length \u0026times; width: 5.8 \u0026times; 5.8 cm). The two close arms were placed opposite to each other and were surrounded by 15.5-cm-high walls. The height of the maze was 55 cm above the ground. The image-capturing camera was placed directly above the central square of the EPM. During the test, the mouse was placed in the central square, and its head faced one of the open arms. Each mouse was allowed 5 min of free exploration. The total distance traveled, percentage of time spent in the open arms, and percentage of open arm entries were determined using Mouse EthoVision XT (Noldus, Holland). In each mouse, the percentage of open arm entries was estimated as follows: number of open arm entries/number of total arm entries \u0026times; 100%; the percentage of time spent in the open arms was estimated as follows: time spent in open arms/time spent in all arms \u0026times; 100%. After each test, the testing device was cleaned and disinfected with 75% ethanol.\u003c/p\u003e\n\u003ch3\u003eRecombinant adeno-associated virus (rAAV) injection\u003c/h3\u003e\n\u003cp\u003eThe surgery and rAAV injection were performed as described previously. Briefly, after inducing anesthesia with 4% isoflurane inhalant, the mice were placed in the brain stereotaxic apparatus. After exposing the skull, the corresponding rAAVs were injected into the vlPAG at the rate of 30 nL/min. Following this injection, the head skin of each mouse was carefully sutured. The postoperative treatment was identical to that of embedding of the guide cannula. The following rAAVs were used: rAAV2/5-GfaABC1D-cyto-GCaMP6f-SV40 pA (100 nL/injection, 1.20 \u0026times; 10\u003csup\u003e13\u003c/sup\u003e vg/mL; Brain VTA, China) was used for fluorescent visualization of calcium activity of astrocytes in the vlPAG; rAAV2/5-GfaABC1D-hM3D(Gq)-mCherry-SV40 pA (170 nL/injection, 5.93 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg/mL; Brain VTA) was used to homogenetically activate the calcium signaling of astrocytes in the vlPAG; rAAV2/5-GfaABC1D-hPMCA2w/b-mCherry-SV40 pA (170 nL/injection, 5.27 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg/mL; Brain VTA) was used to specifically intercept calcium signaling of astrocytes in the vlPAG; and rAAV2/5-GfaABC1D-mCherry-SV40 pA (170 nL/injection, 5.27 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg/mL; Brain VTA) was used as a control for the above rAAVs. All animals underwent recovery for at least 21 days for ensuring complete expression of rAAVs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecalcium imaging of vlPAG astrocytes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe calcium activity of vlPAG astrocytes was monitored and analyzed by \u003cem\u003ein vitro\u003c/em\u003e calcium imaging as described previously. Under 4% isoflurane-induced anesthesia, mice injected with rAAV2/5-GfaABC1D-cyto-GCaMP6f-SV40 pA were rapidly decapitated, and their brains were removed and rapidly transferred into oxygenated modified artificial cerebrospinal fluid (ACSF, including [in mM]: NaCl, 125; KCl, 2.5; NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.25; MgCl\u003csub\u003e2\u003c/sub\u003e, 2; CaCl\u003csub\u003e2\u003c/sub\u003e, 2; glucose, 25; and NaHCO\u003csub\u003e3\u003c/sub\u003e, 25; pH 7.4). By using a vibrating slicer (1000 plus; Vibratome Company, St. Louis, MO, USA), 300-\u0026micro;m-thick coronal slices containing vlPAG were cut. The vlPAG astrocytes were imaged using continuous fluorescence excitation with a 488-nm light source (Leica, Germany). The imaging sessions were conducted at the rate of 1 frame/s. Brain slice processing and astrocyte imaging were performed under oxygenated ACSF incubation. The bath solution for imaging contained the following materials individually or mixed: DAMGO (1 \u0026micro;M), CTAP (10 \u0026micro;M), and/or tetrodotoxin (TTX, 1 \u0026micro;M; 554412, Sigma-Aldrich, USA). TTX and CTAP were added to the bath 10 min before the start of recording. Imaging data were acquired and analyzed using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eMorphological assessment of mutant mouse lines\u003c/h3\u003e\n\u003cp\u003eTo confirm the knockdown efficiency of MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus;, MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus;, and MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus; mice, fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) with RNAscope and immunofluorescence were used as described previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, mice were deeply anesthetized with 4% isoflurane inhalant and subjected to cardiovascular perfusion with 0.9% saline for 5 min. The brains were rapidly removed and embedded in an optimal cutting temperature (OCT) compound (SAKURA Tissue-Tek, Japan) at \u0026minus;\u0026thinsp;22\u0026deg;C. A CM1950 freezing microtome (Leica, Germany) was utilized for cutting fresh frozen sections (16 \u0026micro;m) containing the vlPAG region (coronal plane). After fixation with 4% paraformaldehyde (PFA) for 30 min at 4\u0026deg;C, the sections were dehydrated using three grades of ethanol (50%, 75%, and 100%) for 5 min each at 25\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor fluorescence staining of MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus;, MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus; mice and their littermates, FISH alone was used. The sections were pretreated with hydrogen dioxide and protease IV for 10 and 15 min, respectively. The sections were incubated with probes for \u003cem\u003evglut2\u003c/em\u003e (416631-C1, ACD, USA) conjugated to Atto 520, \u003cem\u003eOprm1\u003c/em\u003e (544731-C2, ACD) conjugated to Atto 570, and \u003cem\u003eGAT\u003c/em\u003e (424548-C3, ACD) conjugated to Atto 650. \u003cem\u003eIn situ\u003c/em\u003e hybridization was performed in a HybEZTM oven (ACD) by using an RNAscope Multiplex Fluorescent Reagent Kit (ACD) in accordance with the manufacturer\u0026rsquo;s protocol. Finally, the sections were mounted in a DAPI-containing anti-fade mounting medium. Both FISH with RNAscope and immunofluorescence were conducted for fluorescence staining of MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; mice and littermates. Fluorescence labeling of \u003cem\u003eOprm1\u003c/em\u003e was conducted following the protocol for FISH. The fluorescence labeling of astrocytes was performed by sequentially incubating the sections with rabbit anti-GFAP primary antibodies (1:500, 16825-1-AP, Proteintech, China) at 4\u0026deg;C (12 h) and dylight488-conjugated goat anti-rabbit antibodies (1:500, A23240, Abbkine, China) at room temperature (2 h). Subsequently, the sections were mounted with a DAPI-containing anti-fade mounting medium when both Oprm1 and astrocytes were labeled. A fluorescence microscope (Zeiss, Germany) was used to acquire confocal images of all sections, and cells showing positive labeling were enumerated.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMorphological examination for confirmation of the specific expression of rAAVs\u003c/h2\u003e \u003cp\u003eTo confirm the specific expression of rAAVs coupled with the GfaABC1D promoter in vlPAG astrocytes, immunofluorescence was performed as reported previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, mice injected with rAAVs were anesthetized by 4% isoflurane inhalant, before transcardial perfusion with 0.9% sodium chloride solution (Kelun, China) and 4% PFA fixative (BL539A, Biosharp, China). The brains were removed, post-fixed with 4% PFA fixative for 24 h, and then immersed in 30% sucrose phosphate-buffered saline (PBS) solution at 4\u0026deg;C (48 h). Subsequently, the brains were embedded in an OCT compound at -22\u0026deg;C. Fresh frozen sections (16 \u0026micro;m) containing the vlPAG region (coronal plane) were cut. For immunofluorescence assay, the sections were incubated for 60 min with PBS supplemented with 10% non-immune donkey serum (T8200, SolarBio, China) and 0.5% Triton X-100 (BL939A, Biosharp, China). The sections were sequentially incubated with rabbit anti-GFAP antibodies (1:300, 16825-1-AP, Proteintech) at 4\u0026deg;C (12 h) and dylight680-conjugated goat anti-rabbit antibodies (1:500, A23720, Abbkine) at room temperature (2 h). After labeling, the sections were confocally imaged using a fluorescence microscope to acquire confocal images of the sections, and cells showing positive labeling were enumerated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed in their original form or as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. GraphPad Prism 9.0 was utilized for data analysis. Comparison of two groups was achieved through unpaired or paired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test, and comparison of multiple groups was performed with one-way analysis of variance (ANOVA) and Sidak\u0026rsquo;s multiple comparison test. Statistical significance was considered at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003evlPAG MOR activation triggered anxiety-like behavior\u003c/h2\u003e \u003cp\u003eMORs play a role in modulating emotional responses; however, the specific brain mechanisms underlying this effect remain unclear. To determine how MORs expressed on the vlPAG modulate anxiety-like behavior, we conducted bilateral insertion of guide cannulas into the vlPAG of mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B) to specifically activate vlPAG MORs through pharmacological administration. The cannula position was confirmed morphologically (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Next, we determined the effect of intravenous DAMGO, saline, or DAMGO\u0026thinsp;+\u0026thinsp;CTAP on anxiety-like behaviors. We found that the total distance traveled by mice in the EPM tests was comparable between the three mice groups, suggesting that locomotor activity was not affected by DAMGO (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). However, intra-vlPAG DAMGO induced a significant anxiogenic performance, as shown by the lower percentage of open arm entries and time spent in the open arms in control mice than in those with intra-vlPAG saline administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, E, F). These effects were reversed by CTAP (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, E, F), thus indicating the functional role of vlPAG MORs in modulating anxiety responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMOR\u003csub\u003eAstro\u003c/sub\u003e, but