Astrocytic Signaling Research Articles

Astrocytes mediate migraine-related intracranial meningeal mechanical hypersensitivity.

The genesis of the headache phase in migraine with aura is thought to be mediated by cortical spreading depression (CSD) and the subsequent activation and sensitization of primary afferent neurons that innervate the intracranial meninges and their related large vessels. Yet, the exact mechanisms underlying this peripheral meningeal nociceptive response remain poorly understood. We investigated the relative contribution of cortical astrocytes to CSD-evoked meningeal nociception using extracellular single-unit recording of meningeal afferent activity and 2-photon imaging of cortical astrocyte calcium activity, in combination with 2 pharmacological approaches to inhibit astrocytic function. We found that fluoroacetate and l-α-aminoadipate, which inhibit astrocytes through distinct mechanisms, suppressed CSD-evoked afferent mechanical sensitization, but did not affect afferent activation. Pharmacological inhibition of astrocytic function, which ameliorated meningeal afferents' sensitization, reduced basal astrocyte calcium activity but had a minimal effect on the astrocytic calcium wave during CSD. We propose that calcium-independent signaling in cortical astrocytes plays an important role in driving the sensitization of meningeal afferents and the ensuing intracranial mechanical hypersensitivity in migraine with aura.

Dopamine controls neuronal spontaneous calcium oscillations via astrocytic signal

Dopamine is a neuromodulator and neurotransmitter responsible for a number of physiological processes. Dysfunctions of the dopamine metabolism and signalling are associated with neurological and psychiatric diseases. Here we report that in primary co-culture of neurons and astrocytes dopamine-induces calcium signal in astrocytes and suppress spontaneous synchronous calcium oscillations (SSCO) in neurons. Effect of dopamine on SSCO in neurons was dependent on calcium signal in astrocytes and could be modified by inhibition of dopamine-induced calcium signal or by stimulation of astrocytic calcium rise with ATP. Ability of dopamine to suppress SSCO in neurons was independent on D1- or D2- like receptors but dependent on GABA and alpha-adrenoreceptors. Inhibitor of monoaminoxidase bifemelane blocked effect of dopamine on astrocytes but also inhibited the effect dopamine on SSCO in neurons. These findings suggest that dopamine-induced calcium signal may stimulate release of neuromodulators such as GABA and adrenaline and thus suppress spontaneous calcium oscillations in neurons.

Larger Connection Radius Increases Hub Astrocyte Number in a 3-D Neuron–Astrocyte Network Model

Astrocytes - a prominent glial cell type in the brain - form networks that tightly interact with the brain's neuronal circuits. Thus, it is essential to study the modes of such interaction if we aim to understand how neural circuits process information. Thereby, calcium elevations, the primary signal in astrocytes, propagate to the adjacent neighboring cells and directly regulate neuronal communication. It is mostly unknown how the astrocyte network topology influences neuronal activity. Here, we used a computational model to simulate planar and 3D neuron-astrocyte networks with varying topologies. We investigated the number of active nodes, the shortest path, and the mean degree. Furthermore, we applied a graph coloring analysis that highlights the network organization between different network structures. With the increase of the maximum distance between two connected astrocytes, the information flow is more centralized to the most connected cells. Our results suggest that activity-dependent plasticity and the topology of brain areas might alter the amount of astrocyte controlled synapses.

Sonic Hedgehog receptor Patched deficiency in astrocytes enhances glucose metabolism in mice

ObjectiveAstrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. MethodsWe analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc−/− (YFP-Ptc−/−) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. ResultsShh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc−/− mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc−/− mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. ConclusionsHere, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.

Design of an Imaging Probe to Monitor Real-Time Redistribution of L-type Voltage-Gated Calcium Channels in Astrocytic Glutamate Signaling.

