RESUMO
Neurotransmitters and neuromodulators have a wide range of key roles throughout the nervous system. However, their dynamics in both health and disease have been challenging to assess, owing to the lack of in vivo tools to track them with high spatiotemporal resolution. Thus, developing a platform that enables minimally invasive, large-scale and long-term monitoring of neurotransmitters and neuromodulators with high sensitivity, high molecular specificity and high spatiotemporal resolution has been essential. Here, we review the methods available for monitoring the dynamics of neurotransmitters and neuromodulators. Following a brief summary of non-genetically encoded methods, we focus on recent developments in genetically encoded fluorescent indicators, highlighting how these novel indicators have facilitated advances in our understanding of the functional roles of neurotransmitters and neuromodulators in the nervous system. These studies present a promising outlook for the future development and use of tools to monitor neurotransmitters and neuromodulators.
Assuntos
Neurotransmissores , HumanosRESUMO
The purinergic signaling molecule adenosine (Ado) modulates many physiological and pathological functions in the brain. However, the exact source of extracellular Ado remains controversial. Here, utilizing a newly optimized genetically encoded GPCR-Activation-Based Ado fluorescent sensor (GRABAdo), we discovered that the neuronal activity-induced extracellular Ado elevation is due to direct Ado release from somatodendritic compartments of neurons, rather than from the axonal terminals, in the hippocampus. Pharmacological and genetic manipulations reveal that the Ado release depends on equilibrative nucleoside transporters but not the conventional vesicular release mechanisms. Compared with the fast-vesicular glutamate release, the Ado release is slow (~40 s) and requires calcium influx through L-type calcium channels. Thus, this study reveals an activity-dependent second-to-minute local Ado release from the somatodendritic compartments of neurons, potentially serving modulatory functions as a retrograde signal.
Assuntos
Adenosina , Neurônios , Adenosina/farmacologia , Proteínas de Transporte de Nucleosídeos/genética , Transdução de Sinais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismoRESUMO
As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.
Assuntos
Dependovirus , Microglia , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Transdução GenéticaRESUMO
Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor nitrobenzylthioinosine reduced dopamine release and promoted A1R-mediated inhibition, and, conversely, virally mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor [GPCR-activation based (GRAB)-Ado], we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mm) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.SIGNIFICANCE STATEMENT Dopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A1 receptors (A1Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A1Rs on dopamine axons inhibit dopamine release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease.
Assuntos
Astrócitos , Dopamina , Transportador Equilibrativo 1 de Nucleosídeo , Etanol , Núcleo Accumbens , Receptor A1 de Adenosina , Adenosina/farmacologia , Agonistas do Receptor A1 de Adenosina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Dopamina/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Etanol/farmacologia , Feminino , Masculino , Camundongos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Receptor A1 de Adenosina/metabolismoRESUMO
Astrocytes participate in information processing by releasing neuroactive substances termed gliotransmitters, including ATP. Individual astrocytes come into contact with thousands of synapses with their ramified structure, but the spatiotemporal dynamics of ATP gliotransmission remains unclear, especially in physiological brain tissue. Using a genetically encoded fluorescent sensor, GRABATP1.0 , we discovered that extracellular ATP increased locally and transiently in absence of stimuli in neuron-glia co-cultures, cortical slices, and the anesthetized mouse brain. Spontaneous ATP release events were tetrodotoxin-insensitive but suppressed by gliotoxin, fluorocitrate, and typically spread over 50-250 µm2 area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca2+ transients, and intracellular Ca2+ buffering with BAPTA-AM did not affect ATP event frequency. Clustering analysis revealed that these events followed multiple distinct kinetics, and blockade of exocytosis only decreased a minor group of slow events. Overall, astrocytes spontaneously release ATP through multiple mechanisms, mainly in non-vesicular and Ca2+ -independent manners, thus potentially regulating hundreds of synapses all together.
