Introduction

Major depression is one of the most prevalent mood disorders worldwide. Individuals with depression display a variety of symptoms marked by prolonged depressed mood and diminished interest in activities [1, 2]. Although decades of clinical and basic research have provided significant insights into the pathophysiology of depression, the underlying neural basis remains unclear [3, 4]. Current treatment strategies are not sufficient to alleviate the burden caused by depression. For example, classic antidepressant drugs targeting traditional neurotransmitter systems, like serotonin, have delayed onsets [5]. Novel fast-acting antidepressants, such as ketamine, offer rapid relief from depressive symptoms but face restricted clinical use due to adverse effects, including safety in long-term treatment [6]. The future development of antidepressant medications still requires a better understanding of the neural mechanisms involved in depression and the identification of effective molecular targets.

In the central nervous system, hemichannels formed by connexins (Cxs) or pannexins are essential for neuron-glia communication and maintenance of brain homeostatic balance [7, 8]. In normal conditions, glial Cx-based hemichannels have relatively low activity. Under physiological conditions, neuronal pannexin hemichannels are permeable to small molecules like ATP and are preferentially permeable to Cl– [9, 10]. However, exacerbated neuronal hemichannel activity has been observed in diverse pathological conditions [51]. Overall, D4 did not change depressive-like z scores in unstressed mice (Vehicle, 0.00 ± 0.16; D4, − 0.24 ± 0.20) (Fig. 2A, G). Repeated LPS challenge induced significantly higher depressive-like z scores in mice, suggesting the expression of depressive-like behavior. This behavioral effect induced by LPS was reversed by D4 (Saline/vehicle, 0.00 ± 0.09; LPS/vehicle, 1.15 ± 0.11; LPS/D4, 0.28 ± 0.12) (Fig. 2N). Taken together, these results demonstrate that blocking preferentially Cx hemichannels with D4 exerts antidepressant-like effects in mice.

D4 suppresses LPS-induced astrocytic activation and hemichannel activity

Reactive glial cells are hallmarks of neuroinflammation and have been implicated in the pathophysiology of depression [52,53,54,55]. Having demonstrated the antidepressant-like properties of D4, we first investigated the effect of D4 on LPS-mediated inflammation. We injected adult mice intraperitoneally with a low dose of LPS (0.75 mg/kg, i.p.) or saline (control) daily for 1 week. Mice were orally fed with either D4 (5 mg/kg, p.o.) or vehicle after each LPS injection. Four hours after the last LPS injection, plasma and hippocampi were extracted for ELISA assay. We found that repeated LPS injections significantly increased pro-inflammatory cytokine IL-1β levels in the plasma and hippocampi. Notably, D4 significantly attenuated LPS-induced increases in the plasma and hippocampal IL-1β levels (Additional file 4: Fig. S4). We next tested whether D4 can affect reactive glia in LPS-induced depressive-like mice. After LPS injections and D4 treatment, mice were sacrificed after the last behavioral testing. We used GFAP as a marker to label astrocytes. Injections of LPS significantly increased astrocyte density in the hippocampus. Importantly, administration of D4 significantly decreased the number of GFAP+ astrocytes in the hippocampus (Saline/vehicle, 400.6 ± 26.3; LPS/vehicle, 464.6 ± 16.7; LPS/D4, 343.5 ± 17.5) (Fig. 3A, B). However, LPS-mediated hippocampal reactive microgliosis (Iba1+ microglia) was unaffected by D4 (Saline/vehicle, 85.2 ± 12.8; LPS/vehicle, 152.9 ± 21.0; LPS/D4, 149.2 ± 17.8) (Fig. 3C, D).

