Astrocytic Gap Research Articles

Improvement of astrocytic gap junction involves the anti-depressive effect of celecoxib through inhibition of NF-κB

ContextCelecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been shown to exhibit anti-depressive effects in clinical trials. However, the direct mechanism underlying its effect on neuroinflammation remains unclear. Neuroinflammatory reaction from astrocytes leads to depression, and our previous study found that gap junction disorder between astrocytes aggravated neuroinflammatory reaction in depressed mice. ObjectiveTo investigate the potential mechanism of celecoxib's effects on astrocytic gap junctions during the central nervous inflammation-induced depression. Materials & methodsStereotaxic injection of lipopolysaccharide (LPS) into the prefrontal cortex (PFC) to establish a model of major depressive disorder (MDD). Celecoxib was administrated into PFC 15 min after LPS injection. The depressive performance was tested by tail suspension test and forced swimming test, and the levels of proinflammation cytokines were determined at mRNA and protein levels. Resting-state functional connection (rsFC) was employed to assess changes in the default mode network (DMN). Additionally, astrocytic gap junctions were also determined by lucifer yellow (LY) diffusion and transmission electron microscope (TEM), and the expression of connexin 43 (Cx43) was measured by western blotting, quantitative polymerase chain reaction, and immunofluorescence. ResultsLPS injection induced significant depressive performance, which was ameliorated by celecoxib treatment. Celecoxib also improved rsFC in the DMN. Furthermore, celecoxib improved astrocytic gap junctions as evidenced by increased LY diffusion, shortened gap junction width, and normalized levels of phosphorylated Cx43. Celecoxib also blocked the phosphorylation of p65, and inhibition of p65 abolished the improvement of Cx43. Discussion & conclusionAnti-depressive effects of celecoxib are mediated, at least in part, by the inhibition of nuclear factor- kappa B (NF-κB) and the subsequent improvement of astrocytic gap junction function

Ultrastructure of neuron-glia interaction in the norm and experimental pathology

Long-term experience was gained in analyzing synapses and their glial surroundings in normal, natural, and experimental models of functional plasticity and brain pathology development. However, most studies in this area rely on electrophysiological techniques combined with fluorescent imaging. Notably, fine synapse structure and astrocytic processes cannot be resolved using light or some electron microscopy techniques. Studies on experimental brain pathology using volume electron microscopy methods [1] were previously restricted due to their high labor intensity. However, automated methods for sample preparation and analysis, using machine vision and artificial intelligence, significantly simplified this task. Using transmission electron microscopy methods and 3D reconstructions, this study examined the Str. radiatum CA1 hippocampal region of rat brains in a chronic lithium-pilocarpine model of epilepsy. The results indicate a decrease in synaptic number along with an increase in their size and a reduction in astrocytic isolation of the active zones. A decrease in glial ensheathment of enlarged active zones and facilitation of neurotransmitter diffusion to active synapses may have a multiplicative effect on epileptiform activity growth and excitotoxicity. The simplification of the astrocytes meshwork in the somatosensory cortex’s layer 2/3 is comparable to that of layer 1. This decrease in layer 1’s inhibitory action enables pyramidal neurons in layer 2/3 to potentially exhibit epileptiform activity. Thus, the superficial cortical layers’ structural-functional aspects can be used as a natural cellular model in epilepsy development studies. Reduction of Ca2+ events in astrocyte processes in lithium-pilocarpine induced epilepsy may result from the low buffer capacity of Ca2+ ions in the smooth endoplasmic reticulum (sER) and/or impaired Ca2+ wave transmission through the gap junctions between astrocytic processes. High resolution is necessary to analyze the gap junctions, and special methods are required for sER visualization in perisynaptic astrocytic processes. We developed original sER staining methods [2] to quantitatively evaluate gap junctions and sER cisternae within astrocytic meshworks in layers 1 and 2/3 of the somatosensory cortex. In layer 1, the area of gap junctions in relation to the volume of an astrocyte was twice as high as in layer 2/3. The proportion of sER volume differed between layer 1 and layer 2/3 tissues. Specifically, the total sER cisternae volume in layer 2/3 was twice as high as in layer 1, relative to the volume of astrocytic processes [3]. Additionally, a doubling of single astrocytic gap junction area concomitant with decreased calcium events was observed. The results suggest a normal balance between Ca2+ stores (sER) and gap junctions, whose disruption may contribute to the development of seizures.