Assuntos
Astrócitos , Sinapses , Camundongos , Animais , Astrócitos/metabolismo , Sinapses/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologiaRESUMO
To simulate the nonlinear stress-strain curve of rocks under static loads and contribute to the design and construction of rock engineering structures, a constitutive model has been proposed based on the elastic modulus E decreasing with the increase in longitudinal cracks. This constitutive equation offers numerous advantages, with the most noteworthy being that the simulation of stress-strain curves for rocks necessitates only three equations (Eqs 1-3) and four parameters (A, k0, C and εs). Following this, we employ the constitutive equation to analyze the stress distribution around a thick-walled cylinder and explore the impact of its four parameters on the stress distribution surrounding the thick-walled cylinder. Parameter A primarily affects the range of the plastic zone and the magnitude of the maximum tangential stress; parameter C mainly influences the magnitude of the maximum tangential stress; parameter εs mainly affects the range of the plastic zone and the magnitude of the maximum tangential stress; parameter k0 primarily influences the magnitude of the maximum tangential stress. We got the similar results with Bray model, but distribution of stress around the tunnel are different present that the shape of stress-strain curves are different.
Assuntos
Estresse Mecânico , Modelos Teóricos , Módulo de ElasticidadeRESUMO
The CC chemokine ligand 2 (CCL2, also known as MCP-1) and its cognate receptor CCR2 have well-characterized roles in chemotaxis. CCL2 has been previously shown to promote excitatory synaptic transmission and neuronal excitability. However, the detailed molecular mechanism underlying this process remains largely unclear. In cultured hippocampal neurons, CCL2 application rapidly upregulated surface expression of GluA1, in a CCR2-dependent manner, assayed using SEP-GluA1 live imaging, surface GluA1 antibody staining, and electrophysiology. Using pharmacology and reporter assays, we further showed that CCL2 upregulated surface GluA1 expression primarily via Gαq- and CaMKII-dependent signaling. Consistently, using i.p. injection of lipopolysaccharide to induce neuroinflammation, we found upregulated phosphorylation of S831 and S845 sites on AMPA receptor subunit GluA1 in the hippocampus, an effect blocked in Ccr2-/- mice. Together, these results provide a mechanism through which CCL2, and other secreted molecules that signal through G-protein coupled receptors, can directly regulate synaptic transmission.
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Injuries to the brain result in tunable cell responses paired with stimulus properties, suggesting the existence of intrinsic processes that encode and transmit injury information; however, the molecular mechanism of injury information encoding is unclear. Here, using ATP fluorescent indicators, we identify injury-evoked spatiotemporally selective ATP dynamics, Inflares, in adult mice of both sexes. Inflares are actively released from astrocytes and act as the internal representations of injury. Inflares encode injury intensity and position at their population level through frequency changes and are further decoded by microglia, driving changes in their activation state. Mismatches between Inflares and injury severity lead to microglia dysfunction and worsening of injury outcome. Blocking Inflares in ischemic stroke in mice reduces secondary damage and improves recovery of function. Our results suggest that astrocytic ATP dynamics encode injury information and are sensed by microglia.
Assuntos
Trifosfato de Adenosina , Astrócitos , Lesões Encefálicas , Microglia , Animais , Microglia/metabolismo , Astrócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Camundongos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Masculino , Feminino , Camundongos Endogâmicos C57BLRESUMO
Meaningful auditory memories are formed in adults when acoustic information is delivered to the auditory cortex during heightened states of attention, vigilance, or alertness, as mediated by neuromodulatory circuits. Here, we identify that, in awake mice, acoustic stimulation triggers auditory thalamocortical projections to release adenosine, which prevents cortical plasticity (i.e., selective expansion of neural representation of behaviorally relevant acoustic stimuli) and perceptual learning (i.e., experience-dependent improvement in frequency discrimination ability). This sound-evoked adenosine release (SEAR) becomes reduced within seconds when acoustic stimuli are tightly paired with the activation of neuromodulatory (cholinergic or dopaminergic) circuits or periods of attentive wakefulness. If thalamic adenosine production is enhanced, then SEAR elevates further, the neuromodulatory circuits are unable to sufficiently reduce SEAR, and associative cortical plasticity and perceptual learning are blocked. This suggests that transient low-adenosine periods triggered by neuromodulatory circuits permit associative cortical plasticity and auditory perceptual learning in adults to occur.