Fig. 3
figure 3

D4 reduces astrocytic proliferation and hemichannel activity induced by repeated low-dose LPS injections. A Immunostaining for astrocyte (GFAP) in the hippocampus. GFAP, red; DAPI, blue. Scale bar, 100 µm. B Quantification of GFAP+ astrocytes in the hippocampus. n = 15 hippocampal sections from 5 mice for all groups. C Immunostaining for microglia (Iba1) in the hippocampus. Iba1, red; DAPI, blue. Scale bar, 100 µm. D Quantification of Iba1+ cells in the hippocampus. n = 15 hippocampal sections from 5 mice for all groups. E Representative images showing astrocytes (GFAP) and CBF uptake in the hilus of the hippocampal DG from the LPS-injected mice. GFAP, red; CBF, green; DAPI, blue. Scale bar, 10 µm. F Quantification of GFAP+CBF+ area in the stained DG sections. Saline/vehicle, n = 8 sections from 3 mice; LPS/vehicle, n = 10 DG sections from 5 mice; LPS/D4, n = 10 DG sections from 4 mice. G Representative images showing microglia (Iba1) and CBF uptake in the hilus of the hippocampal DG the LPS-injected mice. Iba1, red; CBF, green; DAPI, blue. Scale bar, 10 µm. H Quantification of Iba1+CBF+ area in the stained DG sections. Saline/vehicle, n = 9 sections from 3 mice; LPS/vehicle, n = 25 DG sections from 9 mice; LPS/D4, n = 18 DG sections from 6 mice. I Representative images showing neurons (Nissl) and CBF uptake in the granule cell layer of the hippocampal DG the LPS-injected mice. Nissl, magenta; CBF, green; DAPI, blue. Scale bar, 10 µm. J Quantification of Nissl+CBF+ area in the stained DG sections. Saline/vehicle, n = 8 sections from 3 mice; LPS/vehicle, n = 11 DG sections from 5 mice; LPS/D4, n = 9 DG sections from 4 mice. Saline/vehicle (Ctrl), white; LPS/vehicle, red; LPS/D4, blue. The open circle shows the value from each DG. One-way ANOVA followed by LSD post hoc test (B). Kruskal–Wallis test with Bonferroni correction (D, F, H, J). *p < 0.05, **p < 0.01, ***p < 0.001

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To directly interrogate whether D4 has a stronger impact on the hemichannel activity of astrocytes or microglia, we co-stained for GFAP or Iba1 after CBF dye loading in LPS mice. In the DG, D4 drastically suppressed LPS-induced CBF uptake in GFAP-positive astrocytes compared to saline/vehicle control (Saline/vehicle, 100.0 ± 23.9%; LPS/vehicle, 609.6 ± 145.6%; LPS/D4, 122.2 ± 67.9%), suggesting that D4 inhibits dye uptake in astrocytes triggered by LPS (Fig. 3E, F). By contrast, D4 only had a minimal effect on CBF uptake evoked by LPS in the Iba1-positive microglia (Saline/vehicle, 100.0 ± 14.4%; LPS/vehicle, 246.1 ± 46.0%; LPS/D4, 207.5 ± 26.3%) (Fig. 3G, H). In addition, we found that D4 significantly reduced CBF uptake induced by LPS in Nissl-positive neurons in the hippocampal DG (Saline/vehicle, 100.0 ± 20.6%; LPS/vehicle, 1566.0 ± 461.3%; LPS/D4, 236.5 ± 97.8%) (Fig. 3I, J).

Together, these data suggest that D4 can preferentially block astrocytic Cx hemichannels, and that LPS-induced increases in membrane permeability of microglia might be mediated by other pathways not inhibited by D4, such as pannexin1 hemichannels [7].

D4 improves depressive-like behaviors in mice subjected to chronic restraint stress

To further validate the antidepressant-like potentials of D4, we asked whether D4 could reverse behavioral changes induced by CRS, a stress paradigm used for develo** depressive-like phenotypes in mice [56, 57]. We first assessed the behavioral effects of D4 on unstressed control mice using an alternative testing protocol. Mice were orally fed with either a vehicle or D4 (5 mg/kg) 16–18 h before each behavioral test (Fig. 4A). We found no differences in the OFT center time (Vehicle, 31.8 ± 3.0 s; D4, 31.2 ± 3.7 s) and total distance (Vehicle, 47.4 ± 2.4 m; D4, 48.2 ± 2.0 m) between vehicle and D4 treated mice (Fig. 4B, C). The latter could confirm that D4 had no detectable effect on anxiety-like behavior and locomotor activity in healthy control mice. In the TST, D4 induced a significant reduction in immobility (by 34.5%) at 18 h after treatment (Vehicle, 48.1 ± 9.5 s; D4, 31.5 ± 6.1 s) (Fig. 4D). D4-treated mice showed a small but insignificant decrease (by 17.8%) in FST immobility (Vehicle, 96.1 ± 6.5 s; D4, 79.1 ± 12.6 s) (Fig. 4E). In the SPT, treatment with D4 did not change sucrose preference in unstressed mice (Vehicle, 70.3 ± 6.7%; D4, 64.0 ± 6.2%) (Fig. 4F).