Higenamine exerts antidepressant effect by improving the astrocytic gap junctions and inflammatory response

BackgroundDepression is a refractory psychiatric disorder closely associated with dysfunction of the gap junctions (GJs) between astrocytes as well as neuroinflammation. Higenamine (Hig) is a potent cardiotonic ingredient in Fuzi (i.e., Aconitum carmichaeli Debx.) with anti-inflammatory and antioxidant effects, which has a significant protective effect on damaged nerve cells and has great potential for the treatment of neuropsychiatric diseases. MethodsRats were stimulated by chronic unpredictable stress (CUS) for 28 days while given Hig (5, 10, 20 mg/kg) and then analyzed behaviorally by the open field test, sucrose preference test, and forced swimming test. Changes in astrocyte GJs function and morphology were observed by dye transfer and transmission electron microscopy, respectively. Expression and phosphorylation of connexin 43 (Cx43) were analyzed by Western blot. Also, considering the close relationship between depression and neuroinflammation, we determined the inflammatory response in serum with ELISA kits and analyzed the expression of inflammation-related proteins with Western blot. ResultsHig ameliorated CUS-induced depression-like behavior in rats. Hig administration improved gap junctional dysfunction in astrocytes, reduced gap junctional gaps and elevated the expression of Cx43 and decreased the phosphorylation of Cx43. Meanwhile, Hig administration was also able to attenuate the inflammatory response that occurs after CUS in rats. LimitationsFor the role of Cx43 in depression, we did not validate it more deeply in animal models with knockout Cx43. In addition, GJs dysfunction might be associated with the inflammatory response seen in depression, but this needs to be further investigated. ConclusionsHig ameliorates depression and exerts its antidepressant effect possibly by improving the dysfunctional GJs between astrocytes and the inflammatory response.

Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

Ca2+-dependent rapid uncoupling of astrocytes upon brief metabolic stress.

Astrocytic gap junctional coupling is a major element in neuron-glia interaction. There is strong evidence that impaired coupling is involved in neurological disorders. Reduced coupling was, e.g., demonstrated for core regions of ischemic stroke that suffer from massive cell death. In the surrounding penumbra, cells may recover, but recovery is hampered by spreading depolarizations, which impose additional metabolic stress onto the tissue. Spreading depolarizations are characterized by transient breakdown of cellular ion homeostasis, including pH and Ca2+, which might directly affect gap junctional coupling. Here, we exposed acute mouse neocortical tissue slices to brief metabolic stress and examined its effects on the coupling strength between astrocytes. Changes in gap junctional coupling were assessed by recordings of the syncytial isopotentiality. Moreover, quantitative ion imaging was performed in astrocytes to analyze the mechanisms triggering the observed changes. Our experiments show that a 2-minute perfusion of tissue slices with blockers of glycolysis and oxidative phosphorylation causes a rapid uncoupling in half of the recorded cells. They further indicate that uncoupling is not mediated by the accompanying (moderate) intracellular acidification. Dampening large astrocytic Ca2+ loads by removal of extracellular Ca2+ or blocking Ca2+ influx pathways as well as a pharmacological inhibition of calmodulin, however, prevent the uncoupling. Taken together, we conclude that astrocytes exposed to brief episodes of metabolic stress can undergo a rapid, Ca2+/calmodulin-dependent uncoupling. Such uncoupling may help to confine and reduce cellular damage in the ischemic penumbra in vivo.

Astrocytes as a target for therapeutic strategies in epilepsy: current insights.

Astrocytes are specialized non-neuronal glial cells of the central nervous system, contributing to neuronal excitability and synaptic transmission (gliotransmission). Astrocytes play a key roles in epileptogenesis and seizure generation. Epilepsy, as a chronic disorder characterized by neuronal hyperexcitation and hypersynchronization, is accompanied by substantial disturbances of glial cells and impairment of astrocytic functions and neuronal signaling. Anti-seizure drugs that provide symptomatic control of seizures primarily target neural activity. In epileptic patients with inadequate control of seizures with available anti-seizure drugs, novel therapeutic candidates are needed. These candidates should treat epilepsy with anti-epileptogenic and disease-modifying effects. Evidence from human and animal studies shows that astrocytes have value for developing new anti-seizure and anti-epileptogenic drugs. In this review, we present the key functions of astrocytes contributing to neuronal hyperexcitability and synaptic activity following an etiology-based approach. We analyze the role of astrocytes in both development (epileptogenesis) and generation of seizures (ictogenesis). Several promising new strategies that attempted to modify astroglial functions for treating epilepsy are being developed: (1) selective targeting of glia-related molecular mechanisms of glutamate transport; (2) modulation of tonic GABA release from astrocytes; (3) gliotransmission; (4) targeting the astrocytic Kir4.1-BDNF system; (5) astrocytic Na+/K+/ATPase activity; (6) targeting DNA hypo- or hypermethylation of candidate genes in astrocytes; (7) targeting astrocytic gap junction regulators; (8) targeting astrocytic adenosine kinase (the major adenosine-metabolizing enzyme); and (9) targeting microglia-astrocyte communication and inflammatory pathways. Novel disease-modifying therapeutic strategies have now been developed, such as astroglia-targeted gene therapy with a broad spectrum of genetic constructs to target astroglial cells.