not MOR\u003csub\u003eGABA\u003c/sub\u003e or MOR\u003csub\u003eGlut\u003c/sub\u003e, is involved in vlPAG-MOR-induced anxiety-like behavior\u003c/h2\u003e \u003cp\u003eIn the vlPAG, MORs exhibit a high expression level in different cell types such as astrocytes, GABAergic neurons, and glutaminergic neurons [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To assess the distinct effects of vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e, MOR\u003csub\u003eGABA\u003c/sub\u003e, and MOR\u003csub\u003eGlut\u003c/sub\u003e on the vlPAG-MOR elicited anxiety-like behavior, three mouse lines specifically lacking MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus;, MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus;, and MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus;, respectively, and their controls (MOR\u003csub\u003eAstro\u003c/sub\u003e+/+, MOR\u003csub\u003eGABA\u003c/sub\u003e+/+, or MOR\u003csub\u003eGlut\u003c/sub\u003e+/+, respectively) were generated according to previously reported methods. To further confirm the validity of MOR deletions, the absence of astrocytic MORs of MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B), GABAergic MORs of MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus; mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), and glutamatergic MORs of MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus; mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) were morphologically detected by combined FISH with RNAscope and immunofluorescence technology. We inserted guide cannulas into the bilateral vlPAG of these mice and tested anxiety-like behavior through the EPM test after intra-vlPAG DAMGO or saline administration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring assessment of the effects of vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e on the vlPAG-MOR-elicited anxiety-like behavior, EPM tests showed that the total distance traveled by mice was slightly different among the four mice groups, suggesting that genetic absence of MOR\u003csub\u003eAstro\u003c/sub\u003e did not affect locomotor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; and MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ mice with intra-vlPAG saline administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E) showed no significant differences in the percentage of open arm entries and time spent in the open arms, indicating that the absence of MOR\u003csub\u003eAstro\u003c/sub\u003e did not alter the basal emotional responses of mice. However, the anxiogenic effects induced by vlPAG MORs were significantly reversed by MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus;, as shown by the reduced percentage of open arm entries and time spent in the open arms of MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ mice with intra-vlPAG DAMGO administration compared to that of MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ mice with intra-vlPAG saline administration; in contrast, no significant differences were noted in the percentage of open arm entries and time spent in the open arms between MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; mice with intra-vlPAG DAMGO administration and MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; mice with intra-vlPAG saline administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). A remarkable difference was found in the percentage of open arm entries and time spent in the open arms between MOR\u003csub\u003eAstro\u003c/sub\u003e+/+ mice administered with intra-vlPAG DAMGO administration and MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; mice with intra-vlPAG DAMGO administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Thus, MORs\u003csub\u003eAstro\u003c/sub\u003e have a crucial role in vlPAG-MOR-induced anxiety-like behavior.\u003c/p\u003e \u003cp\u003eWe then assessed the effects of vlPAG MOR\u003csub\u003eGABA\u003c/sub\u003e on vlPAG-MOR-elicited anxiety-like behavior and found that the genetic absence of MOR\u003csub\u003eGABA\u003c/sub\u003e did not affect locomotor activity; this was confirmed by the observation that the total distance traveled by mice was slightly different among the four mice groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The percentage of open arm entries and time spent in the open arms displayed no significant differences between MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus; and MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ mice with intra-vlPAG saline administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), indicating that the absence of MOR\u003csub\u003eGABA\u003c/sub\u003e did not alter the basal emotional responses of mice. Both MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus; and MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ mice were anxiogenic after intra-vlPAG DAMGO administration, and no significant difference was noted between MOR\u003csub\u003eGABA\u003c/sub\u003e+/+ mice with intra-vlPAG DAMGO administration and MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus; mice with intra-vlPAG DAMGO administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Thus, MOR\u003csub\u003eGABA\u003c/sub\u003e hardly participates in vlPAG-MOR-induced anxiety-like behavior.