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Astrocyte-mediated control of cerebral blood flow
Abstract
Local increase in blood flow during neural activity forms the basis for functional brain imaging, but its mechanism remains poorly defined. Here we show that cortical astrocytes in vivo possess a powerful mechanism for rapid vasodilation. We imaged the activity of astrocytes labeled with the calcium (Ca(2+))-sensitive indicator rhod-2 in somatosensory cortex of adult mice. Photolysis of caged Ca(2+) in astrocytic endfeet ensheathing the vessel wall was associated with an 18% increase in arterial cross-section area that corresponded to a 37% increase in blood flow. Vasodilation occurred with a latency of only 1-2 s, and both indomethacin and the cyclooxygenase-1 inhibitor SC-560 blocked the photolysis-induced hyperemia. These observations implicate astrocytes in the control of local microcirculation and suggest that one of their physiological roles is to mediate vasodilation in response to increased neural activity.
... Forepaw stimulation, which has been widely used in brain functional studies with fMRI [28,29] and optical imaging [30], results in increases in CBF and cerebral blood volume (CBV) in the somatosensory cortex to meet the energetic demands evoked by the stimulation as a result of neurovascular coupling (NVC). Studies assessing the role of astrocytes in NVC have measured intracellular Ca 2+ as a marker of activity and have shown that Ca 2+ elevation in astrocytes is associated with release of vasoactive compounds that might drive CBF/CBV changes in NVC [31][32][33][34]. ...
... This suggests that at baseline astrocytes, but not neurons, mediate vascular tone, which is consistent with previous reports with two-photon microscopy. [34] During a cocaine challenge, the inhibition of astrocytic activation prevented the CBFv decreases triggered by cocaine-induced vasoconstriction. Different from baseline, inhibition of astrocyte activation also blunted neuronal activation by cocaine. ...
Cocaine affects both cerebral blood vessels and neuronal activity in brain. Cocaine can also disrupt astrocytes, which modulate neurovascular coupling—a process that regulates cerebral hemodynamics in response to neuronal activation. However, separating neuronal and astrocytic effects from cocaine’s direct vasoactive effects has been challenging, partially due to limitations of neuroimaging techniques able to differentiate vascular from neuronal and glial effects at high temporal and spatial resolutions. Here, we used a newly-developed multi-channel fluorescence and optical coherence Doppler microscope (fl-ODM) that allows for simultaneous measurements of neuronal and astrocytic activities (reflected by the intracellular calcium changes in neurons Ca²⁺N and astrocytes Ca²⁺A, respectively) alongside their vascular interactions in vivo to address this challenge. Using green and red genetically-encoded Ca²⁺ indicators differentially expressed in astrocytes and neurons, fl-ODM enabled concomitant imaging of large-scale astrocytic and neuronal Ca²⁺ fluorescence and 3D cerebral blood flow velocity (CBFv) in vascular networks in the mouse cortex. We assessed cocaine’s effects in the prefrontal cortex (PFC) and found that the CBFv changes triggered by cocaine were temporally correlated with astrocytic Ca²⁺A activity. Chemogenetic inhibition of astrocytes during the baseline state resulted in blood vessel dilation and CBFv increases but did not affect neuronal activity, suggesting modulation of spontaneous blood vessel’s vascular tone by astrocytes. Chemogenetic inhibition of astrocytes during a cocaine challenge prevented its vasoconstricting effects alongside the CBFv decreases, but it also attenuated the neuronal Ca²⁺N increases triggered by cocaine. These results document a role of astrocytes both in regulating vascular tone and consequently blood flow, at baseline and for modulating the vasoconstricting and neuronal activation responses to cocaine in the PFC. Strategies to inhibit astrocytic activity could offer promise for ameliorating vascular and neuronal toxicity from cocaine misuse.
... On the one hand, neurons release glutamate (Glu; throughout this manuscript the three-letter code for amino acid abbreviations will be used referring to the L-isomers) and nitric oxide (NO), the latter exerting its vasodilating effects on the arterioles. On the other hand, astrocytes increase their Ca 2+ concentrations [5][6][7], which have been reported to show vasodilation or vasoconstriction effects and to spread in waves between astrocytes. The combined actions of increased Ca 2+ levels and decreased oxygen supply, which induce glycolytic production and release of lactate from astrocytes, in turn, lead to prostaglandin E2 accumulation and vasodilation [8], which simultaneously correlates with the elevation of extracellular adenosine levels to block vasoconstriction [9,10]. ...
... Amino acids and peptides are depicted in a salmon background, whereas green was the color used for other metabolites. Cytoplasmic (dark blue), mitochondrial (orange), and peroxisomal (red) enzymes involved in these pathways are: AGT, alanine glyoxylate aminotransferase (EC 2. 6 ...
The metabolism and intercellular transfer of glutathione or its precursors may play an important role in cellular defense against oxidative stress, a common hallmark of neurodegeneration. In the 1990s, several studies in the Neurobiology field led to the widely accepted notion that astrocytes produce large amounts of glutathione that serve to feed neurons with precursors for glutathione synthesis. This assumption has important implications for health and disease since a reduction in this supply from astrocytes could compromise the capacity of neurons to cope with oxidative stress. However, at first glance, this shuttling would imply a large energy expenditure to get to the same point in a nearby cell. Thus, are there additional underlying reasons for this expensive mechanism? Are neurons unable to import and/or synthesize the three non-essential amino acids that are the glutathione building blocks? The rather oxidizing extracellular environment favors the presence of cysteine (Cys) as cystine (Cis), less favorable for neuronal import. Therefore, it has also been proposed that astrocytic GSH efflux could induce a change in the redox status of the extracellular space nearby the neurons, locally lowering the Cis/Cys ratio. This astrocytic glutathione release would also increase their demand for precursors, stimulating Cis uptake, which these cells can import, further impacting the local decline of the Cis/Cys ratio, in turn, contributing to a more reduced extracellular environment and subsequently favoring neuronal Cys import. Here, we revisit the experimental evidence that led to the accepted hypothesis of astrocytes acting as suppliers of neuronal glutathione precursors, considering recent data from the Human Protein Atlas. In addition, we highlight some potential drawbacks of this hypothesis, mainly supported by heterogeneous cellular models. Finally, we outline additional and more cost-efficient possibilities by which astrocytes could support neuronal glutathione levels, including its shuttling in extracellular vesicles.
... Astrocytes comprise a large and heterogeneous group of glial cells that perform various essential functions in the CNS. They play a role in brain development and metabolism, control the CNS microenvironment, modulate synaptic transmission and neurotransmitter release [102], and have an important function in controlling systemic circulation and maintaining cerebral blood flow [103,104]. Astrocytes are also crucial for providing the structural and functional integrity of the nervous system. The astrocyte processes are strategically positioned to make numerous contacts with neuronal synapses, on one side, and brain capillaries on the other. ...
... Signal transmission within NVC involves astrocytic Ca 2+ signaling, which can be triggered by the activation of mGLURs [146] or the TRPV4 channels. The mGLUR activation in astrocytes induces rises in [Ca 2+ ] i which in turn trigger the synthesis of AA and its metabolites such as EETs or prostaglandin-2 (PGE2), both possessing the vasodilatory effect [104,146]. These can both either act directly on the smooth muscle cells, or they can modulate astrocytic K + channels and influence K + release into the ECS [147,148]. ...
Transient receptor potential cation channels subfamily V member 4 (TRPV4) are non-selective cation channels expressed in different cell types of the central nervous system. These channels can be activated by diverse physical and chemical stimuli, including heat and mechanical stress. In astrocytes, they are involved in the modulation of neuronal excitability, control of blood flow, and brain edema formation. All these processes are significantly impaired in cerebral ischemia due to insufficient blood supply to the tissue, resulting in energy depletion, ionic disbalance, and excitotoxicity. The polymodal cation channel TRPV4, which mediates Ca2+ influx into the cell because of activation by various stimuli, is one of the potential therapeutic targets in the treatment of cerebral ischemia. However, its expression and function vary significantly between brain cell types, and therefore, the effect of its modulation in healthy tissue and pathology needs to be carefully studied and evaluated. In this review, we provide a summary of available information on TRPV4 channels and their expression in healthy and injured neural cells, with a particular focus on their role in ischemic brain injury.
... Vascular endothelial cells are adjacent to each other and form a barrier by junctional proteins such as Claudin-5, and pericytes localize around vascular endothelial cells. Astrocyte terminals also cover more than approximately 90% of capillaries and mediate communication between neurons and blood vessels [5,6]. Aging occurs in the various organs, and as the aging process advances, various biological, chemical, and physical functions undergo alterations in the molecules and cells of these organs. ...
Brain aging causes a wide variety of changes at the molecular and cellular levels, leading to the decline of cognitive functions and increased vulnerability to neurodegenerative disorders. The research aimed at understanding the aging of the brain has made much progress in recent decades. Technological innovations such as single-cell RNA-sequencing (scRNA-seq), proteomic analyses, and spatial transcriptomic analyses have facilitated the research on the dynamic changes occurring within neurons, glia, and other cells along with their impacts on intercellular communication during aging. In this review, we introduce recent trends of how neurons and glia change during aging and discuss the impact on the brain microenvironment such as the blood-brain barrier (BBB).