Assuntos
Córtex Auditivo , Animais , Camundongos , Córtex Auditivo/fisiologia , Adenosina , Aprendizagem/fisiologia , Estimulação Acústica , SomRESUMO
Microglial calcium signaling is rare in a baseline state but strongly engaged during early epilepsy development. The mechanism(s) governing microglial calcium signaling are not known. By developing an in vivo uridine diphosphate (UDP) fluorescent sensor, GRABUDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP can signal through the microglial-enriched P2Y6 receptor to increase calcium activity during epileptogenesis. P2Y6 calcium activity is associated with lysosome biogenesis and enhanced production of NF-κB-related cytokines. In the hippocampus, knockout of the P2Y6 receptor prevents microglia from fully engulfing neurons. Attenuating microglial calcium signaling through calcium extruder ("CalEx") expression recapitulates multiple features of P2Y6 knockout, including reduced lysosome biogenesis and phagocytic interactions. Ultimately, P2Y6 knockout mice retain more CA3 neurons and better cognitive task performance during epileptogenesis. Our results demonstrate that P2Y6 signaling impacts multiple aspects of myeloid cell immune function during epileptogenesis.
Assuntos
Sinalização do Cálcio , Epilepsia , Camundongos Knockout , Microglia , Fagocitose , Receptores Purinérgicos P2 , Animais , Microglia/metabolismo , Microglia/imunologia , Camundongos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/genética , Sinalização do Cálcio/fisiologia , Epilepsia/metabolismo , Epilepsia/imunologia , Epilepsia/genética , Difosfato de Uridina/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Hipocampo/metabolismo , Neuroimunomodulação/fisiologiaRESUMO
Optochemistry, an emerging pharmacologic approach in which light is used to selectively activate or deactivate molecules, has the potential to alleviate symptoms, cure diseases, and improve quality of life while preventing uncontrolled drug effects. The development of in-vivo applications for optochemistry to render brain cells photoresponsive without relying on genetic engineering has been progressing slowly. The nucleus accumbens (NAc) is a region for the regulation of slow-wave sleep (SWS) through the integration of motivational stimuli. Adenosine emerges as a promising candidate molecule for activating indirect pathway neurons of the NAc expressing adenosine A2A receptors (A2ARs) to induce SWS. Here, we developed a brain-permeable positive allosteric modulator of A2ARs (A2AR PAM) that can be rapidly photoactivated with visible light (λ > 400 nm) and used it optoallosterically to induce SWS in the NAc of freely behaving male mice by increasing the activity of extracellular adenosine derived from astrocytic and neuronal activity.
Assuntos
Adenosina , Núcleo Accumbens , Receptor A2A de Adenosina , Sono de Ondas Lentas , Animais , Núcleo Accumbens/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Masculino , Receptor A2A de Adenosina/metabolismo , Receptor A2A de Adenosina/genética , Camundongos , Adenosina/metabolismo , Adenosina/farmacologia , Regulação Alostérica , Sono de Ondas Lentas/fisiologia , Sono de Ondas Lentas/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Luz , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Humanos , Agonistas do Receptor A2 de Adenosina/farmacologiaRESUMO
Acute brain slices represent a workhorse model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organization and functioning in acute brain slices. We reveal time-dependent microglial phenotype changes influenced by complex extracellular ATP dynamics through P2Y12R and CX3CR1 signalling, which is sustained for hours in ex vivo mouse brain slices. Downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses over time. Importantly, functional microglia modulate synapse sprouting, while microglial dysfunction results in markedly impaired ripple activity both ex vivo and in vivo. Collectively, our data suggest that microglia are modulators of complex neuronal networks with important roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.