Role of Impaired Astrocyte Gap Junction Coupling in Epileptogenesis.

The gap-junction-coupled astroglial network plays a central role in the regulation of neuronal activity and synchronisation, but its involvement in the pathogenesis of neuronal diseases is not yet understood. Here, we present the current state of knowledge about the impact of impaired glial coupling in the development and progression of epilepsy and discuss whether astrocytes represent alternative therapeutic targets. We focus mainly on temporal lobe epilepsy (TLE), which is the most common form of epilepsy in adults and is characterised by high therapy resistance. Functional data from TLE patients and corresponding experimental models point to a complete loss of astrocytic coupling, but preservation of the gap junction forming proteins connexin43 and connexin30 in hippocampal sclerosis. Several studies further indicate that astrocyte uncoupling is a causal event in the initiation of TLE, as it occurs very early in epileptogenesis, clearly preceding dysfunctional changes in neurons. However, more research is needed to fully understand the role of gap junction channels in epilepsy and to develop safe and effective therapeutic strategies targeting astrocytes.

Retraction Note: Comparing astrocytic gap junction of genetic absence epileptic rats with control rats: an experimental study.

Retraction Note: Comparing astrocytic gap junction of genetic absence epileptic rats with control rats: an experimental study.

Ocular stress enhances contralateral transfer of lenadogene nolparvovec gene therapy through astrocyte networks

Lenadogene nolparvovec (GS010) was developed to treat a point mutation in mitochondrial ND4 that causes Leber hereditary optic neuropathy. GS010 delivers human cDNA encoding wild-type ND4 packaged into an rAAV2/2 vector that transduces retinal ganglion cells, to induce allotopic expression of hybrid mitochondrial ND4. GS010 clinical trials improved best-corrected visual acuity (BCVA) up to 5 years after treatment. Interestingly, unilateral treatment improved BCVA bilaterally. Subsequent studies revealed GS010 DNA in visual tissues contralateral to the injected eye, suggesting migration. Here we tested whether unilateral intraocular pressure (IOP) elevation could influence the transfer of viral ND4 RNA in contralateral tissues after GS010 delivery to the IOP-elevated eye and probed a potential mechanism mediating translocation in mice. We found IOP elevation enhanced viral ND4 RNA transcripts in contralateral visual tissues, including retinas. Using conditional transgenic mice, we depleted astrocytic gap junction connexin 43 (Cx43), required for distant redistribution of metabolic resources between astrocytes during stress. After unilateral IOP elevation and GS010 injection, Cx43 knockdown eradicated ND4 RNA transcript detection in contralateral retinal tissues, while transcript was still detectable in optic nerves. Overall, our study indicates long-range migration of GS010 product to contralateral visual tissues is enhanced by Cx43-linked astrocyte networks.

Downregulation of the Astroglial Connexin Expression and Neurodegeneration after Pilocarpine-Induced Status Epilepticus

Astrocytic networks and gap junctional communication mediated by connexins (Cxs) have been repeatedly implicated in seizures, epileptogenesis, and epilepsy. However, the effect of seizures on Cx expression is controversial. The present study focused on the response of Cxs to status epilepticus (SE), which is in turn an epileptogenic insult. The expression of neuronal Cx36 and astrocytic Cx30 and Cx43 mRNAs was investigated in the brain of rats in the first day after pilocarpine-induced SE. In situ hybridization revealed a progressive decrease in Cx43 and Cx30 mRNA levels, significantly marked 24 h after SE onset in neocortical areas and the hippocampus, and in most thalamic domains, whereas Cx36 mRNA did not exhibit obvious changes. Regional evaluation with quantitative real-time-RT-PCR confirmed Cx43 and Cx30 mRNA downregulation 24 h after SE, when ongoing neuronal cell death was found in the same brain regions. Immunolabeling showed at the same time point marked a decrease in Cx43, microglia activation, and interleukin-1β induction in some microglial cells. The data showed a transient downregulation of astroglial Cxs in the cortical and thalamic areas in which SE triggers neurodegenerative events in concomitance with microglia activation and cytokine expression. This could potentially represent a protective response of neuroglial networks to SE-induced acute damage.