... Astrocytes are a major subtype of non-neuronal cells that populate all regions of the brain 7 and modulate diverse neuronal functions including neurotransmitter clearance, ionic homeostasis, cerebral blood flow, and synaptic development and plasticity [8][9][10][11][12][13][14][15][16][17][18][19] . These glia cells are electrically quiescent but show robust intracellular calcium dynamics [20][21][22][23] . ...
Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic nigrostriatal inputs, which causes striatal network dysfunction and leads to pronounced motor deficits. Recent evidence highlights astrocytes as a potential local source of striatal network modulation. However, it remains unknown how dopamine loss affects striatal astrocyte activity and whether astrocyte activity regulates behavioral deficits in PD. We addressed these questions by performing astrocyte-specific calcium recordings and manipulations using in vivo fiber photometry and chemogenetics. We find that locomotion elicits astrocyte calcium activity over a slower timescale than neurons. Unilateral dopamine depletion reduced locomotion-related astrocyte responses. Chemogenetic activation facilitated astrocyte activity, and improved asymmetrical motor deficits and open field exploratory behavior in dopamine lesioned mice. Together, our results establish a novel role for functional striatal astrocyte signaling in modulating motor function in PD and highlight non-neuronal targets for potential PD therapeutics.
... Cortés-Campos et al. (26) demonstrated that tanycytes express MCT1 and MCT4 and both involved in influx and efflux of lactate, and also revealed that MCT1 express on the apical membrane and and cellular processes of the tanycytes while the MCT4 in the apical membrane. Takano et al. (44) revealed that the ECs, endothelial cells and the astrocytes express both MCT1 and lower MCT4. Chiry et al. (45) clearly demonstrated MCT1 in astrocytes and their end feet adjoining capillary while the restricted expression of MCT4 in astrocytes in certain brain regions are shown. ...
Background
Circumventricular organs (CVOs) are specialized structures border the brain ventricles and lack the blood-brain barrier. These CVOs are lined by specialized ependymal cells (ECs) called tanycyte. The sulcus medianus organum (SMO) locates at the floor of the 4th ventricle at the rostral part of the sulcus medianus (SM).
Objective
To explore the expression of the monocarboxylate transporter 1 and monocarboxylate transporter 4 (MCT1 & MCT4) in the tanycytes of the median eminence (ME) and tanycyte-like cells of the SMO to add a functional evidence for describing the SMO as another CVOs and to start a roadmap for the citing the SMO specifically as a sensory or a secretory CVO.
Methods
Ten adult male rats (Rattus norvegicus albinus), aged 3-6 months with 300±50 g, were used to study the histological characteristics of ECs in ME and SMO with Hematoxylin & Eosin and to explore the immunohistochemical expression of MCT1 & MCT4 in ME and SMO.
Results
The ependymal cells were arranged in in 2-3 layers in the depth of SMO region and single layer in the ME region as seen with H&E stains. Immunohistochemical expression of MCT1 & MCT4 using Aperioscope image analysis in tanycytes of the ME is higher than that in tanycyte-like cells of the SMO with significant differences between the two regions as proved by t-test.
Conclusion
the SMO has different structural and functional properties compared to ME suggesting that the SMO may be a sensory CVO.
... There are two basic types of astrocytes; the protoplasmic and fibrous astrocytes were first described in the 19th century by using the Golgi staining (Sofroniew andVinters, 2010a, Oberheim et al., 2006). Protoplasmic astrocytes mainly distributed in gray matter, they have avoided cell bodies with highly branched bushy processes that may extend and exhibit endfeet enveloping the synapses and far blood vessels where it plays important role in blood-brain barrier formation (Nishiyama et al., 2002, Takano et al., 2006. This type has connections with synapses with its processes which have multiple receptors for neurotransmitters, transporters for metabolites and diverse ion channels that all play diverse roles in the formation and modulation of synaptic functions (Pfrieger, 2010, Bélanger andMagistretti, 2009). ...
The neocortex is a multi-layered structure that commands the brain higher functions in mammals generally, and in humans, it is essential for consciousness and cognition. The question about how the human brain works still without an answer where it is related to the underlying brain architecture-function relationship. Particularly, the structure of the neocortex that determines in larger part the brain network dynamics. In this work, some cortical regions were studied to reveal its architecture features unique to the human brain. Such features were previously reported on the transcriptome level for one cortical region in humans compared to other primates like chimpanzees and macaque. Using immunohistochemistry, some of these human-unique features were investigated in the current study over two human cortical regions; supramarginal gyrus and anterior cingulate cortex to show the human-specific regional differences over the cyto-expression patterns of these markers in terms of abundance, cell types and cortical layer specificity. Despite having high histological heterogeneity, this approach did not reveal big differences between studied regions over the investigated markers, suggesting that differences may present on the level function of each marker rather than the cyto-expression one.
In a more comprehensive manner, the whole transcriptome over cortical layers two to six was studied and analyzed in the anterior prefrontal cortex and primary visual cortex areas of the human neocortex using unsupervised sectioning followed by RNA sequencing. This approach revealed differences in the gene expression level of the human-specific cortical layers markers previously described and also for the whole transcriptome between the cortical layers of the studied regions. The most considerable differences were observed in internal layers of the neocortex specifically layer three and five that previously reported to harbor the highest-change in cortical layers-genes specificity between human and other primates, suggesting that these layers may be the sites for unique molecular or architecture differences between human and other primates as well as sites of differences between human neocortex regions.
... Importantly, 21 ADEM genes are overlapped with DAM genes. Of these genes, Axl, Cd74, 6 Cst7, Cybb, Fth1, Spp1, Lpl, and H2-D1 are observed in both physiological and pathophysiological in the aging brain, indicating that the ADEM gene is highly correlated with the etiology of neurodegeneration. In addition, CEBPβ and MEF2C, which regulate microglial homeostasis and reactivity in the aging brain, are potential key mediators that link ADEM genes to characteristic subpopulations. ...
Brain aging causes a wide variety of changes at molecular and cellular levels, leading to the decline of cognitive functions and increased vulnerability to neurodegenerative disorders. The research aimed at understanding the aging of the brain has made much progress in recent decades. Technological innovations such as single-cell RNA-sequencing (scRNA-seq), proteomic analyses, and spatial transcriptomic analyses have facilitated the research on the dynamic changes occurring within neurons, glia, and other cells along with their impacts on intercellular communication during aging. In this review, we introduce recent trends of how neurons and glia change during aging and discuss the impact of the brain microenvironment such as the blood-brain barrier (BBB).
... In our method, the bioheat transfer simulation was used for ∆T estimate. One important parameter that distinguishes the bioheat model from the phantom is the blood perfusion, which is a complicate issue affected by several physiological processes 22 . To assess its effect, we used three methods to determine the perfusion value: ...
Radio frequency induced heating of medical implants during magnetic resonance imaging (MRI) poses a serious threat to patient safety, and in vivo assessment of heating permits individualized on-site safety assessment. Based on the proton resonance frequency (PRF) thermometry, we developed a method for the in vivo evaluation of implant heating. It combined PRF signals around the implants with bioheat transfer law to reduce the interference of metal artifacts and to estimate the RF heating at implant surface. To apply this idea, we proposed a PRF-based test module consisting of processes of thermometry-heating-thermometry and verified its feasibility in phantom. Then, we validated this module for electrodes in the pig brain and investigated the effect of its parameters, as a result, the heating assessment could be achieved in approximately 2 min with a mean difference to probe measurement of 0.6 °C. Finally, we demonstrated the clinical translation in a patient with a conventional deep brain stimulation device and derived the individualized safe RF condition under 3.0T MRI. This study presents a practical solution for the in-human safety assessment of implants during MRI, which can be beneficial for both clinical and research purposes.
... 32 33 Astrocyte-mediated neurovascular coupling signals could stimulate neurons, leading to a calcium efflux and release of vasoactive substances onto vessels. [34][35][36][37] Increasing evidence showed that astrocytes are major cell type affected by ultrasound. 18 38 At the same time, we observed significant changes of HMGB1 and CAMK2N1 in ischaemic mice after ultrasound only in astrocytes, but not in other cells, which intrigue us to conduct cellular level experiments and related validation in astrocytes. ...
Background
Low-intensity focused ultrasound stimulation (LIFUS) has been developed to enhance neurological repair and remodelling during the late acute stage of ischaemic stroke in rodents. However, the cellular and molecular mechanisms of neurological repair and remodelling after LIFUS in ischaemic stroke are unclear.
Methods
Ultrasound stimulation was treated in adult male mice 7 days after transient middle cerebral artery occlusion. Angiogenesis was measured by laser speckle imaging and histological analyses. Electromyography and fibre photometry records were used for synaptogenesis. Brain atrophy volume and neurobehaviour were assessed 0–14 days after ischaemia. iTRAQ proteomic analysis was performed to explore the differentially expressed protein. scRNA-seq was used for subcluster analysis of astrocytes. Fluorescence in situ hybridisation and Western blot detected the expression of HMGB1 and CAMK2N1.