Fig. 4
figure 4

D4 improves depressive-like behaviors induced by chronic restraint stress (CRS). A Schematic of drug treatment and behavioral testing. B Center time and (C) total distance in the OFT. D Time immobile in the TST. E Time immobile in the FST. F Sucrose preference in the SPT. G Depressive-like z score of the unstressed mice treated with vehicle or D4. n = 13 mice (5 male and 8 female) for each group. H Schematic of the design of CRS, drug treatment, and behavioral testing. I Center time and J total distance in the OFT. K Time immobile in the TST. L Time immobile in the FST. M Sucrose preference in the SPT. N Normalized depressive-like z score of control/vehicle, CRS/vehicle, and CRS/D4 treated mice. Control/vehicle, n = 10 mice, 5 male and 5 female; CRS/vehicle, n = 18 mice, 9 male and 9 female; CRS/D4, n = 19 mice, 10 male and 9 female. The filled dot indicates the value of each mouse. Data are mean ± s.e.m. Unpaired Student's t-test (B, C, E–G). Mann–Whitney U test (D). One-way ANOVA followed by LSD post hoc test (L–N). Kruskal–Wallis test with Bonferroni correction (I–K). *p < 0.05, **p < 0.01, ***p < 0.001

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Next, we tested whether D4 could affect depressive-like behaviors in the mice subject to CRS. In the CRS-induced mouse model of depression, mice were exposed to 6-h restraint stress daily for 5 weeks. After the CRS paradigm, mice were orally treated with either vehicle or D4 (5 mg/kg, p.o.) eighteen hours before each behavioral test (Fig. 4H). In the OFT, we observed no significant differences in center time (Control/vehicle, 49.8 ± 7.1 s; CRS/vehicle, 48.2 ± 5.4 s; CRS/D4, 41.1 ± 4.7 s) or total distance (Control/vehicle, 35.4 ± 1.8 m; CRS/vehicle, 38.0 ± 1.9 m; CRS/D4, 39.2 ± 1.2 m) across groups following only a single dose of D4 (Fig. 4I, J). We then measured behavioral despair in the TST and FST. D4 significantly reduced LPS-induced increases in the TST immobility in CRS-exposed mice (Control/vehicle, 57.8 ± 9.6 s; CRS/vehicle, 108.4 ± 10.4; CRS/D4, 58.5 ± 9.4 s) (Fig. 4K). Again, D4 induced a significant decrease in FST immobility time in CRS-exposed mice 18 h after treatment (Control/vehicle, 85.7 ± 9.2 s; CRS/vehicle, 129.5 ± 8.5; CRS/D4, 95.2 ± 8.1 s) (Fig. 4K). In the SPT, CRS led to a reduction in the sucrose preference. D4 treatment also significantly increased the sucrose preference of CRS-exposed mice (Control/vehicle, 69.9 ± 6.9%; CRS/vehicle, 51.1 ± 6.2%; CRS/D4, 67.0 ± 4.5%) (Fig. 4L).

Next, we compared depressive-like behavior using the depressive-like z scores. Oral administration of D4 in a different treatment paradigm did not affect depressive-like z scores in the unstressed control mice (Vehicle, 0.00 ± 0.12; D4, − 0.22 ± 0.15) (Fig. 4G). Indeed, mice exposed to CRS exhibited significantly higher depressive-like z scores than the control/vehicle group. The CRS-induced depressive-like phenotype in mice was again ameliorated by D4 (Control/vehicle, 0.00 ± 0.14; CRS/vehicle, 1.02 ± 0.15; CRS/D4, 0.22 ± 0.13) (Fig. 4N). Given that two doses of D4 were administered before the beneficial effects in TST were observed, the earliest positive effects exerted by D4 were approximately two days. These results could consolidate the antidepressant-like effects of D4 and demonstrate its fast-acting potential using a different mouse model of depression.