Unravelling Contributions of Astrocytic Connexin 43 to the Functional Activity of Brain Neuron-Glial Networks under Hypoxic State In Vitro.

Brain hypoxia remains an Achilles’ heel for public health that must be urgently addressed. Hypoxic damage affects both neurons and glial cells, particularly astrocytes, which are in close dynamic bi-directional communication, and are organized in plastic and tightly regulated networks. However, astroglial networks have received limited attention regarding their influence on the adaptive functional rearrangements of neural networks to oxygen deficiency. Herein, against the background of astrocytic Cx43 gap junction blockade by the selective blocker Gap19, we evaluated the features of spontaneous calcium activity and network characteristics of cells in primary cultures of the cerebral cortex, as well as the expression levels of metabotropic glutamate receptors 2 (mGluR2) and 5 (mGluR5) in the early and late periods after simulated hypoxia in vitro. We showed that, under normoxic conditions, blockade of Cx43 leads to an increase in the expression of metabotropic glutamate receptors mGluR2 and mGluR5 and long-term modulation of spontaneous calcium activity in primary cortical cultures, primarily expressed in the restructuring of the functional architectonics of neuron–glial networks through reducing the level of correlation between cells in the network and the percentage of existing correlated connections between cells. Blocking Cx43 during hypoxic injury has a pronounced neuroprotective effect. Together with the increased expression of mGluR5 receptors, a decrease in mGluR2 expression to the physiological level was found, which suggests the triggering of alternative molecular mechanisms of cell adaptation to hypoxia. Importantly, the blockade of Cx43 in hypoxic damage contributed to the maintenance of both the main parameters of the spontaneous calcium activity of primary cortical cultures and the functional architectonics of neuron–glial networks while maintaining the profile of calcium oscillations and calcium signal communications between cells at a highly correlated level. Our results demonstrate the crucial importance of astrocytic networks in functional brain adaptation to hypoxic damage and could be a promising target for the development of rational anti-hypoxic therapy.

Enhancement of astrocytic gap junctions Connexin43 coupling can improve long-term isoflurane anesthesia-mediated brain network abnormalities and cognitive impairment.

Astrocytes are connected by gap junctions Connexin43 (GJs-Cx43) forming an extensive intercellular network and maintain brain homeostasis. Perioperative neurocognitive disorder (PND) occurs frequently after anesthesia/surgery and worsens patient outcome, but the neural circuit mechanisms remain unclear. This study aimed to determine the effects of the GJs-Cx43-mediated astrocytic network on PND and ascertain the underlying neural circuit mechanism. Male C57BL/6 mice were treated with long-term isoflurane exposure to construct a mouse model of PND. We also exposed primary mouse astrocytes to long-term isoflurane exposure to simulate the conditions of in vivo cognitive dysfunction. Behavioral tests were performed using the Y-maze and fear conditioning (FC) tests. Manganese-enhanced magnetic resonance imaging (MEMRI) and resting-state functional magnetic resonance imaging (rs-fMRI) were used to investigate brain activity and functional connectivity. Western blot and flow cytometry analysis were used to assess protein expression. Reconfiguring the astrocytic network by increasing GJs-Cx43 expression can modulate 22 subregions affected by PND in three ways: reversed activation, reversed inhibition, and intensified activation. The brain functional connectivity analysis further suggests that PND is a brain network disorder that includes sleep-wake rhythm-related brain regions, contextual and fear memory-related subregions, the hippocampal-amygdala circuit, the septo-hippocampal circuit, and the entorhinal-hippocampal circuit. Notably, remodeling the astrocytic network by upregulation of GJs-Cx43 can partially reverse the abnormalities in the above circuits. Pathophysiological degeneration in hippocampus is one of the primary hallmarks of PND pathology, and long-term isoflurane anesthesia contributes to oxidative stress and neuroinflammation in the hippocampus. However, promoting the formation of GJs-Cx43 ameliorated cognitive dysfunction induced by long-term isoflurane anesthesia through the attenuation of oxidative stress in hippocampus. Enhancing GJs-Cx43 coupling can improve brain network abnormalities and cognitive impairment induced by long-term isoflurane anesthesia, its mechanisms might be associated with the regulation of oxidative stress and neuroinflammation.