Results
Optimal ultrasound stimulation increased cerebral blood flow, and improved neurobehavioural outcomes in ischaemic mice (p<0.05). iTRAQ proteomic analysis revealed that the expression of HMGB1 increased and CAMK2N1 decreased in the ipsilateral hemisphere of the brain at 14 days after focal cerebral ischaemia with ultrasound treatment (p<0.05). scRNA-seq revealed that this expression pattern belonged to a subcluster of astrocytes after LIFUS in the ischaemic brain. LIFUS upregulated HMGB1 expression, accompanied by VEGFA elevation compared with the control group (p<0.05). Inhibition of HMGB1 expression in astrocytes decreased microvessels counts and cerebral blood flow (p<0.05). LIFUS reduced CAMK2N1 expression level, accompanied by increased extracellular calcium ions and glutamatergic synapses (p<0.05). CAMK2N1 overexpression in astrocytes decreased dendritic spines, and aggravated neurobehavioural outcomes (p<0.05).
Conclusion
Our results demonstrated that LIFUS promoted angiogenesis and synaptogenesis after focal cerebral ischaemia by upregulating HMGB1 and downregulating CAMK2N1 in a subcluster of astrocytes, suggesting that LIFUS activated specific astrocyte subcluster could be a key target for ischaemic brain therapy.
... In addition, snRNA-seq analyses in vascular cells showed that mural cells (smooth muscle cells and pericytes) were activated, whereas vascular-associated astrocytes were suppressed in SwDI/TKO mice. Since these cells are involved in the regulation of neurovascular structure and functions (Takano et al., 2005;Hill et al., 2015;Sweeney, Ayyadurai and Zlokovic, 2016), transcriptomic changes in each of the cell types likely contributed to the pathological phenotype in SwDI/TKO mice. The results also demonstrate that lack of TREM2 not only affects microglia but also modifies the response of various other cells types in the brain. ...
Alzheimer's disease (AD) is a progressive neurodegenerative disease, and it is the most common cause of dementia worldwide. Recent genome-wide association studies (GWAS) identified TREM2 (triggering receptor expressed on myeloid cells 2) as one of the major risk factors for AD. TREM2 is a surface receptor expressed on microglia and largely mediates microglial functions and immune homeostasis in the brain. The functions of TREM2 in AD pathogenesis, including in the formation of the key pathology parenchymal amyloid-β (Aβ) plaques, have been investigated by introducing Trem2 deficiency in AD mouse models. However, the role of TREM2 in cerebrovascular amyloidosis, in particular cerebral amyloid angiopathy (CAA) remains unexplored. CAA features Aβ deposition along the cerebral vessels, signifying an intersection between AD and vascular dysfunction. Using a well-characterized CAA-prone, transgenic mouse model of AD, Tg-SwDI (SwDI), we found that loss of TREM2 led to a marked increase in overall Aβ load in the brain, but a dramatic decrease in CAA in microvessel-rich regions, along with reduced microglial association with CAA. Transcriptomic analysis revealed that in the absence of Trem2, microglia were activated but trapped in transition to the fully reactive state. Like microglia, perivascular macrophages were activated with upregulation of cell junction related pathways in Trem2-deficient SwDI mice. In addition, vascular mural cells and astrocytes exhibited distinct responses to Trem2 deficiency, contributing to the pathological changes in the brain of Trem2-null SwDI mice. Our study provides the first evidence that TREM2 differentially modulates parenchymal and vascular Aβ pathologies, which may have significant implications for both TREM2- and Aβ-targeting therapies for AD.
... The astrocytes support the neuronal function through their roles in potassium buffering and osmotic and glutamatergic homeostasis. The astrocyte end-feet also participate in the BBB formation and NVC through the release of COX-1 metabolites [132][133][134]. ...
Cerebral small vessel disease (cSVD) refers to the age-dependent pathological processes involving the brain small vessels and leading to vascular cognitive impairment, intracerebral hemorrhage, and acute lacunar ischemic stroke. Despite the significant public health burden of cSVD, disease-specific therapeutics remain unavailable due to the incomplete understanding of the underlying pathophysiological mechanisms. Recent advances in neuroimaging acquisition and processing capabilities as well as findings from cSVD animal models have revealed critical roles of several age-dependent processes in cSVD pathogenesis including arterial stiffness, vascular oxidative stress, low-grade systemic inflammation, gut dysbiosis, and increased salt intake. These factors interact to cause a state of endothelial cell dysfunction impairing cerebral blood flow regulation and breaking the blood brain barrier. Neuroinflammation follows resulting in neuronal injury and cSVD clinical manifestations. Impairment of the cerebral waste clearance through the glymphatic system is another potential process that has been recently highlighted contributing to the cognitive decline. This review details these mechanisms and attempts to explain their complex interactions. In addition, the relevant knowledge gaps in cSVD mechanistic understanding are identified and a systematic approach to future translational and early phase clinical research is proposed in order to reveal new cSVD mechanisms and develop disease-specific therapeutics.
... Astrocytic endfeet is crucial for the structure of the bloodbrain barrier and the regulation of cerebral blood flow [30,31]. Endfeet of cortical astrocytes almost fully cover the blood vessels by P20 [25]. ...
Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.
... Astrocytes, with their extensive blood vessel coverage and ability to sense neuronal activity, represent an optimal candidate cell type for mediating functional hyperemia. Indeed, astrocytic Ca 2+ elevation has been implicated in the modulation of local cerebral blood flow by multiple independent groups [11][12][13][14]. However, this role of astrocytic Ca 2+ has been questioned since IP 3 receptor type-2 knockout mice (IP 3 R2-KO), in which large astrocytic Ca 2+ elevations are diminished, display a similar extent of functional hyperemia [15][16][17]. ...
Activation of Gq-type G protein-coupled receptors (GPCRs) gives rise to large cytosolic Ca2+ elevations in astrocytes. Previous in vitro and in vivo studies have indicated that astrocytic Ca2+ elevations are closely associated with diameter changes in the nearby blood vessels, which astrocytes enwrap with their endfeet. However, the causal relationship between astrocytic Ca2+ elevations and blood vessel diameter changes has been questioned, as mice with diminished astrocytic Ca2+ signaling show normal sensory hyperemia. We addressed this controversy by imaging cortical vasculature while optogenetically elevating astrocyte Ca2+ in a novel transgenic mouse line, expressing Opto-Gq-type GPCR Optoα1AR (Astro-Optoα1AR) in astrocytes. Blue light illumination on the surface of the somatosensory cortex induced Ca2+ elevations in cortical astrocytes and their endfeet in mice under anesthesia. Blood vessel diameter did not change significantly with Optoα1AR-induced Ca2+ elevations in astrocytes, while it was increased by forelimb stimulation. Next, we labeled blood plasma with red fluorescence using AAV8-P3-Alb-mScarlet in Astro-Optoα1AR mice. We were able to identify arterioles that display diameter changes in superficial areas of the somatosensory cortex through the thinned skull. Photo-stimulation of astrocytes in the cortical area did not result in noticeable changes in the arteriole diameters compared with their background strain C57BL/6. Together, compelling evidence for astrocytic Gq pathway-induced vasodiameter changes was not observed. Our results support the notion that short-term (<10 s) hyperemia is not mediated by GPCR-induced astrocytic Ca2+ signaling.
... [129][130][131] In addition, the activity of glial cells plays a key role in neurovascular coupling and has been associated with distinct components of the hemodynamic signal. [132][133][134][135] Most striking are instances in which neuronal activity is decoupled from local blood flow, suggesting complex, highly dynamic coupling that can vary based on sensory and cognitive context. 125,127,128,132,[136][137][138][139][140] Indeed, rather than solely representing a spatially and temporally blurred proxy for neuronal activity, hemodynamic signals may be linked to other aspects of brain function by responding to and influencing neuromodulatory signals (neurotransmitters, neuropeptides, NO 2 ) and non-neuronal cells, such as glia. ...
The brain enables adaptive behavior via the dynamic coordination of diverse neuronal signals across spatial and temporal scales: from fast action potential patterns in microcircuits to slower patterns of distributed activity in brain-wide networks. Understanding principles of multiscale dynamics requires simultaneous monitoring of signals in multiple, distributed network nodes. Combining optical and electrical recordings of brain activity is promising for collecting data across multiple scales and can reveal aspects of coordinated dynamics invisible to standard, single-modality approaches. We review recent progress in combining opto- and electrophysiology, focusing on mouse studies that shed new light on the function of single neurons by embedding their activity in the context of brain-wide activity patterns. Optical and electrical readouts can be tailored to desired scales to tackle specific questions. For example, fast dynamics in single cells or local populations recorded with multi-electrode arrays can be related to simultaneously acquired optical signals that report activity in specified subpopulations of neurons, in non-neuronal cells, or in neuromodulatory pathways. Conversely, two-photon imaging can be used to densely monitor activity in local circuits while sampling electrical activity in distant brain areas at the same time. The refinement of combined approaches will continue to reveal previously inaccessible and under-appreciated aspects of coordinated brain activity.