\u003c/p\u003e \u003cp\u003eSimilar to MOR\u003csub\u003eGABA\u003c/sub\u003e, the assessment of how vlPAG MOR\u003csub\u003eGABA\u003c/sub\u003e affects vlPAG-MOR-elicited anxiety-like behavior revealed no influence of the genetic absence of MOR\u003csub\u003eGlut\u003c/sub\u003e on locomotor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). No significant variations were noted in the percentage of open arm entries and time spent in the open arms between MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus; and MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ mice following intra-vlPAG saline administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E), thus indicating that the absence of MOR\u003csub\u003eGlut\u003c/sub\u003e did not alter the basal emotional responses of mice. Both MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus; and MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ mice were anxiogenic after intra-vlPAG DAMGO administration, and there was no significant difference between MOR\u003csub\u003eGlut\u003c/sub\u003e+/+ mice with intra-vlPAG DAMGO administration and MOR\u003csub\u003eGlu\u003c/sub\u003e\u0026minus;/\u0026minus; mice with intra-vlPAG DAMGO administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Thus, MOR\u003csub\u003eGlut\u003c/sub\u003e barely participates in vlPAG-MOR-induced anxiety-like behavior.\u003c/p\u003e \u003cp\u003eTaken together, our results indicate that MOR\u003csub\u003eAstro\u003c/sub\u003e, but not MOR\u003csub\u003eGABA\u003c/sub\u003e or MOR\u003csub\u003eGlut\u003c/sub\u003e, is involved in vlPAG-MOR-induced anxiety-like behavior.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003evlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e induces anxiety-like behavior through astrocytic calcium signaling\u003c/h2\u003e \u003cp\u003eNext, to investigate the cellular mechanisms underlying MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent anxiety-like behavior, we focused on the role of vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e activation and its effects on intracellular calcium signaling in astrocytes. The activation of MOR\u003csub\u003eAstro\u003c/sub\u003e can trigger calcium signaling in the CNS [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], GCamp6f was expressed in PAG astrocytes by using a viral vector with the GfaABC1D promoter for calcium imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Immunohistochemical assay confirmed the predominant expression of GCamp6f in astrocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). The treatment of vlPAG slices with the specific MOR agonist DAMGO (1 \u0026micro;M) significantly increased calcium levels in vlPAG astrocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, F); this was not observed in the control group perfused with DAMGO-free ACSF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The increase in calcium levels was inhibited by CTAP (10 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) but not by TTX (1 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), suggesting that this increase was specifically mediated by MOR\u003csub\u003eAstro\u003c/sub\u003e activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the involvement of astrocytic calcium signaling in the vlPAG in modulating anxiogenic performance, we examined whether direct activation of PAG astrocytes alone is sufficient to produce anxiety-like behavior. Designer receptors exclusively activated by designer drugs (DREADDs) coupled with the GfaABC1D promotor virus were used, which efficiently activated the calcium signals of astrocytes, as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We injected rAAV5-GfaABC1D-hM3Dq-mCherry-WPRE-pA into the vlPAG targeting astrocytes (Astro\u003csub\u003ehM3Dq\u003c/sub\u003e), with rAAV5-GfaABC1D-mCherry-WPRE-pA (Astro\u003csub\u003econtrol\u003c/sub\u003e) as the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Immunofluorescence results showed that the mCherry report proteins and GFAP were mostly co-labeled (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C), indicating that hM3Dq rAAVs were specifically expressed in PAG astrocytes. The intraperitoneal administration of 1 mg/kg CNO remarkably caused anxiety-like behavior in Astro\u003csub\u003ehM3Dq\u003c/sub\u003e mice; this was confirmed by the lower percentage of open arm entries and time spent in the open arms as compared to those of Astro\u003csub\u003econtrol\u003c/sub\u003e mice with CNO administration or Astro\u003csub\u003ehM3Dq\u003c/sub\u003e mice with saline administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). The total distance traveled by mice was comparable between these three mice groups, suggesting no influence of Astro\u003csub\u003ehM3D\u003c/sub\u003eq on locomotor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Thus, the direct activation of vlPAG astrocytic calcium signaling elicits anxiety-like behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined whether MOR\u003csub\u003eAstro\u003c/sub\u003e-triggered calcium signaling is the cellular mechanism underlying the vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent anxiety-like