Assuntos
Trifosfato de Adenosina , Encéfalo , Receptor 1 de Quimiocina CX3C , Microglia , Neurônios , Receptores Purinérgicos P2Y12 , Sinapses , Animais , Microglia/metabolismo , Trifosfato de Adenosina/metabolismo , Camundongos , Neurônios/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y12/genética , Encéfalo/metabolismo , Sinapses/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Masculino , Transdução de SinaisRESUMO
Flickering light stimulation has emerged as a promising non-invasive neuromodulation strategy to alleviate neuropsychiatric disorders. However, the lack of a neurochemical underpinning has hampered its therapeutic development. Here, we demonstrate that light flickering triggered an immediate and sustained increase (up to 3 h after flickering) in extracellular adenosine levels in the primary visual cortex (V1) and other brain regions, as a function of light frequency and intensity, with maximal effects observed at 40 Hz frequency and 4000 lux. We uncovered cortical (glutamatergic and GABAergic) neurons, rather than astrocytes, as the cellular source, the intracellular adenosine generation from AMPK-associated energy metabolism pathways (but not SAM-transmethylation or salvage purine pathways), and adenosine efflux mediated by equilibrative nucleoside transporter-2 (ENT2) as the molecular pathway responsible for extracellular adenosine generation. Importantly, 40 Hz (but not 20 and 80 Hz) light flickering for 30 min enhanced non-rapid eye movement (non-REM) and REM sleep for 2-3 h in mice. This somnogenic effect was abolished by ablation of V1 (but not superior colliculus) neurons and by genetic deletion of the gene encoding ENT2 (but not ENT1), but recaptured by chemogenetic inhibition of V1 neurons and by focal infusion of adenosine into V1 in a dose-dependent manner. Lastly, 40 Hz light flickering for 30 min also promoted sleep in children with insomnia by decreasing sleep onset latency, increasing total sleep time, and reducing waking after sleep onset. Collectively, our findings establish the ENT2-mediated adenosine signaling in V1 as the neurochemical basis for 40 Hz flickering-induced sleep and unravel a novel and non-invasive treatment for insomnia, a condition that affects 20% of the world population.
Assuntos
Distúrbios do Início e da Manutenção do Sono , Humanos , Criança , Animais , Camundongos , Sono , Transdução de Sinais , Adenosina , AstrócitosRESUMO
Astrocytes play a crucial role in regulating sleep-wake behavior, and adenosine signaling is generally thought to be involved. Here we show multiple lines of evidence supporting that modulation of the sleep-wake behavior by astrocyte Ca2+ activity could occur without adenosine signaling. In the basal forebrain and the brainstem, two brain regions that are known to be essential for sleep-wake regulation, chemogenetically-induced astrocyte Ca2+ elevation significantly modulated the sleep-wake cycle. Although astrocyte Ca2+ level positively correlated with the amount of extracellular adenosine, as revealed by a genetically encoded adenosine sensor, we found no detectable change in adenosine level after suppressing astrocyte Ca2+ elevation, and transgenic mice lacking one of the major extracellular ATP-adenosine conversion enzymes showed similar extracellular adenosine level and astrocyte Ca2+-induced sleep modulation. Furthermore, astrocyte Ca2+ is dependent primarily on local neuronal activity, causing brain region-specific regulation of the sleep-wake cycle. Thus, neural activity-dependent astrocyte activity could regulate the sleep-wake behavior independent of adenosine signaling.
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Microglial calcium signaling is rare in a baseline state but shows strong engagement during early epilepsy development. The mechanism and purpose behind microglial calcium signaling is not known. By developing an in vivo UDP fluorescent sensor, GRABUDP1.0, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP signals to the microglial P2Y6 receptor for broad increases in calcium signaling during epileptogenesis. UDP-P2Y6 signaling is necessary for lysosome upregulation across limbic brain regions and enhances production of pro-inflammatory cytokines-TNFα and IL-1ß. Failures in lysosome upregulation, observed in P2Y6 KO mice, can also be phenocopied by attenuating microglial calcium signaling in Calcium Extruder ("CalEx") mice. In the hippocampus, only microglia with P2Y6 expression can perform full neuronal engulfment, which substantially reduces CA3 neuron survival and impairs cognition. Our results demonstrate that calcium activity, driven by UDP-P2Y6 signaling, is a signature of phagocytic and pro-inflammatory function in microglia during epileptogenesis.
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A fundamental challenge in fluorescence microscopy is the photon shot noise arising from the inevitable stochasticity of photon detection. Noise increases measurement uncertainty and limits imaging resolution, speed and sensitivity. To achieve high-sensitivity fluorescence imaging beyond the shot-noise limit, we present DeepCAD-RT, a self-supervised deep learning method for real-time noise suppression. Based on our previous framework DeepCAD, we reduced the number of network parameters by 94%, memory consumption by 27-fold and processing time by a factor of 20, allowing real-time processing on a two-photon microscope. A high imaging signal-to-noise ratio can be acquired with tenfold fewer photons than in standard imaging approaches. We demonstrate the utility of DeepCAD-RT in a series of photon-limited experiments, including in vivo calcium imaging of mice, zebrafish larva and fruit flies, recording of three-dimensional (3D) migration of neutrophils after acute brain injury and imaging of 3D dynamics of cortical ATP release. DeepCAD-RT will facilitate the morphological and functional interrogation of biological dynamics with a minimal photon budget.