... While astroglial endfeet are separated from endothelial cells and pericytes by the basal lamina, extensive signaling occurs between astrocytes, pericytes and endothelial cells [14][15][16][17][18] . Moreover, a recent study reported that a large portion of the astrocyte proteome is dedicated to astroglial endfeet, highlighting the complex nature of this cellular compartment 19 . ...
Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation.
... Plasma labeling is typically performed by intravenous injection of fluorescent tracers (e.g., Texas Red, Fluorescein Isothiocyanate) conjugated to large dextran (size 80 to 2,000 kDa), whereby the vasculature and the flow therein can be visualized after a few seconds. In combination with twophoton microscopy [3], fluorescent plasma tracers have enabled investigation of cerebral vessels in normal and disease model rodents [4][5][6][7][8][9]. Convenient as these tracers are, major limitations include tracer leakage within few hours from administration, stress of repeated injections for longer experiments and introduction of biases on blood flow due to a higher blood viscosity. ...
Albumin, a protein produced by liver hepatocytes, represents the most abundant protein in blood plasma. We have previously engineered a liver-targeting adeno-associated viral vector (AAV) that expresses fluorescent protein-tagged albumin to visualize blood plasma in mice. While this approach was versatile for imaging in adult mice, transgene expression vanishes when AAV is administered in neonates due to dilution of the episomal AAV genome in the rapidly growing liver. Here, we use CRISPR/Cas9 genome editing to insert the fluorescent protein mNeonGreen (mNG) gene into the albumin (Alb) locus of hepatocytes to produce fluorescently labeled albumin (Alb-mNG). We constructed a CRISPR AAV that includes ~1 kb homologous arms around Alb exon 14 to express Alb-mNG. Subcutaneous injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days. The expression levels of Alb-mNG were persistent for at least three months and were so robust that vasomotion and capillary blood flow could be assessed transcranially in early postnatal mice. This knock-in approach provides powerful means for micro- and macroscopic imaging of cerebral vascular dynamics in postnatal and adult mice.
... In organisms, calcium signals propagate through secondary messengers to cause intercellular calcium waves (ICW) that coordinate concerted action in whole tissues 8 . ICW play direct or indirect roles in processes ranging from muscle contraction 9 , potentiation of neuronal firing 10 , blood vessel dilation 11 , digestion 12 and breathing 13 . Dysfunctional calcium signalling contributes to disease states ranging from cancer to cardiovascular disease and neurodegenerative disorders 14,15 . ...
Intercellular calcium waves (ICW) are complex signalling phenomena that control many essential biological activities, including smooth muscle contraction, vesicle secretion, gene expression and changes in neuronal excitability. Accordingly, the remote stimulation of ICW could result in versatile biomodulation and therapeutic strategies. Here we demonstrate that light-activated molecular machines (MM)—molecules that perform mechanical work on the molecular scale—can remotely stimulate ICW. MM consist of a polycyclic rotor and stator that rotate around a central alkene when activated with visible light. Live-cell calcium-tracking and pharmacological experiments reveal that MM-induced ICW are driven by the activation of inositol-triphosphate-mediated signalling pathways by unidirectional, fast-rotating MM. Our data suggest that MM-induced ICW can control muscle contraction in vitro in cardiomyocytes and animal behaviour in vivo in Hydra vulgaris. This work demonstrates a strategy for directly controlling cell signalling and downstream biological function using molecular-scale devices.
... Whisker stimulation activates barrel cortex neurons along with an increase in local blood flow 4 , and the magnitude of functional hyperemia is determined by the stimulation parameters 5,6 . To validate our model and optimize our stimulation protocol, we applied different Article https://doi.org/10.1038/s41593-023-01327-2 ...
Functional hyperemia, also known as neurovascular coupling, is a phenomenon that occurs when neural activity increases local cerebral blood flow. Because all biological activity produces metabolic waste, we here sought to investigate the relationship between functional hyperemia and waste clearance via the glymphatic system. The analysis showed that whisker stimulation increased both glymphatic influx and clearance in the mouse somatosensory cortex with a 1.6-fold increase in periarterial cerebrospinal fluid (CSF) influx velocity in the activated hemisphere. Particle tracking velocimetry revealed a direct coupling between arterial dilation/constriction and periarterial CSF flow velocity. Optogenetic manipulation of vascular smooth muscle cells enhanced glymphatic influx in the absence of neural activation. We propose that impedance pumping allows arterial pulsatility to drive CSF in the same direction as blood flow, and we present a simulation that supports this idea. Thus, functional hyperemia boosts not only the supply of metabolites but also the removal of metabolic waste.
... In this case, astrocytic Ca 2þ signals are triggered by opening of P2X 1 purinoceptors; increase in cytosolic Ca 2þ activates phospholipase-D2, production of arachidonic acid, with subsequent activation of cyclooxygenase-1 (COX-1), and release of prostaglandin E 2, which, acting through EP4 receptors in pericytes, initiates dilatation of capillaries. 218 Functional hyperaemia mediated by astrocyte-derived prostaglandins, 219 as well as by eicosanoids, 220 was characterised in vivo in the cortex. It has to be noted, however, that the ability of capillaries to rapidly dilatate remains debatable, 207 while pericytes seem to control the capillary tone in a slow time scale. ...
... Astrocytes differ from neurons both in function and morphology: they do not fire action potentials and are characterized by a complex surface of nanoscopic processes (Verkhratsky & Nedergaard, 2018). Astrocytes have specialized processes that envelop neuronal synapses (Bushong et al., 2002), endfeet that connect them to vasculature (Takano et al., 2006), and differential spatial organization of intracellular components (Calì et al., 2019). Like neurons, whose branching structure influences the biophysical properties of neuronal networks (Cuntz et al., 2007), astrocytes have unique branching geometries that differ from astrocyte subtype and brain region to other (Khakh & Sofroniew, 2015). ...
Understanding functions of astrocytes can be greatly enhanced by building and simulating computational models that capture their morphological details. Novel computational tools enable utilization of existing morphological data of astrocytes and building models that have appropriate level of details for specific simulation purposes. In addition to analyzing existing computational tools for constructing, transforming, and assessing astrocyte morphologies, we present here the CellRemorph toolkit implemented as an add-on for Blender, a 3D modeling platform increasingly recognized for its utility for manipulating 3D biological data. To our knowledge, CellRemorph is the first toolkit for transforming astrocyte morphologies from polygonal surface meshes into adjustable surface point clouds and vice versa, precisely selecting nanoprocesses, and slicing morphologies into segments with equal surface areas or volumes. CellRemorph is an open-source toolkit under the GNU General Public License and easily accessible via an intuitive graphical user interface. CellRemorph will be a valuable addition to other Blender add-ons, providing novel functionality that facilitates the creation of realistic astrocyte morphologies for different types of morphologically detailed simulations elucidating the role of astrocytes both in health and disease.
... The involvement of astrocytes in NVC was supported by another study performed in the somatosensory cortex (SSCx) of anesthetized adult mice, where Ca 2+ uncaging in astrocyte elicited a local hyperemic response that was sensitive to specific COX-1 inhibitors. Similarly, triggering neuronal activity via extracellular electrical stimulation induced Ca 2+ responses in astrocytes and resulted in arteriole vasodilatation, significantly reduced by mGluR antagonists and COX-1 inhibitors (Takano et al., 2006). ...
The brain is a highly energy demanding organ, which accounts in humans for the 20% of total energy consumption at resting state although comprising only 2% of the body mass. The necessary delivery of nutrients to brain parenchyma is ensured by the cerebral circulatory system, through the exchange of glucose and oxygen (O2) at the capillary level. Notably, a tight spatial and temporal correlation exists between local increases in neuronal activity and the subsequent changes in regional cerebral blood flow. The recognized concept of neurovascular coupling (NVC), also named functional hyperemia, expresses this close relationship and stands at the basis of the modern functional brain imaging techniques. Different cellular and molecular mechanisms have been proposed to mediate this tight coupling. In this context, astrocytes are ideally positioned to act as relay elements that sense neuronal activity through their perisynaptic processes and release vasodilator agents at their endfeet in contact with brain parenchymal vessels. Two decades after the astrocyte involvement in neurovascular coupling has been proposed, we here review the experimental evidence that contributed to unraveling the molecular and cellular mechanisms underlying cerebral blood flow regulation. While traveling through the different controversies that moved the research in this field, we keep a peculiar focus on those exploring the role of astrocytes in neurovascular coupling and conclude with two sections related to methodological aspects in neurovascular research and to some pathological conditions resulting in altered neurovascular coupling.
... Astrocytes are central nervous system (CNS)-restricted glial cells that tile the entire CNS [1][2][3], and as an integral part of the blood-brain barrier (BBB), they regulate the blood flow through the brain [4]. Their best known functions are to regulate synaptic formation and neuronal activity. ...
Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs.
... Astrocytes perform this vital function via spontaneous Ca 2+ signals within endfeet that completely ensheathe capillaries 31,32 . Indeed, Ca 2+ signals in astrocytic endfeet can alter vasoconstriction and dilation 32,33 , mediate vascular repair after injury 34 , regulate brain volume 35 , alter aging-related cognitive behavior via intracellular IP3R2specific Ca 2+ signals 36 , as well as maintain NVU coupling 37 . Thus, at a functional level, astrocytic endfoot Ca 2+ signals govern critical aspects of BBB integrity and NVU function. ...
Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases the risk for neurodegeneration. Among various cells that participate in BBB and NVU function, calcium signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with altered calcium signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed GCaMP6f in DLS astrocytes of young (3-4 months), middle-aged (12-15 months) and aging (20-30 months) mice. Compared to endfeet in young mice, DLS endfeet in aging mice demonstrated decreased calreticulin expression, and alterations to both spontaneous membrane-associated and mitochondrial calcium signals. While young mice required both extracellular and endoplasmic reticulum calcium sources for endfoot signals, middle-aged and aging mice showed heavy dependence on endoplasmic reticulum calcium. Thus, astrocytic endfeet show significant changes in calcium buffering and sources throughout the lifespan, which is important for understanding mechanisms by which aging impairs the BBB and NVU.
... Nevertheless, they play multiple roles in the CNS physiology. Within the human brain, astrocytes have different essential roles including support and guidance for migrating neurons, regulation of the cerebral blood flow [4] and maintenance of neurotransmission homeostasis [5]. In addition, astrocytes are involved in the inflammatory response, in blood brain barrier formation and in synapse regulation [6] and plasticity [7]. ...
Astrocytes coordinate several homeostatic processes of the central nervous system and play essential roles for normal brain development and response to disease conditions. Protocols for the conversion of human induced pluripotent stem cells (hiPSCs) into mature astrocytes have opened to the generation of in vitro systems to explore astrocytes’ functions in living human cell contexts and patient-specific settings. In this study, we present an optimized monolayer procedure to commit hiPSC-derived cortical progenitors into enriched populations of cortical astrocyte progenitor cells (CX APCs) that can be further amplified and efficiently differentiated into mature astrocytes. Our optimized system provides a valid tool to explore the role of these cells in neurodevelopmental and neuropsychiatric diseases, opening it up to applications in drug development and biomarkers discovery/validation.
... Astrocyte endfeet also express proteins related to blood flow regulation (Figure 1e). They contain phospholipase A2 (PLA2), phospholipase D2 (PLD2), cyclooxygenase 1 (COX1), and prostaglandin E2 synthase (PGES), which synthesize the vasodilatory agent PGE 2 (Gordon et al. 2008, Mishra et al. 2016, Takano et al. 2006, and the cytochrome P450 ω-hydroxylase 4A (CYP4A), which synthesizes vasoconstrictive 20-hydroxyeicosatetraenoic acid (20-HETE) (Gonzalez-Fernandez et al. 2020). Cytochrome P450 2C11 (CYP2C11) epoxygenase produces epoxyeicosatrienoic acids (EETs) and is strongly expressed in endfeet on arterioles to induce vasodilation (Peng et al. 2002) but is completely absent in endfeet on capillaries (Mishra et al. 2016), suggesting possible vascular segment-specific heterogeneity. ...
Astrocyte endfeet enwrap the entire vascular tree within the central nervous system, where they perform important functions in regulating the blood-brain barrier (BBB), cerebral blood flow, nutrient uptake, and waste clearance. Accordingly, astrocyte endfeet contain specialized organelles and proteins, including local protein translation machinery and highly organized scaffold proteins, which anchor channels, transporters, receptors, and enzymes critical for astrocyte-vascular interactions. Many neurological diseases are characterized by the loss of polarization of specific endfoot proteins, vascular dysregulation, BBB disruption, altered waste clearance, or, in extreme cases, loss of endfoot coverage. A role for astrocyte endfeet has been demonstrated or postulated in many of these conditions. This review provides an overview of the development, composition, function, and pathological changes of astrocyte endfeet and highlights the gaps in our knowledge that future research should address.
Expected final online publication date for the Annual Review of Neuroscience, Volume 46 is July 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Astrocytes, as an abundant resident cell type in the CNS, play crucial roles in managing homeostasis by supplying neurotrophic support and sustaining synaptic integrity and function [31][32][33][34][35]. Astrocytes become reactive with a significant change in morphology, loss of normal astrocyte function, and alteration of gene expression under pathological circumstances [16,36]. In the last decades, the biological and physiological importance of astrocytes has come into the limelight [37][38][39][40]. Thus, determining the mechanisms underlying astrocyte activity could unveil their potential therapeutic potential for treating neurological disorders. ...
Background
Limited progress in terms of an effective treatment for spinal cord injury (SCI) emphasizes the urgent need for novel therapies. As a vital central nervous system component, the resident astrocytes play crucial roles in regulating recovery after SCI. In this study, recovery after SCI was compared following the transplantation of either A1 or A2 astrocytes. A1 astrocytes are harmful as they upregulate the neurotoxic classical complement cascade genes. Conversely, A2 astrocytes are characterized as neuroprotective as they upregulate the production of many neurotrophic factors.
Methods
We used different supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4 to generate A1 and A2 astrocytes. We detected the influence of astrocytes on neurons by co-culturing A1 and A2 astrocytes with neurons. We transplanted astrocytes into the lesion site of the spinal cord and assessed lesion progression, neural restoration, glia formation and locomotor recovery.
Results
Astrocytes were polarized into A1 and A2 phenotypes following culture in the supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4, respectively. Furthermore, co-culturing A2 astrocytes with neurons significantly suppressed glutamate-induced neuronal apoptosis and promoted the degree of neuron arborization. Transplantation of these A2 astrocytes into the lesion site of the spinal cord of mice significantly improved motor function recovery, preserved spared supraspinal pathways, decreased glia scar deposition, and increased neurofilament formation at the site of injury compared to the transplantation of A1 astrocytes. Additionally, enhanced A2 astrocytes with potentially beneficial A2-like genes were also detected in the A2 group. Moreover, luxol fast blue staining and electron microscopy indicated increased preservation of myelin with organized structure after transplantation of A2 astrocytes than of A1 astrocytes.
Conclusions
A2 astrocyte transplantation could be a promising potential therapy for SCI.
Graphical Abstract
... Astrocytes are the most abundant glial cells which display many metabolic functions. Particularly they regulate ion and water homeostasis [51,52], blood flow to meet neuronal energy demand [53,54], supplying the building blocks of neurotransmitters [55] and energy substrates to neurons [1,56]. Additionally, astrocytes participate in synapse formation, elimination, functional maturation, and plasticity during brain development and in adulthood [51,57] as well as brain repair after injury [58]. ...
The brain is an organ that consumes a lot of energy. In the brain, energy is required for synaptic transmission, numerous biosynthetic processes and axonal transport in neurons, and for many supportive functions of glial cells. The main source of energy in the brain is glucose and to a lesser extent lactate and ketone bodies. ATP is formed at glucose catabolism via glycolysis and oxidative phosphorylation in mitochondrial electron transport chain (ETC) within mitochondria being the main source of ATP. With age, brain's energy metabolism is disturbed, involving a decrease in glycolysis and mitochondrial dysfunction. The latter is accompanied by intensified generation of reactive oxygen species (ROS) in ETC leading to oxidative stress. Recently, we have found that crucial changes in energy metabolism and intensity of oxidative stress in the mouse brain occur in middle age with minor progression in old age. In this review, we analyze the metabolic changes and functional causes that lead to these changes in the aging brain.
Neuroscientific research on emotion has developed dramatically over the past decade. The cognitive neuroscience of human emotion, which has emerged as the new and thriving area of 'affective neuroscience', is rapidly rendering existing overviews of the field obsolete. This handbook provides a comprehensive, up-to-date and authoritative survey of knowledge and topics investigated in this cutting-edge field. It covers a range of topics, from face and voice perception to pain and music, as well as social behaviors and decision making. The book considers and interrogates multiple research methods, among them brain imaging and physiology measurements, as well as methods used to evaluate behavior and genetics. Editors Jorge Armony and Patrik Vuilleumier have enlisted well-known and active researchers from more than twenty institutions across three continents, bringing geographic as well as methodological breadth to the collection. This timely volume will become a key reference work for researchers and students in the growing field of neuroscience.
Neuroscientific research on emotion has developed dramatically over the past decade. The cognitive neuroscience of human emotion, which has emerged as the new and thriving area of 'affective neuroscience', is rapidly rendering existing overviews of the field obsolete. This handbook provides a comprehensive, up-to-date and authoritative survey of knowledge and topics investigated in this cutting-edge field. It covers a range of topics, from face and voice perception to pain and music, as well as social behaviors and decision making. The book considers and interrogates multiple research methods, among them brain imaging and physiology measurements, as well as methods used to evaluate behavior and genetics. Editors Jorge Armony and Patrik Vuilleumier have enlisted well-known and active researchers from more than twenty institutions across three continents, bringing geographic as well as methodological breadth to the collection. This timely volume will become a key reference work for researchers and students in the growing field of neuroscience.