behavior. For this purpose, we bilaterally delivered rAAV5-GfaABC1D-hPMCA2w/b-mCherry-WPRE-SV40 into the PAG (Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), which specifically extruded cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e and reduced Ca\u003csup\u003e2+\u003c/sup\u003e oscillations in PAG astrocytes, as described previously [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The rAAV5-GfaABC1D-mCherry-WPRE-SV40 virus was also injected into the PAG to generate control mice (Astro\u003csub\u003econtrol\u003c/sub\u003e). The results of immunofluorescence assay showed that the mCherry report proteins and GFAP were mostly co-labeled (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C), indicating that hPMCA2w/b rAAVs were specifically expressed in PAG astrocytes. We inserted guide cannulas into the bilateral vlPAG of these mice and tested their anxiety-like behavior through EPM tests following intra-vlPAG DAMGO or saline administration. Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e and Astro\u003csub\u003econtrol\u003c/sub\u003e mice with intra-vlPAG saline administration showed no apparent differences in the percentage of open arm entries and time spent in the open arms (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F); this finding indicated that Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e did not alter the basal emotional responses of mice. However, the anxiogenic performance elicited by vlPAG MORs were significantly reversed by Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e, as shown by the decreased percentage of open arm entries and time spent in the open arms of Astro\u003csub\u003econtrol\u003c/sub\u003e mice with intra-vlPAG DAMGO administration as compared to those of Astro\u003csub\u003econtrol\u003c/sub\u003e mice with intra-vlPAG saline administration. In contrast, Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e mice with intra-vlPAG DAMGO administration and Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e mice with intra-vlPAG saline administration showed no significant differences in the two abovementioned parameters (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). The percentage of open arm entries as well as the time spent in the open arms displayed significant differences between Astro\u003csub\u003econtrol\u003c/sub\u003e mice with intra-vlPAG DAMGO administration and Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e mice with intra-vlPAG DAMGO administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). These four mice groups showed comparable total distance traveled, suggesting that the locomotor activity was not influenced by Astro\u003csub\u003ehPMCA2w/b\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Thus, astrocytic calcium signaling is the cellular mechanism underlying vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent anxiety-like behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, the obtained results indicate that vlPAG MOR\u003csub\u003eAstro\u003c/sub\u003e elicits anxiety-like behavior through astrocytic calcium signaling.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we examined the role of PAG MORs in modulating anxiety-like behavior. We observed that the local activation of PAG MORs through microinjection of DAMGO in the PAG elicited an apparent MOR-dependent anxiety-like behavior in the EPM tests. Surprisingly, neither MOR\u003csub\u003eGlut\u003c/sub\u003e nor MOR\u003csub\u003eGABA\u003c/sub\u003e was involved in modulating this form of MOR-dependent anxiety-like behavior. In contrast, in the EPM test, conditional knockdown of MOR\u003csub\u003eAstro\u003c/sub\u003e reversed the DAMGO-induced reduction in the percentage of open arm entries and time spent in the open arms. By using calcium imaging and chemogenetic technologies, we further demonstrated the critical role of MOR\u003csub\u003eAstro\u003c/sub\u003e in inducing astrocytic calcium signaling during the modulation of MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent anxiety-like behavior. Our study demonstrates a novel MOR\u003csub\u003eAstro\u003c/sub\u003e-dependent emotional response in the PAG, which reveals a possible solution for treating emotional dysfunctions during MOR-targeted management in clinical settings.\u003c/p\u003e \u003cp\u003eThe functional role of MORs in analgesia has been widely studied, with corresponding theories implemented in the area of clinical pain management [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, several clinical studies have cautioned about the increased risks of emotional dysfunction among patients who use opioid drugs during pain management [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition to pain modulation, MORs are extensively involved in emotional responses [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In clinical settings, patients with major depressive disorder display lower MOR availability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Indeed, according to previous studies, blocking the effect of MORs by oral administration of naltrexone increased the remarkable panic provocation of the volunteers under 35% CO\u003csub\u003e2\u003c/sub\u003e inhalation stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, anxiety and depression disorders are also enhanced in individuals with long-term opioid-related drug use [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These conflicting reports suggest that the role of MORs in modulating emotional responses may be bidirectional. MORs are extensively expressed in several emotion-related brain areas that control different aspects of the transmission of emotive information. The modulation of emotional behavior by MORs in different brain regions exhibit region-specificity. For example, MORs expressed on the basolateral amygdala and dorsal raphe nucleus induce anxiolytic effects upon their activation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], whereas MORs expressed on the central amygdala and lateral septum elicit anxiety-like behavior upon their activation in rodents [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, the precise mechanism by which different local brain regions of MORs modulate emotional responses is advantageous for risk aversion to emotional dysfunctions during MOR-targeted pain management. The PAG is a critical brainstem region that controls the activation of the descending pain inhibitory pathway. MORs are densely expressed in the PAG [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As shown in rodent studies, both endogenous and exogenous activation of these MORs induce remarkable analgesia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The PAG also efficiently participates in the modulation of emotional responses such as aversive and defensive behaviors [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Homogenetically activating PAG vglut2-positive excitatory neurons elicits anxiety-like behaviors in open field and light/dark tests [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Here, we found that activated PAG MORs caused a decrease in the percentage of open arm entries and time spent in the open arms by mice; moreover, these effects were reversed by pharmacological blockage of PAG MORs. Thus, PAG MORs have a functional role in modulating anxiety-related behavior, which may explain the elevated emotional disorders of patients using opioid-related drugs.\u003c/p\u003e \u003cp\u003eThe most conspicuous results of our work were that the anxiety-like behaviors elicited by the activation of PAG MORs were primarily contributed by PAG MOR\u003csub\u003eAstro\u003c/sub\u003e, whereas MOR\u003csub\u003eGlut\u003c/sub\u003e and MOR\u003csub\u003eGABA\u003c/sub\u003e only had a negligible effect on anxiety-related responses. Several morphological studies have confirmed that MORs are distributed in various types of neurocytes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, their functional modulatory role in the CNS is not fully understood. By developing three lines of MOR-knockdown mice, including MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus;, MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus;, and MOR\u003csub\u003eAstro\u003c/sub\u003e\u0026minus;/\u0026minus; [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we detected the functional role of divergent cell types of PAG MORs on the modulation of anxiety-like behaviors. We found that anxiety-like behaviors induced by the local activation of vlPAG MORs were reversed by the conditional knockdown of MOR\u003csub\u003eAstro\u003c/sub\u003e. In contrast, anxiety-like behaviors were marginally modulated in MOR\u003csub\u003eGlut\u003c/sub\u003e\u0026minus;/\u0026minus; or MOR\u003csub\u003eGABA\u003c/sub\u003e\u0026minus;/\u0026minus; mice. These results are exhilarating because of confirmation of the functional role of PAG MOR\u003csub\u003eGABA\u003c/sub\u003e in pain modulation. As an inhibitory G protein-coupled receptor (GiPCR), the activation of MOR\u003csub\u003eGABA\u003c/sub\u003e could decrease GABAergic neuron excitability and subsequently weaken the tonic inhibition from GABAergic neurons to PAG-rostral ventromedial medulla (RVM) excitatory projections to elicit analgesic effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our results demonstrated the previously unappreciated role of MOR\u003csub\u003eGABA\u003c/sub\u003e in modulating anxiety-related responses, with MOR\u003csub\u003eAstro\u003c/sub\u003e displaying critical effects in these responses. Thus, theoretically, the targeted interception of the activation of MOR\u003csub\u003eAstro\u003c/sub\u003e would reduce the aversive emotional responses without affecting the analgesic effects during PAG MOR activation. This would provide a potential possibility to address the elevated emotional disorders of patients using opioid-related drugs targeting astrocytes of the PAG. We also found that MOR\u003csub\u003eGlut\u003c/sub\u003e slightly influenced the modulation of anxiety-like behaviors. This finding aligned with the observation that the chemogenetic activation of GiPCR signaling in vGlut2-positive neurons of the PAG minimally contributed to emotional responses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Beyond the modulation of pain and emotion, the PAG is associated with several other brain-related functions, including respiratory, cardiovascular, and sleep and wakefulness regulation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thus, MOR expression on PAG glutamatergic neurons might have a role in modulating autonomic nervous system function or sleep-wake reactions.