Connexin 43 gap junction-mediated astrocytic network reconstruction attenuates isoflurane-induced cognitive dysfunction in mice

BackgroundPostoperative cognitive dysfunction (POCD) is a common complication following anesthesia and surgery. General anesthetic isoflurane has potential neurotoxicity and induces cognitive impairments, but the exact mechanism remains unclear. Astrocytes form interconnected networks in the adult brain through gap junctions (GJs), which primarily comprise connexin 43 (Cx43), and play important roles in brain homeostasis and functions such as memory. However, the role of the GJ-Cx43-mediated astrocytic network in isoflurane-induced cognitive dysfunction has not been defined.Methods4-month-old male C57BL/6 mice were exposure to long-term isoflurane to induce cognitive impairment. To simulate an in vitro isoflurane-induced cognitive dysfunction‐like condition, primary mouse astrocytes were subjected to long-term isoflurane exposure. Cognitive function was assessed by Y-maze and fear conditioning tests. Western blot was used to determine the expression levels of different functional configurations of Cx43. The morphology of the GJs-Cx43 was evaluated by immunofluorescence staining. Levels of IL-1β and IL-6 were examined by ELISA. The ability of GJs-Cx43-mediated intercellular communication was examined by lucifer yellow dye transfer assay. Ethidium bromide uptake assays were used to measure the activity of Cx43 hemichannels. The ultrastructural morphology of astrocyte gap junctions and tripartite synapse were observed by transmission electron microscopy.ResultsAfter long-term isoflurane anesthesia, the GJs formed by Cx43 in the mouse hippocampus and primary mouse astrocytes were significantly reduced, GJs function was impaired, hemichannel activity was enhanced, the levels of IL-1β and IL-6 were increased, and mice showed significant cognitive impairment. After treatment with the novel GJ-Cx43 enhancer ZP1609, GJ-Cx43-mediated astrocytic network function was enhanced, neuroinflammation was alleviated, and ameliorated cognition dysfunction induced by long-term isoflurane exposure. However, ZP1609 enhances the astrocytic network by promoting Cx43 to form GJs without affecting hemichannel activity. Additionally, our data showed that long-term isoflurane exposure does not alter the structure of tripartite synapse.ConclusionOur results reveal a novel mechanism of the GJ-Cx43-mediated astrocytic network involved in isoflurane-induced neuroinflammation and cognitive impairments, which provides new mechanistic insight into the pathogenesis of POCD and identifies potential targets for its treatment.

Decoupling astrocytes in adult mice impairs synaptic plasticity and spatial learning.

The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks by gap junction coupling, mainly composed of two gap junction channel proteins, connexin 30 (Cx30) and connexin 43 (Cx43). To circumvent developmental perturbations and to test whether astrocytic gap junction coupling is required for hippocampal neural circuit function and behavior, we generate and study inducible, astrocyte-specific Cx30 and Cx43 double knockouts. Surprisingly, disrupting astrocytic coupling in adult mice results in broad activation of astrocytes and microglia, without obvious signs of pathology. We show that hippocampal CA1 neuron excitability, excitatory synaptic transmission, and long-term potentiation are significantly affected. Moreover, behavioral inspection reveals deficits in sensorimotor performance and a complete lack of spatial learning and memory. Together, our findings establish that astrocytic connexins and an intact astroglial network in the adult brain are vital for neural homeostasis, plasticity, and spatial cognition.

Does astrocyte gap junction protein expression differ during development in absence epileptic rats?