D4 reduces CRS-mediated hemichannel activity and reactive glia

Chronic stress-induced changes in glial cell density and functions have been proposed to contribute to mood disorders, including depression [58]. Previous work reported that restraint stress increases glial activation and hemichannel activity in the hippocampus, which may be associated with behavioral deficits induced by chronic stress [39]. In the LPS-induced mouse model of depression, we found that D4 decreases hemichannel-mediated dye uptake (Fig. 1) and the number of reactive astrocytes in LPS-induced depressive-like mice (Fig. 3). To corroborate these results in the CRS-induced mouse model of depression, we measured hemichannel activity in the hippocampal DG from mice treated with either the vehicle or D4 by using CBF uptake. Consistent with results from the LPS model, treatments with D4 induced significant reductions in the number of GFAP+ astrocytes (Control/vehicle, 100.0 ± 23.0%; CRS/vehicle, 418.6 ± 62.0%; CRS/D4, 199.7 ± 48.8%), as well as a decrease in the CBF uptake in GFAP+ astrocytes (Control/vehicle, 100.0 ± 39.0%; CRS/vehicle, 444.5 ± 81.7%; CRS/D4, 267.6 ± 38.8) in the hippocampal DG (Fig. 5A–C). Compared to vehicle-treated unstressed control mice, CRS led to an increase in the DG Iba1+ microglia. CRS mice treated with D4 also showed a significant decrease in the number of Iba1+ microglia (Control/vehicle, 100.0 ± 39.0%; CRS/vehicle, 303.9 ± 37.6%; CRS/D4, 160.3 ± 16.5), and a tendency to reduce CBF uptake in the Iba1+ microglia (Control/vehicle, 100.0 ± 31.4%; CRS/vehicle, 417.2 ± 58.6%; CRS/D4, 347.8 ± 57.6) in the hippocampal DG (Fig. 5D–F). In the CRS-exposed mice, D4 also reduced CBF uptake (Control/vehicle, 100.0 ± 31.4%; CRS/vehicle, 917.6 ± 221.1%; CRS/D4, 341.4 ± 77.4) and dye uptake in Nissl+ neurons (Control/vehicle, 100.0 ± 12.1%; CRS/vehicle, 329.1 ± 51.1%; CRS/D4, 147.1 ± 43.1) in the hippocampal DG from mice subjected to CRS (Fig. 5G–I). Thus, these results suggest that D4 can suppress dye uptake through hemichannel and glial activation induced by CRS, indicating potential mechanisms that contribute to antidepressant-like behavioral outcomes of D4.

Fig. 5
figure 5

D4 decreases CRS-mediated hemichannel activity and glial activation. A Representative images showing astrocyte (GFAP) and CBF in the DG from the CRS-exposed mice. GFAP, red; CBF, green; DAPI, blue. Scale bar, 10 µm. B Quantification of GFAP+ astrocytes and (C) GFAP+CBF+ area in the stained DG sections. Control/vehicle, n = 9 DG sections from 3 mice; CRS/vehicle, n = 15 DG sections from 5 mice; CRS/D4, n = 13 DG sections from 5 mice. D Representative images showing microglia (Iba1) and CBF in the DG from the CRS-exposed mice. Iba1, red; CBF, green; DAPI, blue. Scale bar, 10 µm. E Quantification of Iba1+ microglia and F Iba1+CBF+ area in the stained DG sections. Control/vehicle, n = 8 DG sections from 4 mice; CRS/vehicle, n = 15 DG sections from 5 mice; CRS/D4, n = 15 DG sections from 5 mice. G Representative images showing CBF uptake in the hippocampal DG from the CRS-exposed mice treated with vehicle (Left) and D4 (Right). Nissl, magenta; CBF, green; DAPI, blue. Scale bar, 20 µm. H Quantification of CBF+ area and I Nissl+CBF+ area in the hippocampal DG sections. Control/vehicle, n = 8 DG sections from 3 mice; CRS/vehicle, n = 12 DG sections from 5 mice; CRS/D4, n = 13 DG sections from 5 mice. The open circle represents the value from each section. Control/vehicle (Ctrl), white; CRS/vehicle, red; CRS/D4, blue. Kruskal–Wallis test with Bonferroni correction (B, C, E, H, I). One-way ANOVA followed by LSD post hoc test (F). *p < 0.05, **p < 0.01, ***p < 0.001