\u003c/p\u003e \u003cp\u003eBy using \u003cem\u003ein vitro\u003c/em\u003e calcium imaging combined with chemical genetics [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we further demonstrated that the PAG MOR\u003csub\u003eAstro\u003c/sub\u003e elicits anxiety-like behavior through an astrocytic calcium signaling mechanism. As confirmed previously, astrocytic GiPCR activation elevates calcium ion levels in astrocytes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the nucleus accumbens, the activation of MOR\u003csub\u003eAstro\u003c/sub\u003e by DAMGO also elicited calcium oscillations in astrocytes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In agreement with these studies, we found that the activation of PAG MORs induced significant enhancement of calcium levels in astrocytes, and this phenomenon was not blocked by TTX, indicating that this increase was specifically mediated by MOR\u003csub\u003eAstro\u003c/sub\u003e activation. The chemical activation of calcium signaling in PAG astrocytes further confirmed that calcium signaling in PAG astrocytes efficiently induces anxiogenic behavior. In contrast, anxiety-like behaviors elicited by the activation of PAG MORs were reversed by specific interception of astrocytic calcium signaling. As reported earlier, astrocytic calcium signaling activation can promote the release of gliotransmitters and subsequently modulate neural excitability through the control of synaptic transmission [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, the activation of MOR\u003csub\u003eAstro\u003c/sub\u003e seemed to modulate synaptic transmission through astrocytic calcium signaling, thereby controlling the release of gliotransmitters in the PAG to elicit anxiogenic effects. However, further research is required to support our hypothesis.\u003c/p\u003e \u003cp\u003eAlthough our study has certain strengths, there are also some limitations. First, male mice alone were used because female mice show nominal differences in the MOR-dependent modulation of emotional responses compared to male mice. Additional investigations with female mice are required for improved understanding of the role of PAG MORs in the modulation of emotional responses. Second, we confirmed only the action of the MOR\u003csub\u003eAstro\u003c/sub\u003e-induced astrocytic calcium signaling mechanism on the modulation of vlPAG MOR-induced anxiety-like behavior. However, the role of astrocytes in modulating brain behaviors is always accomplished by regulating neural activity through control of synaptic transmission or intrinsic excitability of neurons. Further electrophysiological studies should focus on explaining the astrocytic control of the neural mechanisms of PAG MOR-induced anxiogenic effects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were carried out by following the guidelines of the Chinese Council on Animal Care. The study protocol was approved by the Animal Protection Committee of Shaanxi Normal University and the Animal Care Committee of The First Affiliated Hospital of Xi\u0026apos;an Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYinan Du and Zhiqiang Liu designed experiments, conceived the project and prepared the manuscript. Yinan Du performed behavior test, relevant surgery and in vitro calcium imaging. Aozhuo Zhang, Zhiwei Li and Yukui Zhao performed morphological test and statistical analysis. Other authors helped to write and refine the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (NSFC) (grants 91949105, 82071516) and the Science and Technology Innovation Team Project of Xi\u0026apos;an Medical University (grants 2021TD14).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCuitavi J, Torres-P\u0026eacute;rez JV, Lorente JD, Campos-Jurado Y, Andr\u0026eacute;s-Herrera P, Polache A, et al. Crosstalk between mu-opioid receptors and neuroinflammation: Consequences for drug addiction and pain. 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Front Cell Neurosci. 2024;18:1347491.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKennedy SE, Koeppe RA, Young EA, Zubieta JK. Dysregulation of endogenous opioid emotion regulation circuitry in major depression in women. Arch Gen Psychiatry. 2006;63(11):1199\u0026ndash;208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsquivel G, Fern\u0026aacute;ndez-Torre O, Schruers KR, Wijnhoven LL, Griez EJ. The effects of opioid receptor blockade on experimental panic provocation with CO2. J Psychopharmacol. 2009;23(8):975\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePickar D, Cohen MR, Naber D, Cohen RM. Clinical studies of the endogenous opioid system. Biol Psychiatry. 1982;17(11):1243\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasaki K, Fan LW, Tien LT, Ma T, Loh HH, Ho IK. 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J Neurosci. 2016;36(29):7580\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBazargani N, Attwell D. Astrocyte calcium signaling: the third wave. Nat Neurosci. 2016;19(2):182\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoenaga J, Araque A, Kofuji P, Herrera Moro Chao D. Calcium signaling in astrocytes and gliotransmitter release. Front Synaptic Neurosci. 