Neuroscientific research on emotion has developed dramatically over the past decade. The cognitive neuroscience of human emotion, which has emerged as the new and thriving area of 'affective neuroscience', is rapidly rendering existing overviews of the field obsolete. This handbook provides a comprehensive, up-to-date and authoritative survey of knowledge and topics investigated in this cutting-edge field. It covers a range of topics, from face and voice perception to pain and music, as well as social behaviors and decision making. The book considers and interrogates multiple research methods, among them brain imaging and physiology measurements, as well as methods used to evaluate behavior and genetics. Editors Jorge Armony and Patrik Vuilleumier have enlisted well-known and active researchers from more than twenty institutions across three continents, bringing geographic as well as methodological breadth to the collection. This timely volume will become a key reference work for researchers and students in the growing field of neuroscience.
Neurovascular coupling (NVC) ensures sufficient and targeted blood flow during increased neuronal activity. Astrocytic participation in NVC has long been debated, likely due to the intricacy of the intracellular Ca2+ fluxes and the diversity of their regulatory capacities. As astrocyte signaling changes with brain states, we focused on their involvement in voluntary sensing in freely behaving mice. We used 2-photon microscopy to record cellular and vascular activity in the whisker barrel cortex of awake head-fixed animals. The NVC initiated by volitional whisking in the resting mouse was compared to the whisking preceding locomotion and experimenter-evoked whisker deflections. We developed an analysis method to detect early, subcellular astrocytic activity and found it corresponded with neuronal and vascular responses under all three conditions. After the depletion of noradrenaline (NA), the early astrocytic Ca2+ response to volitional whisking was only moderately reduced and primarily in astrocytic processes closest to the blood vessels. Meanwhile, the dilation of 1st order capillaries was also reduced. Together, these findings demonstrate significant disruptions in the focal regulation of cerebral blood flow, potentially limiting the sustenance of activated neurons. This disruption appeared to translate into behavioral aberrations, as NA-depleted mice exhibited an extended period of exploratory whisking prior to locomotion. Remarkably, NA-depletion did not alter cellular or blood flow responses to locomotion or experimenter-evoked whisking. Our study confirms an astrocytic contribution to NVC, which is relevant during volitional sensing. It also suggests that self-directed sensory processing depends on an appropriate NVC response, which itself depends on NA and astrocyte activity.
Mild cognitive impairment (MCI) is common in people with chronic kidney disease (CKD) and its prevalence increases with progressive loss of kidney function. MCI is characterized by a decline in cognitive performance greater than expected for an individual age and education level but with minimal impairment of instrumental activities of daily living. Deterioration can affect one or several cognitive domains (attention, memory, executive functions, language, and perceptual motor or social cognition). Given the increasing prevalence of kidney disease, more and more people with CKD will also develop MCI causing an enormous disease burden for these individuals, their relatives and society. However, the underlying pathomechanisms are poorly understood and current therapies mostly aim at supporting patients in their daily life. This illustrates the urgent need to elucidate the pathogenesis, and potential therapeutic targets and test novel therapies in appropriate preclinical models. Here, we will outline the necessary criteria for experimental modelling of cognitive disorders in CKD. We discuss the use of mice, rats and zebrafish as model systems and present valuable techniques through which kidney function and cognitive impairment can be assessed in this setting. Our objective is to enable researchers to overcome hurdles and accelerate preclinical research aimed at improving therapy of people with CKD and MCI.
Neurovascular coupling (NVC) is the functional hyperemia of the brain responding to local neuronal activity. It is mediated by astrocytes and affected by subcortical ascending pathways in the cortex that convey information, such as sensory stimuli and the animal condition. Here, we investigate the influence of the raphe serotonergic system, a subcortical ascending arousal system in animals, on the modulation of cortical NVC and cerebral blood flow (CBF). Raphe serotonergic neurons were optogenically activated for 30 s, which immediately awakened the mice from non-rapid eye movement sleep. This caused a biphasic cortical hemodynamic change: a transient increase for a few seconds immediately after photostimulation onset, followed by a large progressive decrease during the stimulation period. Serotonergic neuron activation increased intracellular Ca ²⁺ levels in cortical pyramidal neurons and astrocytes, demonstrating its effect on the NVC components. Pharmacological inhibition of cortical neuronal firing activity and astrocyte metabolic activity had small hypovolemic effects on serotonin-induced biphasic CBF changes, while blocking 5-HT 1B receptors expressed primarily in cerebral vasculature attenuated the decreasing CBF phase. This suggests that serotonergic neuron activation leading to animal awakening could allow the NVC to exert a hyperemic function during a biphasic CBF response, with a predominant decrease in the cortex.
Dynamic changes in astrocyte Ca²⁺ are recognized as contributors to functional hyperemia, a critical response to increased neuronal activity mediated by a process known as neurovascular coupling (NVC). Although the critical role of glutamatergic signaling in this process has been extensively investigated, the impact of behavioral state, and the release of behavior-associated neurotransmitters, such as norepinephrine and serotonin, on astrocyte Ca²⁺ dynamics and functional hyperemia have received less attention. We used two-photon imaging of the barrel cortex in awake mice to examine the role of noradrenergic and serotonergic projections in NVC. We found that both neurotransmitters facilitated sensory stimulation-induced increases in astrocyte Ca²⁺. Interestingly, while ablation of serotonergic neurons reduced sensory stimulation-induced functional hyperemia, ablation of noradrenergic neurons caused both attenuation and potentiation of functional hyperemia. Our study demonstrates that norepinephrine and serotonin are involved in modulating sensory stimulation-induced astrocyte Ca²⁺ elevations and identifies their differential effects in regulating functional hyperemia.
Components that comprise our brain parenchymal and cerebrovascular structures provide a homeostatic environment for proper neuronal function to ensure normal cognition. Cerebral insults (e.g., ischemia, microbleeds, infection) alter cellular structures and physiologic processes within the neurovascular unit and contribute to cognitive dysfunction. COVID-19 has posed significant complications during acute and convalescent stages on multiple organ systems, including the brain. Cognitive impairment is a prevalent complication in COVID-19 patients, irrespective of severity of acute SARS-CoV-2 infection. Moreover, overwhelming evidence from in vitro, preclinical, and clinical studies have reported SARS-CoV-2-induced pathologies in components of the neurovascular unit that associate with cognitive impairment. Neurovascular unit disruption alters the neurovascular coupling response, a critical mechanism that regulates cerebromicrovascular blood flow to meet the energetic demands of locally active neurons. Normal cognitive processing is achieved through the neurovascular coupling response and involves the coordinated action of brain parenchymal cells (i.e., neurons, glia) and cerebrovascular cell types (i.e., endothelia, smooth muscle cells, pericytes). However, current work on COVID-19-induced cognitive impairment has yet to investigate disruption of neurovascular coupling as a causal factor. Hence, in this review, we aim to describe SARS-CoV-2’s effects on the neurovascular unit and how they can impact neurovascular coupling and contribute to cognitive decline in acute and convalescent stages of the disease. Additionally, we explore potential therapeutic interventions to mitigate COVID-19-induced cognitive impairment. Given the great impact of cognitive impairment associated with COVID-19 on both individuals and public health, the necessity for a coordinated effort from fundamental scientific research to clinical application becomes imperative. This integrated endeavor is crucial for mitigating the cognitive deficits induced by COVID-19 and its subsequent burden in this especially vulnerable population.
The vasculature of the spinal axis reflects the segmental and radicular arteries, which variably supply multiple neuraxial segments. The parenchymal arterioles, capillaries, and venules penetrate the parenchyma through the perivascular Virchow-Robin spaces, and the venous outflow exits through the intradural and extradural vessels and plexi. Species differences in this perfusion structure are compared and contrasted. Specific discussions of rostrocaudal flow patterns, spinal watershed zones, and dorsal root ganglion perfusion are provided. Intraparenchymal perfusion patterns are described along with parenchymal vascular density. The physiology of spinal cord blood flow regulation and perfusion autoregulation, as well as ionic and neurohormonal effects, are discussed in the context of neurogenic (afferent, sympathetic, and parasympathetic) components of neurovascular regulation.
Increased neurotrophic support, including insulin-like growth factor I (IGF-I), is an important aspect of the adaptive response to ischemic insult. However, recent findings indicate that the IGF-I receptor (IGF-IR) in neurons plays a detrimental role in the response to stroke. Thus, we investigated the role of astrocytic IGF-IR on ischemic insults using tamoxifen-regulated Cre deletion of IGF-IR in glial fibrillary acidic protein (GFAP) astrocytes, a major cellular component in the response to injury. Ablation of IGF-IR in astrocytes (GFAP-IGF-IR KO mice) resulted in larger ischemic lesions, greater blood-brain-barrier disruption and more deteriorated sensorimotor coordination. RNAseq detected increases in inflammatory, cell adhesion and angiogenic pathways, while the expression of various classical biomarkers of response to ischemic lesion were significantly increased at the lesion site compared to control littermates. While serum IGF-I levels after injury were decreased in both control and GFAP-IR KO mice, brain IGF-I mRNA expression show larger increases in the latter. Further, greater damage was also accompanied by altered glial reactivity as reflected by changes in the morphology of GFAP astrocytes, and relative abundance of ionized calcium binding adaptor molecule 1 (Iba 1) microglia. These results suggest a protective role for astrocytic IGF-IR in the response to ischemic injury.