Intercellular communication via gap junctions (GJs) has a wide variety of complex and essential functions in the CNS. In the present developmental study, we aimed to quantify the number of astrocytic GJs protein connexin 30 (Cx30) of genetic model of absence epilepsy rats from Strasbourg (GAERS) at postnatal P10, P30, and P60 days in the epileptic focal areas involved in the cortico-thalamic circuit. We compared the results with Wistar rats using immunohistochemistry and western blotting. The number of Cx30 immunopositive astrocytes per unit area were quantified for the somatosensory cortex (SSCx), ventrobasal (VB), and lateral geniculate (LGN) thalamic nuclei of the two strains and Cx30 western blot was applied to the tissue samples from the same regions. Both immunohistochemical and western blot results revealed the presence of Cx30 in all regions studied at P10 in both Wistar and GAERS animals. The SSCx, VB, and LGN of Wistar animals showed progressive increase in the number of Cx30 immunopositive labeled astrocytes from P10 to P30 and reached a peak at P30; then a significant decline was observed from P30 to P60 for the SSCx and VB. However, in GAERS Cx30 immunopositive labeled astrocytes showed a progressive increase from P10 to P60 for all brain regions studied. The immunohistochemical data highly corresponded with western blotting results. We conclude that the developmental disproportional expression of Cx30 in the epileptic focal areas in GAERS may be related to the onset of absence seizures or may be related to the neurogenesis of absence epilepsy.

Astrocytic Gap Junctions Contribute to Aberrant Neuronal Synchronization in a Mouse Model of MeCP2 Duplication Syndrome.

Abnormal synchronous neuronal activity has been widely detected by brain imaging of autistic patients, but its underlying neural mechanism remains unclear. Compared with wild-type mice, our in vivo two-photon imaging showed that transgenic (Tg1) mice over-expressing human autism risk gene MeCP2 exhibited higher neuronal synchrony in the young but lower synchrony in the adult stage. Whole-cell recording of neuronal pairs in brain slices revealed that higher neuronal synchrony in young postnatal Tg1 mice was attributed mainly to more prevalent giant slow inward currents (SICs). Both in vivo and slice imaging further demonstrated more dynamic activity and higher synchrony in astrocytes from young Tg1 mice. Blocking astrocytic gap junctions markedly decreased the generation of SICs and overall cell synchrony in the Tg1 brain. Furthermore, the expression level of Cx43 protein and the coupling efficiency of astrocyte gap junctions remained unchanged in Tg1 mice. Thus, astrocytic gap junctions facilitate but do not act as a direct trigger for the abnormal neuronal synchrony in young Tg1 mice, revealing the potential role of the astrocyte network in the pathogenesis of MeCP2 duplication syndrome.

Involvement of Hippocampal Astrocytic Connexin-43 in Morphine dependence

Repeated exposure to drugs of abuse can lead to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters. Such alterations in neurotransmission modify synaptic plasticity which causes addictive-like behaviors. Our previous study shed light on the involvement of glial cells in morphine-induced behavioral responses. It has been shown that glial cells play an indispensable role in synaptic transmission through the release of gliotransmitter into and uptake of neurotransmitters from the synaptic cleft. Connexin-43 (Cx43), the dominant Cx protein in astrocytes, is the main component of astrocytic gap junctions and hemichannels. It has a critical role in synaptic efficacy through setting the amount of presynaptic gliotransmitter release in physiological conditions. It is probable that addictive substances affecting gliotransmitters release through the alteration of Cx43 function. In this study, we examined the role of the hippocampal-specific astrocytic connexin (Cx43) in morphine-induced behavioral responses. Male rats received subcutaneous (s.c.) morphine sulfate (10 mg/kg) at an interval of 12 h for 9 days. The animals received microinjection of TAT-Gap19 (inhibitor of Cx43) into the CA1 region before each morning morphine administration. The animals were assessed for morphine dependence by monitoring naloxone hydrochloride precipitated withdrawal somatic signs. Results showed that animals receiving TAT-Gap19 before morphine injection demonstrated a significant reduction in several signs of morphine withdrawal such as Activity, Freezing, Chewing, Ptosis, Defecation, Teeth chattering, Writhing, Penis- licking, Head tremor, Scratching, Sniffing, Rearing, and Diarrhea (One way ANOVA, P < 0.001; P < 0.01; P < 0.05). Our findings suggest that hippocampal Cx43 may be involved in morphine-induced behavioral responses. Therefore, gliotransmitter release by astrocytes seems to be a mechanism which is engaged in addictive-like behaviors.

Critical Role of Astrocytic Polyamine and GABA Metabolism in Epileptogenesis.