2023;15:1138577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKofuji P, Araque A. G-protein-coupled receptors in astrocyte-neuron communication. Neuroscience. 2021;456:71\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewman EA. Glial cell regulation of neuronal activity and blood flow in the retina by release of gliotransmitters. Philos Trans R Soc Lond B Biol Sci. 2015;370(1672):20140195.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"behavioral-and-brain-functions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"babf","sideBox":"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)","snPcode":"12993","submissionUrl":"https://submission.nature.com/new-submission/12993/3","title":"Behavioral and Brain Functions","twitterHandle":"@BBF_Journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anxiety, mu-opioid receptors, periaqueductal gray, astrocytes, calcium signaling","lastPublishedDoi":"10.21203/rs.3.rs-6262877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6262877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMu-opioid receptors (MORs) are critical regulators mediating the modulation of several behavioral reactions, including analgesia, addiction, and sedation. Recent studies have reported that MORs are closely associated with mood disorders or anxiety behaviors; however, the underlying neural mechanisms remain unclear. The periaqueductal gray (PAG), a key brain area, participates in the modulation of aversive emotional behaviors. MORs show a high expression in the ventrolateral PAG (vlPAG) region. This study explored the preliminary role of MORs expressed in the vlPAG in modulating emotional behaviors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBilateral administration of DAMGO, an MOR-specific agonist, into the vlPAG of male mice elicited anxiety-like behaviors in elevated plus maze tests. This phenotype was reversed by conditional knockdown of astrocytic MORs. In contrast, glutamatergic or GABAergic MORs were not involved in vlPAG MOR-dependent anxiety-like behaviors. By using \u003cem\u003ein vitro\u003c/em\u003e calcium imaging of vlPAG astrocytes and chemical genetic technologies, we found that vlPAG astrocytic MORs can promote astrocytic calcium signaling, which can efficiently induce anxiety-like behaviors. Accordingly, the interference of astrocytic calcium signaling by viral infection reversed vlPAG-dependent anxiety-like behaviors.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings demonstrated that vlPAG astrocytic, but not glutamatergic or GABAergic, MORs are involved in modulating emotional reactions, and these effects are accomplished by MOR-elicited astrocytic calcium signaling mechanisms. The present study provides a theoretical basis for treating emotional dysfunctions during MOR-targeted management.\u003c/p\u003e","manuscriptTitle":"Role of astrocytic mu-opioid receptors of the ventrolateral periaqueductal gray in modulating anxiety-like responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 05:37:10","doi":"10.21203/rs.3.rs-6262877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-27T03:07:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T04:51:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T18:52:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T14:29:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66442108541332222537922544985181832025","date":"2025-04-06T01:56:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257292254525172291619283315667311072816","date":"2025-04-05T08:28:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212507608853475122006111166065262199015","date":"2025-04-04T00:52:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T01:11:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-21T06:21:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-21T06:19:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Behavioral and Brain Functions","date":"2025-03-19T15:04:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"behavioral-and-brain-functions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"babf","sideBox":"Learn more about [Behavioral and Brain Functions](http://behavioralandbrainfunctions.biomedcentral.com)","snPcode":"12993","submissionUrl":"https://submission.nature.com/new-submission/12993/3","title":"Behavioral and Brain Functions","twitterHandle":"@BBF_Journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf74c5bd-40bc-4641-95e4-be788ec6f417","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:07:22+00:00","versionOfRecord":{"articleIdentity":"rs-6262877","link":"https://doi.org/10.1186/s12993-025-00291-0","journal":{"identity":"behavioral-and-brain-functions","isVorOnly":false,"title":"Behavioral and Brain Functions"},"publishedOn":"2025-07-23 15:58:25","publishedOnDateReadable":"July 23rd, 2025"},"versionCreatedAt":"2025-05-05 05:37:10","video":"","vorDoi":"10.1186/s12993-025-00291-0","vorDoiUrl":"https://doi.org/10.1186/s12993-025-00291-0","workflowStages":[]},"version":"v1","identity":"rs-6262877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6262877","identity":"rs-6262877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}