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D4 restores neuronal activity in CRS-induced depressive-like mice

Alterations in neural activity and brain network connectivity are consistently associated with depression [59,60,61,62]. Glial cells are important for supporting the functions of neurons [63, 64]. In addition, astroglial Cx hemichannels have been shown to regulate behavior by modulating neuronal activity [65, 66]. Therefore, we asked whether the antidepressant-like effects of hemichannel blockade with D4 could be accompanied by changes in neural activity.

After CRS and the battery of four behavioral tests (OFT, TST, FST, and SPT), mice were subjected to one last TST, which acted as an acute stressor, and were sacrificed 1 h later to evaluate neuronal activation (Fig. 6A). Similar to previous results (Fig. 4), CRS increased depressive-like z scores in mice, and D4 treatment (5 mg/kg, p.o.) reduced mouse depressive-like z scores (Depressive-like z score, ctrl/vehicle, 0.00 ± 0.17; CRS/vehicle, 0.86 ± 0.24; CRS/D4, − 0.26 ± 0.21) (Fig. 6B). In the TST, D4 (5 mg/kg, p.o.) decreased immobility time, and significantly increased struggling time compared to CRS/vehicle group (TST immobility, ctrl/vehicle, 85.0 ± 23.3 s; CRS/vehicle, 161.1 ± 9.1 s; CRS/D4, 92.3 ± 36.5 s. TST struggling, ctrl/vehicle, 7.2 ± 3.1 s; CRS/vehicle, 1.1 ± 0.4 s; CRS/D4, 22.3 ± 9.0 s) (Fig. 6C, D). This suggests that D4 reduces depressive-like behaviors in the CRS-exposed mice.

Fig. 6
figure 6

D4 rescues hypofunction of depression-related brain regions in the CRS-induced depressive-like mice. A Schematic of the design of CRS, drug treatment, behavioral testing, and c-Fos immunostaining after tail suspension test (TST). B Depressive-like z score of mice used for the TST and c-Fos experiments. C Time immobile and struggling (D) in the TST. The filled dot shows the value of each mouse. Ctrl/vehicle, white; CRS/vehicle, red; CRS/D4, blue. n = 5 mice for all groups. Data are mean ± s.e.m. One-way ANOVA followed by LSD post hoc test (B). Kruskal–Wallis test with Bonferroni correction (C, D). E Immunostaining for c-Fos expression (red) in the depression-associated brain regions of ctrl/vehicle, CRS/vehicle, and CRS/D4 treated mice (from top to bottom). The nuclei were stained with DAPI (blue). Scale bar, 100 µm. dDG: dorsal dentate gyrus; vDG: ventral dentate gyrus; vCA1: ventral cornu ammonis 1; vSub: ventral subiculum; vEC: ventral entorhinal cortex; LS: lateral septum; Amy: amygdala; NAc: nucleus accumbens. F Violin plot of c-Fos+ cells in each region after the TST. Ctrl/vehicle, white; CRS/vehicle, red; CRS/D4, blue. The open circle indicates the value from each section. n = 15 sections from 5 mice for all groups. One-way ANOVA followed by LSD post hoc test (LS and NAc). Kruskal–Wallis test with Bonferroni correction (dDG, vDG, vCA1, vSub, EC, and Amy). *p < 0.05, **p < 0.01, ***p < 0.001