In the brain, astrocytes are non-excitable cells that undergo rapid morphological changes when stimulated by the excitatory neurotransmitter glutamate. We developed a chemical probe to monitor how glutamate affects the density and distribution of astrocytic L-type voltage-gated calcium channels (LTCC). The imaging probe FluoBar1 was created from a barbiturate ligand modified with a fluorescent coumarin moiety. The probe selectivity was examined with colocalization analyses of confocal fluorescence imaging in U118-MG and transfected COS-7 cells. Living cells treated with 50nM FluoBar1 were imaged in real time to reveal changes in density and distribution of astrocytic LTCCs upon exposure to glutamate. FluoBar1 was synthesized in ten steps. The selectivity of the probe was demonstrated with immunoblotting and confocal imaging of immunostained cells expressing the CaV1.2 isoform of LTCCs proteins. Applying FluoBar1 to astrocyte model cells U118-MG allowed us to measure a fivefold increase in fluorescence density of LTCCs upon glutamate exposure. Imaging probe FluoBar1 allows the real-time monitoring of LTCCs in living cells, revealing for first time that glutamate causes a rapid increase of LTCC membranar density in astrocyte model cells. FluoBar1 may help tackle previously intractable questions about LTCC dynamics in cellular events.

Engineering calcium signaling of astrocytes for neural\u2013molecular computing logic gates

This paper proposes the use of astrocytes to realize Boolean logic gates, through manipulation of the threshold of hbox {Ca}^{2+} ion flows between the cells based on the input signals. Through wet-lab experiments that engineer the astrocytes cells with pcDNA3.1-hGPR17 genes as well as chemical compounds, we show that both AND and OR gates can be implemented by controlling hbox {Ca}^{2+} signals that flow through the population. A reinforced learning platform is also presented in the paper to optimize the hbox {Ca}^{2+} activated level and time slot of input signals T_b into the gate. This design platform caters for any size and connectivity of the cell population, by taking into consideration the delay and noise produced from the signalling between the cells. To validate the effectiveness of the reinforced learning platform, a hbox {Ca}^{2+} signalling simulator was used to simulate the signalling between the astrocyte cells. The results from the simulation show that an optimum value for both the hbox {Ca}^{2+} activated level and time slot of input signals T_b is required to achieve up to 90% accuracy for both the AND and OR gates. Our method can be used as the basis for future Neural–Molecular Computing chips, constructed from engineered astrocyte cells, which can form the basis for a new generation of brain implants.

Calcium signals in astrocytes of the fly brain promote sleep

Astrocytes are morphologically and functionally linked to neuronal synapses, and can regulate the activity of neural circuits, brain function, and behavior. However, the molecular mechanisms by which astrocytes regulate fundamental biological processes such as sleep are not completely understood. Wu and colleagues show that an increase in calcium signals within the processes of astrocytes of the fruit fly brain can promote sleep by upregulating the expression of a monoamine receptor, TyrRII, which in turn activates sleep promoting neurons via the astrocytic release of an interleukin-1 analog, spatzle. This study provides compelling evidence for a novel molecular mechanism by which increases in astrocytic calcium signals can induce sleep by activating sleep promoting neurons in the fly brain.

Alexander disease: diversity of cell population and interactions between neuron and glia

Alexander disease (AxD) is a rare neurodegenerative disorder caused by the mutations in glial fibrillary acidic protein (GFAP) gene. Rosenthal fiber formations in astrocytes are the pathological hallmarks of AxD. Astrocyte dysfunction in the AxD brain is considered to be involved in its pathogenesis. We have previously reported that in AxD model mice aberrant Ca2+ signals in astrocytes were associated with the upregulation of reactive phenotype. Reactive astrocytes are conditions that lead to morphological, functional, and molecular changes by responding to various pathological insults (trauma, inflammation, ischemia), and environmental stimuli. Recent technological advances in single-cell gene expression analysis have revealed that astrocytes have heterogeneity by indicating that they form sub population with different characteristics depending on the brain region, the growth development, aging stage, and the pathological condition. AxD astrocytes are also thought to constitute a heterogeneous population with diverse properties and functions. Moreover, it is presumed that AxD pathogenesis occur due to interactions with neurons and other glial cells, as well as the microenvironment in tissues. Research strategies based on these perspectives will help us understand AxD pathology better and may lead to the elucidation of disease modifiers and clinical diversity.