Accumulating evidence indicate that astrocytes are essential players of the excitatory and inhibitory signaling during normal and epileptiform activity via uptake and release of gliotransmitters, ions, and other substances. Polyamines can be regarded as gliotransmitters since they are almost exclusively stored in astrocytes and can be released by various mechanisms. The polyamine putrescine (PUT) is utilized to synthesize GABA, which can also be released from astrocytes and provide tonic inhibition on neurons. The polyamine spermine (SPM), synthesized form PUT through spermidine (SPD), is known to unblock astrocytic Cx43 gap junction channels and therefore facilitate astrocytic synchronization. In addition, SPM released from astrocytes may also modulate neuronal NMDA, AMPA, and kainate receptors. As a consequence, astrocytic polyamines possess the capability to significantly modulate epileptiform activity. In this study, we investigated different steps in polyamine metabolism and coupled GABA release to assess their potential to control seizure generation and maintenance in two different epilepsy models: the low-[Mg2+] model of temporal lobe epilepsy in vitro and in the WAG/Rij rat model of absence epilepsy in vivo. We show that SPM is a gliotransmitter that is released from astrocytes and significantly contributes to network excitation. Importantly, we found that inhibition of SPD synthesis completely prevented seizure generation in WAG/Rij rats. We hypothesize that this antiepileptic effect is attributed to the subsequent enhancement of PUT to GABA conversion in astrocytes, leading to GABA release through GAT-2/3 transporters. This interpretation is supported by the observation that antiepileptic potential of the Food and Drug Administration (FDA)-approved drug levetiracetam can be diminished by specifically blocking astrocytic GAT-2/3 with SNAP-5114, suggesting that levetiracetam exerts its effect by increasing surface expression of GAT-2/3. Our findings conclusively suggest that the major pathway through which astrocytic polyamines contribute to epileptiform activity is the production of GABA. Modulation of astrocytic polyamine levels, therefore, may serve for a more effective antiepileptic drug development in the future.

Amyloid-β plaques affect astrocyte Kir4.1 protein expression but not function in the dentate gyrus of APP/PS1 mice.

Alzheimer pathology is accompanied by astrogliosis. Reactive astrocytes surrounding amyloid plaques may directly affect neuronal communication, and one of the mechanisms by which astrocytes impact neuronal function is by affecting K+ homeostasis. Here we studied, using hippocampal slices from 9‐month‐old Alzheimer mice (APP/PS1) and wild‐type littermates, whether astrocyte function is changed by analyzing Kir4.1 expression and function and astrocyte coupling in astrocytes surrounding amyloid‐β plaques. Immunohistochemical analysis of Kir4.1 protein in the dentate gyrus revealed localized increases in astrocytes surrounding amyloid‐β plaque deposits. We subsequently focused on changes in astrocyte function by using patch‐clamp slice electrophysiology on both plaque‐ and non‐plaque associated astrocytes to characterize general membrane properties. We found that Ba2+‐sensitive Kir4.1 conductance in astrocytes surrounding plaques was not affected by changes in Kir4.1 protein expression. Additional analysis of astrocyte gap junction coupling efficiency in the dentate gyrus revealed no apparent changes. Quantification of basic features of glutamatergic transmission to granule cells did not indicate disturbed neuronal communication in the dentate gyrus of APP/PS1 mice. Together, these results suggest that astrocytes in the dentate gyrus of APP/PS1 mice maintain their ability to buffer extracellular K+ and attempt to rectify imbalances in K+ concentration to maintain normal neuronal and synaptic function, possibly by localized increases in Kir4.1 protein expression. Our earlier transcriptomic data indicated that chronically activated astrocytes lose their neuronal support function. Here we show that, despite localized increased Kir4.1 protein expression, astrocyte Kir4.1 channel dysfunction is likely not involved in the pathogenesis of Alzheimer's disease.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

Simple SummaryGap junctions are intercellular channels that provide the means for direct transport of small molecules, ions, and water between connected cells. With these functions, gap junctions are essential for the maintenance of astrocytic homeostasis and of particular importance in the context of pathophysiological disbalances. These include the hyperosmolar hyperglycemic syndrome or the pathology after brain trauma. We demonstrate that short-term hyperosmolarity reduces intercellular communication via gap junctions. These functional changes coincide with the transformation of gap junction ultrastructure as evidenced by freeze-fracture replica immunolabeling and transmission electron microscopy. The hyperosmolarity-induced immediate changes in the ultrastructural assembly of connexons, the protein constituents of gap junction channels, have not been described in astrocytes before and are revealing the coherence of structure and function in gap junctions. Phosphorylation of Connexin 43, the main gap junction protein in astrocytes, at amino acid 368 (Serine) might link the two.In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.