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Subsequently, we assessed neuronal activation using c-Fos immunostaining following acute exposure to stress (TST). In mice subjected to CRS, there were significantly fewer c-Fos+ cells in several depression-related brain regions, including the dorsal and ventral hippocampus (dDG, vDG, vCA1, vSub), ventral entorhinal cortex, lateral septum, and nucleus accumbens (NAc). CRS-exposed mice treated with D4 (5 mg/kg, p.o.) exhibited increases in the number of c-Fos+ cells in the hippocampus (dDG, vDG, vCA1), entorhinal cortex, and lateral septum. No significant change in the number of c-Fos+ cells was found in the amygdala (Fig. 6E, F). These data suggest that D4 may improve behavioral deficits in depressive-like mice by restoring neural activity in various brain regions.

Discussion

Hemichannels are important for regulating cellular communication and maintaining homeostasis [8]. Uncontrolled hemichannel activation has been proposed to disrupt neuron-glia communication, promote inflammation, and lead to cell damage in various brain diseases [7]. Recent evidence suggests that elevated hemichannel activity is associated with pathological changes in depression [67]. Therefore, inhibiting hemichannel activity may provide therapeutic benefits, as previously suggested [67,68,69].

Accordingly, in the present study, we demonstrated the rapid, antidepressant-like effects of D4, which reduced depressive-like symptoms in mice exposed to repeated systemic LPS challenges or CRS. We also showed that blocking hemichannels with D4 inhibits stress-mediated reactive astrogliosis and induces a brain-wide restoration of neural activity in the depressive-like mice subjected to CRS, which may contribute to the behavioral effects of D4. Together, our findings indicate that Cx hemichannels can constitute effective therapeutic targets for depression, and that D4 can be a new molecule mediating this therapeutic effect.

The pathogenesis of depression is highly heterogeneous and is associated with intricate interplays across multiple neurotransmitter systems, neuroimmune systems, neural circuity, and brain networks [70,71,72]. Considering that neuroinflammatory responses are likely to affect the ontogeny of the central nervous system [73], outcomes under these conditions could be irreversible, and could generate a radical difference compared to the effects of neuroinflammation that only occurs in adulthood. These differences limit current treatment outcomes and dramatically impede the rational design of successful therapies.

Cx hemichannels have been suggested as potential targets for depression [24, 67, 74]. Several agents, including small molecules, antibodies, and peptides, are available for inhibiting Cx hemichannels, with most studies carried out in vitro [18]. However, the behavioral benefits of hemichannel inhibition in depression in vivo remain elusive. In the present study, using two well-established mouse models of depression, we provide substantive evidence for the first time that D4 treatment can improve stress-induced depressive-like symptoms in adult mice. In addition, adult mice that received either acute or chronic applications of D4 did not show evident side effects. The latter could suggest that D4 is a relatively safe Cx hemichannel inhibitor with therapeutic effects. The antidepressant-like potential of D4 still needs to be substantiated for future translational research and clinical studies. Future work is also required to determine its bioavailability and druggability.

It has been shown that hemichannel activity can increase due to inflammation and chronic stress in adult animals [39, 67]. One of the prevailing hypotheses is that abnormal hemichannel activity may amplify detrimental effects on several cellular processes and promote the pathogenesis of a range of neurological and neurodegenerative diseases [10, 16, 43, 75, 76]. For example, in animal models of brain injury, such as ischemic stroke, hemichannels are activated in response to injury and pro-inflammatory cytokines. Dysregulated hemichannel activity can facilitate excitotoxicity due to the excessive release of gliotransmitters and may lead to neuronal death [77]. During epileptogenesis, seizures increase glial hemichannel activity [16, 78]. In turn, such heightened hemichannel activity promotes seizure generation and propagation through neuroinflammation and neuronal hyperexcitability [15, 79]. In animal studies, currently available hemichannel inhibitors have been used to control diseases like ischemia and epilepsy [12, 15]. Consistent with a previous study [41], we found that a single high-dose LPS injection increases glial hemichannel activity in the hippocampus. In addition, we found that repeated low doses of LPS challenges can increase Cx hemichannel activity in the hippocampal DG. Blocking hemichannels with D4 also prevented LPS-mediated reactive astrogliosis, probably due to reduced hemichannel activity in astrocytes, as evidenced by decreased CBF dye uptake. Thus, decreases in Cx hemichannel activity may contribute to antidepressant-like behavioral effects exerted by D4 in the LPS-induced depression model. These findings are consistent with the hypothesis that inhibition of Cx hemichannels by small molecules or other agents can improve depressive-like behaviors.