Sleep: Astrocytes Take Their Toll on Tired Flies

Calcium signalling in astrocytes modulates sleep, yet how astrocytes communicate with neural circuits that control sleep is unclear. A new study now uncovers a calcium-dependent relay between astrocytes and neurons that promotes sleep homeostasis in fruit flies.

Glycogen Phosphorylase Isoform Regulation of Ventromedial Hypothalamic Nucleus Gluco-Regulatory Neuron 5'-AMP-Activated Protein Kinase and Transmitter Marker Protein Expression.

Brain glycogen is remodeled during metabolic homeostasis and provides oxidizable L-lactate equivalents. Brain glycogen phosphorylase (GP)-brain (GPbb; AMP-sensitive) and -muscle (GPmm; norepinephrine-sensitive) type isoforms facilitate stimulus-specific control of glycogen disassembly. Here, a whole animal model involving stereotactic-targeted delivery of GPmm or GPbb siRNA to the ventromedial hypothalamic nucleus (VMN) was used to investigate the premise that these variants impose differential control of gluco-regulatory transmission. Intra-VMN GPmm or GPbb siRNA administration inhibited glutamate decarboxylate65/67 (GAD), a protein marker for the gluco-inhibitory transmitter γ--aminobutyric acid (GABA), in the caudal VMN. GPbb knockdown, respectively overturned or exacerbated hypoglycemia-associated GAD suppression in rostral and caudal VMN. GPmm siRNA caused a segment-specific reversal of hypoglycemic augmentation of the gluco-stimulatory transmitter indicator, neuronal nitric oxide synthase (nNOS). In both cell types, GP siRNA down-regulated 5′-AMP-activated protein kinase (AMPK) during euglycemia, but hypoglycemic suppression of AMPK was reversed by GPmm targeting. GP knockdown elevated baseline GABA neuron phosphoAMPK (pAMKP) content, and amplified hypoglycemic augmentation of pAMPK expression in each neuron type. GPbb knockdown increased corticosterone secretion in eu- and hypoglycemic rats. Outcomes validate efficacy of GP siRNA delivery for manipulation of glycogen breakdown in discrete brain structures in vivo, and document VMN GPbb control of local GPmm expression. Results document GPmm and/or -bb regulation of GABAergic and nitrergic transmission in discrete rostro-caudal VMN segments. Contrary effects of glycogenolysis on metabolic-sensory AMPK protein during eu- versus hypoglycemia may reflect energy state-specific astrocyte signaling. Amplifying effects of GPbb knockdown on hypoglycemic stimulation of pAMPK infer that glycogen mobilization by GPbb limits neuronal energy instability during hypoglycemia.

Transmembrane Prolyl 4-Hydroxylase is a Novel Regulator of Calcium Signaling in Astrocytes.

Prolyl 4-hydroxylases (P4Hs) have vital roles in regulating collagen synthesis and hypoxia response. A transmembrane P4H (P4H-TM) is a recently identified member of the family. Biallelic loss of function P4H-TM mutations cause a severe autosomal recessive intellectual disability syndrome in humans, but functions of P4H-TM are essentially unknown at cellular level. Our microarray data on P4h-tm−/− mouse cortexes where P4H-TM is abundantly expressed indicated expression changes in genes involved in calcium signaling and expression of several calcium sequestering ATPases was upregulated in P4h-tm−/− primary mouse astrocytes. Cytosolic and intraorganellar calcium imaging of P4h-tm−/− cells revealed that receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE) and calcium re-uptake by mitochondria were compromised. HIF1, but not HIF2, was found to be a key mediator of the P4H-TM effect on calcium signaling. Furthermore, total internal reflection fluorescence (TIRF) imaging showed that calcium agonist-induced gliotransmission was attenuated in P4h-tm−/− astrocytes. This phenotype was accompanied by redistribution of mitochondria from distal processes to central parts of the cell body and decreased intracellular ATP content. Our data show that P4H-TM is a novel regulator of calcium dynamics and gliotransmission.