Layer-dependent functional magnetic resonance imaging (fMRI) offers a compelling avenue for investigating directed functional connectivity (FC). To construct a comprehensive map of brain-wide directed FC, several technical criteria must be met, including sub-mm spatial resolution, adequate temporal resolution, functional sensitivity, global brain coverage, and high spatial specificity. Although gradient echo (GE)–based echo planar imaging (EPI) is commonly used for rapid fMRI acquisition, it faces significant challenges due to the draining-vein effect, particularly when utilizing blood oxygen level-dependent (BOLD) contrast. In this study, we mitigated this effect by incorporating velocity-nulling (VN) gradients into a GE-BOLD fMRI sequence, opting for a 3T magnetic field strength over 7T. We also integrated several advanced techniques, such as simultaneous multi-slice (SMS) acceleration and NORDIC denoising, to enhance temporal resolution, spatial coverage, and signal sensitivity. Collectively, the VN fMRI method exhibited notable spatial specificity, as evidenced by the identification of double-peak activation patterns within the primary motor cortex (M1) during a finger-tapping task. Additionally, the technique demonstrated BOLD sensitivity in the lateral geniculate nucleus (LGN). Furthermore, our VN fMRI technique displayed superior robustness when compared to conventional fMRI approaches across participants. Our findings of directed FC elucidate several layer-specific functional relationships between different brain regions and align closely with existing literature. Given the widespread availability of 3T scanners, this technical advancement has the potential for significant impact across multiple domains of neuroscience research.
Understanding the roles of astrocytic calcium signaling in multiple brain regulatory mechanisms including metabolism, blood flow, neuromodulation and neuroinflammation has remained one of the enduring challenges in glial biology. To delineate astrocytic contribution from concurrent neuronal activity, it is vital to establish robust control and manipulate astrocytes using a technique like optogenetics due to its high cellular specificity and temporal resolution. Lack of an experimental paradigm to induce controlled calcium signaling in astrocytes has hindered progress in the field. To address this, i n this study, we systematically characterize and identify light stimulation paradigms for inducing regulated, on-demand increases in astrocytic calcium in cortical astrocytes in MlC1-ChR2(C128S)-EYFP mice. We identified paradigms 20%, 40% and 60% (of T = 100s) to elicit robust calcium responses upon multiple stimulations, while the 95% paradigm exhibited a response only during the first stimulation. We also quantified several parameters, including peak height, Full Width Half Maximum (FWHM), and latencies, and observe that the 20% paradigm has the highest peak ΔF/F 0 among the paradigms across all stimulations and the lowest FWHM during the first stimulation. Overall, the 20% paradigm is a favorable choice for eliciting robust astrocytic calcium responses in astrocytes while performing multiple stimulations.
Astrocytes are important regulators of blood flow and play a key role in the response to injury and disease in the central nervous system (CNS). Despite having an understanding that structural changes to these cells have consequences for local neurovascular physiology, individual astrocyte morphology remains largely unexplored in the retina. Here, we used MORF3 mice to capture full membranous morphology for over fifteen hundred individual astrocytes in the mouse retina, a highly metabolically active component of the CNS. We demonstrate that retinal astrocytes have been misrepresented as stellate in morphology due to marker use like GFAP and S100β which underestimates cell complexity. We also find that astrocytes contain recurring morphological motifs which are predictive of the underlying neurovascular architecture of the inner retina and suggestive of function. These motifs predict fine sampling and integration of retinal ganglion cell electrical activity with consequences for blood flow regulation. Additionally, our data shows that astrocytes participate in neurovascular interactions to a much greater degree than currently reported. 100% of cells contact the vasculature through one of three mutually exclusive classes of connections. Similarly, 100% of cells contact some neuronal element, be it an RGC axon or soma. Finally, we report that astrocyte morphology depends on retinal eccentricity, with cells appearing compressed near the nerve head and in the periphery. These results reveal a large degree of astrocyte morphological complexity that informs their contribution to neurovascular coupling in the retina.
Astrocytes are diverse brain cells that form large networks communicating via gap junctions and chemical transmitters. Despite recent advances, the functions of astrocytic networks in information processing in the brain are not fully understood. In culture, brain slices, and in vivo, astrocytes, and neurons grow in tight association, making it challenging to establish whether signals that spread within astrocytic networks communicate with neuronal groups at distant sites, or whether astrocytes solely respond to their local environments. A multi‐electrode array (MEA)‐based device called AstroMEA is designed to separate neuronal and astrocytic networks, thus allowing to study the transfer of chemical and/or electrical signals transmitted via astrocytic networks capable of changing neuronal electrical behavior. AstroMEA demonstrates that cortical astrocytic networks can induce a significant upregulation in the firing frequency of neurons in response to a theta‐burst charge‐balanced biphasic current stimulation (5 pulses of 100 Hz × 10 with 200 ms intervals, 2 s total duration) of a separate neuronal‐astrocytic group in the absence of direct neuronal contact. This result corroborates the view of astrocytic networks as a parallel mechanism of signal transmission in the brain that is separate from the neuronal connectome. Translationally, it highlights the importance of astrocytic network protection as a treatment target.
Cognitive decline has emerged as one of the greatest health threats of old age. Meanwhile, aging is the primary risk factor for Alzheimer's disease (AD) and other prevalent neurodegenerative disorders. Developing therapeutic interventions for such conditions demands a greater understanding of the processes underlying normal and pathological brain aging. Despite playing an important role in the pathogenesis and incidence of disease, brain aging has not been well understood at a molecular level. Recent advances in the biology of aging in model organisms, together with molecular- and systems-level studies of the brain, are beginning to shed light on these mechanisms and their potential roles in cognitive decline. This chapter seeks to integrate the knowledge about the neurological mechanisms of age-related cognitive changes that underlie aging.
In this review, we first describe the current understanding of glial-mediated vascular function affecting the role of the blood-brain barrier (BBB) in central nervous system (CNS) disorders. BBB, mainly composed of glial and endothelial cells (ECs), is the protective structure that orchestrates the transport of substances, including ions, molecules, and cells from brain vessels into or out of the CNS. Then, we display the multiple communication between glial and vascular function based on angiogenesis, vascular wrapping, and blood perfusion in the brain. Glial can support microvascular ECs to form a blood network connecting to neurons. Astrocytes, microglia, and oligodendrocytes are the common types of glial surrounding the brain vessel. Glial-vessel interaction is required for the permeability and integrity of BBB. Glial cells surrounding the cerebral blood vessels can transmit communication signals to ECs and regulate the activity of vascular endothelial growth factor (VEGF) or Wnt-dependent endothelial angiogenesis mechanism. In addition, these glial cells monitor the blood flow in the brain via Ca2+/K+-dependent pathways. Finally, we provide a potential research direction for the glial-vessel axis in CNS disorders. Microglial activation can trigger astrocyte activation, which suggests that microglia-astrocyte interaction may play a key role in monitoring cerebral blood flow. Thus, microglia-astrocyte interaction can be the key point of follow-up studies focusing on the microglia-blood mechanism. More investigations focus on the mechanism of how oligodendrocyte progenitor cells communicate and interact with ECs. The direct role of oligodendrocytes in modulating vascular function needs to be explored in the future.
Networks of neurons are the primary substrate of information processing. Conversely, blood vessels in the brain are generally viewed to have physiological functions unrelated to information processing, such as the timely supply of oxygen, and other nutrients to the neural tissue. However, recent studies have shown that cerebral microvessels, like neurons, exhibit tuned responses to sensory stimuli. Tuned neural responses to sensory stimuli may be enhanced with experience-dependent Hebbian plasticity and other forms of learning. Hence it is possible that the microvascular network might also be subject to some form of competitive learning rules during early postnatal development such that its fine-scale structure becomes optimized for metabolic delivery to a given neural micro-architecture. To explore the possibility of adaptive lateral interactions and tuned responses in cerebral microvessels, we modeled the cortical neurovascular network by interconnecting two laterally connected self-organizing networks. The afferent and lateral connections of the neural and vascular networks were defined by trainable weights. By varying the topology of lateral connectivity in the vascular network layer, we observed that the partial correspondence of feature selectivity between neural and hemodynamic responses could be explained by lateral coupling across local blood vessels such that the central domain receives an excitatory drive of more blood flow and a distal surrounding region where blood flow is reduced. Critically, our simulations suggest a new role for feedback from the vascular to the neural network because the radius of vascular perfusion determines whether the cortical neural map develops into a clustered vs. salt-and-pepper organization.
Interoception is the process by which the nervous system regulates internal functions to achieve homeostasis. The role of neurons in interoception has received considerable recent attention, but glial cells also contribute. Glial cells can sense and transduce signals including osmotic, chemical, and mechanical status of extracellular milieu. Their ability to dynamically communicate "listening" and "talking" to neurons is necessary to monitor and regulate homeostasis and information integration in the nervous system. This review introduces the concept of "Glioception" and focuses on the process by which glial cells sense, interpret and integrate information about the inner state of the organism. Glial cells are ideally positioned to act as sensors and integrators of diverse interoceptive signals and can trigger regulatory responses via modulation of the activity of neuronal networks, both in physiological and pathological conditions. We believe that understanding and manipulating glioceptive processes and underlying molecular mechanisms provide a key path to develop new therapies for the prevention and alleviation of devastating interoceptive dysfunctions, among which pain is emphasized here with more focused details.