Assuntos
Fótons , Peixe-Zebra , Animais , Camundongos , Imagem com Lapso de Tempo , Microscopia de Fluorescência , Razão Sinal-RuídoRESUMO
The purinergic transmitter ATP (adenosine 5'-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. Here, we developed a sensitive GPCR activation-based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, has ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including stimuli-induced and spontaneous ATP release in primary hippocampal cultures, injury-induced ATP release in a zebrafish model, and lipopolysaccharides-induced ATP-release events in individual astrocytes in the mouse cortex. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.
Assuntos
Trifosfato de Adenosina , Peixe-Zebra , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Lipopolissacarídeos/farmacologia , CamundongosRESUMO
The prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness to locomotion. We find that PFC astrocytes express receptors for dopamine but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time locked to dopamine release and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in the PFC, contributing to PFC function though neuromodulator receptor crosstalk.
Assuntos
Dopamina , Receptores Adrenérgicos alfa 1 , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Dopamina/metabolismo , Camundongos , Córtex Pré-Frontal/metabolismo , Receptores Adrenérgicos alfa 1/metabolismoRESUMO
BACKGROUND: Major depressive disorder is a devastating psychiatric illness that affects approximately 17% of the population worldwide. Astrocyte dysfunction has been implicated in its pathophysiology. Traumatic experiences and stress contribute to the onset of major depressive disorder, but how astrocytes respond to stress is poorly understood. METHODS: Using Western blotting analysis, we identified that stress vulnerability was associated with reduced astrocytic glucocorticoid receptor (GR) expression in mouse models of depression. We further investigated the functions of astrocytic GRs in regulating depression and the underlying mechanisms by using a combination of behavioral studies, fiber photometry, biochemical experiments, and RNA sequencing methods. RESULTS: GRs in astrocytes were more sensitive to stress than those in neurons. GR absence in astrocytes induced depressive-like behaviors, whereas restoring astrocytic GR expression in the medial prefrontal cortex prevented the depressive-like phenotype. Furthermore, we found that GRs in the medial prefrontal cortex affected astrocytic Ca2+ activity and dynamic ATP (adenosine 5'-triphosphate) release in response to stress. RNA sequencing of astrocytes isolated from GR deletion mice identified the PI3K-Akt (phosphoinositide 3-kinase-Akt) signaling pathway, which was required for astrocytic GR-mediated ATP release. CONCLUSIONS: These findings reveal that astrocytic GRs play an important role in stress response and that reduced astrocytic GR expression in the stressed subject decreases ATP release to mediate stress vulnerability.
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Astrócitos , Transtorno Depressivo Maior , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Transtorno Depressivo Maior/metabolismo , Glucocorticoides/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Córtex Pré-Frontal/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Glucocorticoides/metabolismoRESUMO
Gq-coupled receptors regulate numerous physiological processes by activating enzymes and inducing intracellular Ca2+ signals. There is a strong need for an optogenetic tool that enables powerful experimental control over Gq signaling. Here, we present chicken opsin 5 (cOpn5) as the long sought-after, single-component optogenetic tool that mediates ultra-sensitive optical control of intracellular Gq signaling with high temporal and spatial resolution. Expressing cOpn5 in HEK 293T cells and primary mouse astrocytes enables blue light-triggered, Gq-dependent Ca2+ release from intracellular stores and protein kinase C activation. Strong Ca2+ transients were evoked by brief light pulses of merely 10 ms duration and at 3 orders lower light intensity of that for common optogenetic tools. Photostimulation of cOpn5-expressing cells at the subcellular and single-cell levels generated fast intracellular Ca2+ transition, thus demonstrating the high spatial precision of cOpn5 optogenetics. The cOpn5-mediated optogenetics could also be applied to activate neurons and control animal behavior in a circuit-dependent manner. cOpn5 optogenetics may find broad applications in studying the mechanisms and functional relevance of Gq signaling in both non-excitable cells and excitable cells in all major organ systems.