Therapeutic potential of oxytocin receptor signaling in vascular dementia

AbstractBackgroundWe recently demonstrated a de novo upregulation of the oxytocin receptor (OXTR) in astrocytes located in the peri‐infarct regions of subjects who died with vascular dementia (multi‐infarct subtype). OXTR activation has been linked to antioxidant, anti‐inflammatory, and angiogenic signaling, suggesting that OXTR upregulation is a novel protective target for vascular dementia, mixed dementias, and perhaps even cerebrovascular accidents. To test this hypothesis, we modeled OXTR upregulation in a novel rat model of vascular dementia.MethodFour month‐old spontaneously hypertensive, stroke prone rats are being randomized to receive cortical injections of adeno‐associated virus (AAV, serotype 6) bearing either OXTR or green fluorescent protein (GFP) cDNA under control of the astroglial‐specific GFAP promoter, followed by randomization to receive cortical injections of the vasoconstrictor endothelin‐1 (ET‐1) or saline vehicle (n = 16/group, balanced for sex). Following behavioral testing, rats are being analyzed postmortem for infarction lesion size, cerebrovascular pathology, and several mechanistic indices including markers for cell death, inflammation, and oxidative stress.ResultPreliminary results (n = 6‐8/group) showed that rats receiving AAV‐OXTR and ET‐1 displayed a nonsignificant 40% reduction in mean latency time to target on the Barnes maze compared to rats receiving AAV‐GFP and ET‐1 (p = 0.7). Similarly, OXTR/ET‐1‐treated rats showed a 110% increase in the mean ratio of time spent exploring novel vs. familiar objects on the Novel Object Task compared to GFP/ET‐1‐treated rats (p = 0.08). Postmortem histological analysis revealed a nonsignificant 60% reduction in infarct size in OXTR/ET‐1 compared to GFP/ET‐1 rats (p = 0.08).ConclusionPreliminary data are trending to support the concept that astrocytic OXTR signaling may have a therapeutic benefit for dementia resulting from cerebrovascular accidents.

Methamphetamine Activates Trace Amine Associated Receptor 1 to Regulate Astrocyte Excitatory Amino Acid Transporter-2 via Differential CREB Phosphorylation During HIV-Associated Neurocognitive Disorders

Methamphetamine (METH) use, referred to as methamphetamine use disorder (MUD), results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders (HAND). MUD exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for >90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-κB, transcription factors activated by cAMP, calcium and IL-1β. Second messengers, cAMP and calcium, are triggered by TAAR1 activation, which is upregulated by IL-1β METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference and pharmacological inhibitors targeting or blocking cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Overall, this work revealed METH-induced differential CREB phosphorylation is a critical regulator for EAAT-2 function and may thus serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.

Striatal Astrocytes Shape Behavioral Flexibility via Regulation of the Glutamate Transporter EAAT2

BackgroundStriatal circuits must be modulated for behavioral flexibility, the ability to adapt to environmental changes. Striatal astrocytes contribute to circuit neuromodulation by controlling the activity of ambient neurotransmitters. In particular, extracellular glutamate levels are tightly controlled by the astrocytic glutamate transporter EAAT2, influencing synaptic functioning and neural network activity. However, it remains unclear if EAAT2 responds to environmental cues to specifically shape action control. MethodsTo investigate the relationship between behavioral flexibility and experience-dependent regulation of EAAT2 expression in the dorsal striatum, mice were trained on an instrumental task. We manipulated EAAT2 expression using chemogenetic activation of astrocytic Gq signaling or in vivo morpholinos and determined the ability to adapt to novel environmental contingencies. ResultsThe loss of behavioral flexibility with task overtraining is associated with the upregulation of EAAT2, which results in enhanced glutamate clearance and altered modulation of glutamatergic neurotransmission in the lateral part of the dorsal striatum. Interfering with EAAT2 upregulation in this striatal area preserves behavioral flexibility. ConclusionsAstrocytes are emerging as critical regulators of striatal functions. This work demonstrates that plasticity of EAAT2 expression in the lateral part of the dorsal striatum shapes behavior, thus providing novel mechanistic insights into how flexibility in action control is regulated.