Our finding that blocking Cx hemichannels with D4 restored neuronal activity in CRS-exposed depressive-like mice provides another mechanistic insight into how hemichannel inhibition can contribute to behavioral improvement. Reduced neuronal activity in several regions of the limbic system has been identified in the etiology of depression [80]. Notably, post-mortem and neuroimaging studies have confirmed that the hippocampus is hypoactive in patients with depression [81, 82]. Antidepressant treatments can reverse depression-associated structural and functional changes in the hippocampus [83,84,85].

Similar to previous findings, we found that mice acutely exposed to CRS display significant reductions in neuronal activity in the hippocampus [86] and several other limbic regions, including the entorhinal cortex and the lateral septum. In contrast, D4 treatment reduces depressive-like behavior in the TST and enhances neural activation, as evidenced by the recovery of c-Fos+ cells in these regions. Although our dye uptake and glial density assays were restricted to the hippocampal DG regions, findings from our c-Fos screening results raise the possibility that D4 could impact the hemichannel and neural activity in multiple sites across the brain after systemic application via oral administration. Evidence for this possibility is supported by our recent study showing that oral treatment of D4 with optimized doses is sufficient to reduce seizure-induced glial cell density and hemichannel activity in different subfields of the hippocampus and anterior piriform cortex [16]. Data from clinical and preclinical studies suggest that the route of antidepressant administration may affect therapeutic efficacy and experience, especially for the novel rapid-acting antidepressant such as ketamine [6, 85]. Thus, specific mechanisms by which CRS reduces neuronal activation and D4 regulates neural activity remain to be studied with more sophisticated and precise targeted drug delivery strategies.

Cx43 is predominantly expressed in astrocytes in the adult mouse brain. Resting microglia rarely express Cx43, but its expression level can be increased in activated microglia [75, 87, 88]. Previously, it has been shown that D4 inhibits Cx43 hemichannels but not gap junction channels [29]. In this study, we showed that D4 reduces LPS-induced CBF uptake in astrocytes without affecting dye uptake in microglia. D4-induced reduction in astroglia-mediated hemichannel activity could indirectly reduce pannexin1 hemichannel activity in neurons, as observed under neuroinflammation triggered by a neurotoxic beta-amyloid peptide [89]. Based on existing studies suggesting that hemichannels can regulate gliotransmitter release, synaptic transmission, and neuronal firing [90,91,92,93,94], we speculate that the behavioral effect of D4 is likely mediated by the initial inhibition of astrocytic Cx43 hemichannel activity, which subsequently affects neuron–glia interaction and neuronal function. A limitation of our current study was the lack of detailed comparisons between sexes in their sensitivity to chronic stressors and compound D4. Sex could be a factor that affects the prognosis of depression and the treatment response of antidepressant interventions [2, 5]. It will be interesting to test whether D4 has a sex-specific effect on changes in hemichannel and behavioral activity induced by depressogenic factors. Given that Cx hemichannels can actively regulate neural activity and behavioral states via a cornucopia of cellular processes [64, 65], future studies are needed to elucidate the mechanisms of antidepressant-like behavioral effects of D4.

Conclusion

In conclusion, we have shown that a recently discovered hemichannel inhibitor, namely the small organic molecule D4, can exert antidepressant-like effects in mice subjected to repeated systemic LPS challenges or CRS. These behavioral benefits of D4 are accompanied by the blockade of hemichannel activity reduced inflammatory response, and a brain-wide restoration of neural activity in depressive-like mice. Our findings support the hypothesis that the hemichannel inhibitor is a promising pharmacophore for future antidepressant development focusing on glial cells. A better understanding of D4’s cellular mechanisms may help to identify its therapeutic potential and promote clinical uses of hemichannel inhibitors in mood disorders.