Increased C-X-C Motif Chemokine Ligand 12 Levels in Cerebrospinal Fluid as a Candidate Biomarker in Sporadic Amyotrophic Lateral Sclerosis.

Sporadic amyotrophic lateral sclerosis (sALS) is a fatal progressive neurodegenerative disease affecting upper and lower motor neurons. Biomarkers are useful to facilitate the diagnosis and/or prognosis of patients and to reveal possible mechanistic clues about the disease. This study aimed to identify and validate selected putative biomarkers in the cerebrospinal fluid (CSF) of sALS patients at early disease stages compared with age-matched controls and with other neurodegenerative diseases including Alzheimer disease (AD), spinal muscular atrophy type III (SMA), frontotemporal dementia behavioral variant (FTD), and multiple sclerosis (MS). SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC–MS/MS) for protein quantitation, and ELISA for validation, were used in CSF samples of sALS cases at early stages of the disease. Analysis of mRNA and protein expression was carried out in the anterior horn of the lumbar spinal cord in post-mortem tissue of sALS cases (terminal stage) and controls using RTq-PCR, and Western blotting, and immunohistochemistry, respectively. SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC–MS/MS) revealed 51 differentially expressed proteins in the CSF in sALS. Receiver operating characteristic (ROC) curves showed CXCL12 to be the most valuable candidate biomarker. We validated the values of CXCL12 in CSF with ELISA in two different cohorts. Besides sALS, increased CXCL12 levels were found in MS but were not altered in AD, SMA, and FTD. Therefore, increased CXCL12 levels in the CSF can be useful in the diagnoses of MS and sALS in the context of the clinical settings. CXCL12 immunoreactivity was localized in motor neurons in control and sALS, and in a few glial cells in sALS at the terminal stage; CXCR4 was in a subset of oligodendroglial-like cells and axonal ballooning of motor neurons in sALS; and CXCR7 in motor neurons in control and sALS, and reactive astrocytes in the pyramidal tracts in terminal sALS. CXCL12/CXCR4/CXCR7 axis in the spinal cord probably plays a complex role in inflammation, oligodendroglial and astrocyte signaling, and neuronal and axonal preservation in sALS.

Astrocyte-mediated spike-timing-dependent long-term depression modulates synaptic properties in the developing cortex.

Astrocytes have been shown to modulate synaptic transmission and plasticity in specific cortical synapses, but our understanding of the underlying molecular and cellular mechanisms remains limited. Here we present a new biophysicochemical model of a somatosensory cortical layer 4 to layer 2/3 synapse to study the role of astrocytes in spike-timing-dependent long-term depression (t-LTD) in vivo. By applying the synapse model and electrophysiological data recorded from rodent somatosensory cortex, we show that a signal from a postsynaptic neuron, orchestrated by endocannabinoids, astrocytic calcium signaling, and presynaptic N-methyl-D-aspartate receptors coupled with calcineurin signaling, induces t-LTD which is sensitive to the temporal difference between post- and presynaptic firing. We predict for the first time the dynamics of astrocyte-mediated molecular mechanisms underlying t-LTD and link complex biochemical networks at presynaptic, postsynaptic, and astrocytic sites to the time window of t-LTD induction. During t-LTD a single astrocyte acts as a delay factor for fast neuronal activity and integrates fast neuronal sensory processing with slow non-neuronal processing to modulate synaptic properties in the brain. Our results suggest that astrocytes play a critical role in synaptic computation during postnatal development and are of paramount importance in guiding the development of brain circuit functions, learning and memory.

Effects of O-GlcNAcylation on functional mitochondrial transfer from astrocytes

Mitochondria may be transferred from cell to cell in the central nervous system and this process may help defend neurons against injury and disease. But how mitochondria maintain their functionality during the process of release into extracellular space remains unknown. Here, we report that mitochondrial protein O-GlcNAcylation is a critical process to support extracellular mitochondrial functionality. Activation of CD38-cADPR signaling in astrocytes robustly induced protein O-GlcNAcylation in mitochondria, while oxygen-glucose deprivation and reoxygenation showed transient and mild protein modification. Blocking the endoplasmic reticulum – Golgi trafficking with Brefeldin A or slc35B4 siRNA reduced O-GlcNAcylation, and resulted in the secretion of mitochondria with decreased membrane potential and mtDNA. Finally, loss-of-function studies verified that O-GlcNAc-modified mitochondria demonstrated higher levels of neuroprotection after astrocyte-to-neuron mitochondrial transfer. Collectively, these findings suggest that post-translational modification by O-GlcNAc may be required for supporting the functionality and neuroprotective properties of mitochondria released from astrocytes.

The Vasomotor Response to Dopamine Is Altered in the Rat Model of l-dopa-Induced Dyskinesia.

ABSTRACTBackgroundLevodopa (l‐dopa) is the frontline treatment for motor symptoms of Parkinson's disease. However, prolonged use of l‐dopa results in a motor complication known as levodopa‐induced dyskinesia (LID) in ~50% of patients over 5 years.ObjectivesWe investigated neurovascular abnormalities in a rat model of LID by examining changes in angiogenesis and dopamine‐dependent vessel diameter changes.MethodsDifferences in striatal and nigral angiogenesis in a parkinsonian rat model (6‐OHDA lesion) treated with 2 doses of l‐dopa (saline, 2, and 10 mg/kg/day subcutaneous l‐dopa treatment for 22 days) by 5‐bromo‐2'‐deoxyuridine (BrdU)‐RECA1 co‐immunofluorescence. Difference in the vasomotor response to dopamine was examined with 2‐photon laser scanning microscopy and Dodt gradient imaging.ResultsWe found that the 10 mg/kg l‐dopa dosing regimen induced LID in all animals (n = 5) and induced significant angiogenesis in the striatum and substantia nigra. In contrast, the 2 mg/kg treatment induced LID in 6 out of 12 rats and led to linearly increasing LID severity over the 22‐day treatment period, making this a promising model for studying LID progression longitudinally. However, no significantly different level of angiogenesis was observed between LID versus non‐LID animals. Dopamine‐induced vasodilatory responses were exaggerated only in rats that show LID‐like signs compared to the rest of groups. Additionally, in juvenile rats, we showed that DA‐induced vasodilation is preceded by increased Ca2+ release in the adjacent astrocytes.ConclusionThis finding supports that astrocytic dopamine signaling controls striatal blood flow bidirectionally, and the balance is altered in LID. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Mechanosensory Signaling in Astrocytes.

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca2+ responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y1 receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca2+ responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca2+ signals via P2Y1 receptors and Ca2+ recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits.SIGNIFICANCE STATEMENT There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca2+ responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.

Hydrogen sulfide induces Ca2+ release from the endoplasmic reticulum and suppresses ATP-induced Ca2+ signaling in rat spinal cord astrocytes

Hydrogen sulfide (H2S) has a variety of physiological functions. H2S reportedly increases intracellular Ca2+ concentration ([Ca2+]i) in astrocytes. However, the precise mechanism and functional role of this increase are not known. Here, we examined the effects of H2S on [Ca2+]i in astrocytes from the rat spinal cord and whether H2S affects ATP-induced Ca2+ signaling, which is known to be involved in synaptic function. Na2S (150 μM), an H2S donor, produced a nontoxic increase in [Ca2+]i. The [Ca2+]i increase by Na2S was inhibited by Ca2+ depletion in the endoplasmic reticulum (ER) but not by removal of extracellular Ca2+, indicating that H2S releases Ca2+ from the ER. On the other hand, Na2S inhibited ATP-induced [Ca2+]i increase when Na2S clearly increased [Ca2+]i in the astrocytes, which was not suppressed by a reducing agent. In addition, Na2S had no effect on intracellular cyclic AMP (cAMP) level. These results indicate that oxidative post-translational modification of proteins and cAMP are not involved in the inhibitory effect of H2S on ATP-induced Ca2+ signaling. We conclude that H2S indirectly inhibits ATP-induced Ca2+ signaling by decreasing Ca2+ content in the ER in astrocytes. In this way, H2S may influence intercellular communication between astrocytes and neurons, thereby contributing to neuronal signaling in the nervous system.