Ischemic stroke is one of the most common causes of mortality and disability worldwide. However, treatment efficacy and the progress of research remain unsatisfactory. As the critical support system and essential components in neurovascular units, glial cells and blood vessels (including the blood-brain barrier) together maintain an optimal microenvironment for neuronal function. They provide nutrients, regulate neuronal excitability, and prevent harmful substances from entering brain tissue. The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis, supporting neuronal function, and reacting to injuries. However, most studies have focused on postmortem animals, which inevitably lack critical information about the dynamic changes that occur after ischemic stroke. Therefore, a high-precision technique for research in living animals is urgently needed. Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions. Two-photon fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure, information on multicellular component interactions, and provide images of structure and function in the cranial window. This technique shifts the existing research paradigm from static to dynamic, from flat to stereoscopic, and from single-cell function to multicellular intercommunication, thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain. In this review, we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy, highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain's support systems. We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.
The prostanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) is expressed in selected cerebral cortical neurons and is involved in synaptic signaling. We sought to determine whether COX-2 participates in the increase in cerebral blood flow produced by synaptic activity in the somatosensory cortex. In anesthetized mice, the vibrissae were stimulated mechanically, and cerebral blood flow was recorded in the contralateral somatosensory cortex by a laser-Doppler probe. We found that the COX-2 inhibitor NS-398 attenuates the increase in somatosensory cortex blood flow produced by vibrissal stimulation. Furthermore, the flow response was impaired in mice lacking the COX-2 gene, whereas the associated increase in whisker-barrel cortex glucose use was not affected. The increases in cerebral blood flow produced by hypercapnia, acetylcholine, or bradykinin were not attenuated by NS-398, nor did they differ between wild-type and COX-2 null mice. The findings provide evidence for a previously unrecognized role of COX-2 in the mechanisms coupling synaptic activity to neocortical blood flow and provide an insight into one of the functions of constitutive COX-2 in the CNS.
Analysis of the spatiotemporal coupling between neuronal activity and cerebral blood flow requires the precise measurement of the dynamics of RBC flow in individual capillaries that irrigate activated neurons. Here, we use two-photon microscopy in vivo to image individual RBCs in glomerular capillaries in the rat dorsal olfactory bulb. We find that odor stimulation evokes capillary vascular responses that are odorant- and glomerulus-specific. These responses consist of increases as well as decreases in RBC flow, both resulting from independent changes in RBC velocity or linear density. Finally, measuring RBC flow with micrometer spatial resolution and millisecond temporal resolution, we demonstrate that, in olfactory bulb superficial layers, capillary vascular responses precisely outline regions of synaptic activation.
There are multiple mechanisms by which adenine nucleotides can be released into the extracellular space in brain. Adenine nucleotides are converted extracellularly to adenosine, which then acts on adenosine receptors to elicit physiological responses, but the rate at which this conversion takes place is unknown. In the present experiments, adenine nucleotides were applied to individual hippocampal neurons, and the subsequent activation of a postsynaptic K+ conductance by adenosine A1 receptors was used to determine the rate of adenosine formation. None of the adenine nucleotides tested (cAMP, AMP, ADP, and ATP) activated A1 receptors directly at the concentrations tested (</=200 microM). AMP, ADP, and ATP were all rapidly converted to adenosine, with a T1/2 for ATP conversion to adenosine of approximately 200 msec, and the last step in this pathway (transformation of AMP to adenosine by 5'-nucleotidase) seems to be the rate-limiting step. As we have reported previously, cAMP is converted to adenosine as well, but on a much slower time scale than any of the other nucleotides tested. These experiments demonstrate that fast, localized release of AMP, ADP, or ATP can result in a transient activation of adenosine receptors but that this is unlikely to occur with cAMP. The existence of a highly active ecto-nucleotidase pathway in brain provides a mechanism for the rapid generation of adenosine after the release of adenine nucleotides into the extracellular space.
Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca2+ mobilization and was inhibited by Cl- channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl- channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.
The prostanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) is expressed in selected cerebral cortical neurons and is involved in synaptic signaling. We sought to determine whether COX-2 participates in the increase in cerebral blood flow produced by synaptic activity in the somatosensory cortex. In anesthetized mice, the vibrissae were stimulated mechanically, and cerebral blood flow was recorded in the contralateral somatosensory cortex by a laser-Doppler probe. We found that the COX-2 inhibitor NS-398 attenuates the increase in somatosensory cortex blood flow produced by vibrissal stimulation. Furthermore, the flow response was impaired in mice lacking the COX-2 gene, whereas the associated increase in whisker-barrel cortex glucose use was not affected. The increases in cerebral blood flow produced by hypercapnia, acetylcholine, or bradykinin were not attenuated by NS-398, nor did they differ between wild-type and COX-2 null mice. The findings provide evidence for a previously unrecognized role of COX-2 in the mechanisms coupling synaptic activity to neocortical blood flow and provide an insight into one of the functions of constitutive COX-2 in the CNS.
Astrocytes respond to chemical, electrical and mechanical stimuli with transient increases in intracellular calcium concentration ([Ca2+]i). We now show that astrocytes in situ display intrinsic [Ca2+]i oscillations that are not driven by neuronal activity. These spontaneous astrocytic oscillations can propagate as waves to neighboring astrocytes and trigger slowly decaying NMDA receptor-mediated inward currents in neurons located along the wave path. These findings show that astrocytes in situ can act as a primary source for generating neuronal activity in the mammalian central nervous system.
The amyloid-beta (A beta) peptide, which is derived from the amyloid precursor protein (APP), is involved in the pathogenesis of Alzheimer's dementia and impairs endothelium-dependent vasodilation in cerebral vessels. We investigated whether cerebrovascular autoregulation, i.e., the ability of the cerebral circulation to maintain flow in the face of changes in mean arterial pressure (MAP), is impaired in transgenic mice that overexpress APP and A beta. Neocortical cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry in anesthetized APP(+) and APP(-) mice. MAP was elevated by intravenous infusion of phenylephrine and reduced by controlled exsanguination. In APP(-) mice, autoregulation was preserved. However, in APP(+) mice, autoregulation was markedly disrupted. The magnitude of the disruption was linearly related to brain A beta concentration. The failure of autoregulation was paralleled by impairment of the CBF response to endothelium-dependent vasodilators. Thus A beta disrupts a critical homeostatic mechanism of the cerebral circulation and renders CBF highly dependent on MAP. The resulting alterations in cerebral perfusion may play a role in the brain dysfunction and periventricular white-matter changes associated with Alzheimer's dementia.
Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-D-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 micromol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 +/- 9 to 18 +/- 7%, means +/- SD, n = 8). With 20 micromol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 +/- 9% to 9 +/- 4%, n = 8). Superfusion with 20 micromol/l miconazole decreased the flow response by 67% (from 31 +/- 6% to 10 +/- 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 +/- 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A(2) agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.
Rivastigmine enhances cholinergic activity and has been shown in clinical trials to decrease the rate of deterioration in Alzheimer's disease. It remains unclear where in the brain it exerts its effect. Functional magnetic resonance imaging (fMRI) can be used to measure changes in brain function and relate these to cognition.
To use fMRI to study brain activation with rivastigmine treatment.
The effect on brain activation of a single dose of rivastigmine was tested in seven patients with mild Alzheimer's disease using fMRI during face encoding, and in five patients during a parametric working memory task.
During face encoding, rivastigmine increased bilateral activation in the fusiform gyrus. Brain activation was also enhanced in the prefrontal cortex in a simple working memory task. When working memory load was further increased, not only was increased activation seen, but in certain areas there was also decreased activation.
These findings link the previously observed increase in cognitive performance in Alzheimer's disease after treatment with a cholinesterase inhibitor to altered brain activation. Although the results cannot be generalised to the Alzheimer's disease population at large, they provide evidence that in mild Alzheimer's disease, rivastigmine enhances brain activation in the fusiform and frontal cortices. This is compatible with the concept of cholinergic circuitry.
The cellular mechanisms underlying functional hyperemia--the coupling of neuronal activation to cerebral blood vessel responses--are not yet known. Here we show in rat cortical slices that the dilation of arterioles triggered by neuronal activity is dependent on glutamate-mediated [Ca(2+)](i) oscillations in astrocytes. Inhibition of these Ca(2+) responses resulted in the impairment of activity-dependent vasodilation, whereas selective activation--by patch pipette--of single astrocytes that were in contact with arterioles triggered vessel relaxation. We also found that a cyclooxygenase product is centrally involved in this astrocyte-mediated control of arterioles. In vivo blockade of glutamate-mediated [Ca(2+)](i) elevations in astrocytes reduced the blood flow increase in the somatosensory cortex during contralateral forepaw stimulation. Taken together, our findings show that neuron-to-astrocyte signaling is a key mechanism in functional hyperemia.
Patients with probable Alzheimer's disease (AD) have difficulty understanding verbs. To investigate the neural basis for this deficit, the authors used functional magnetic resonance imaging to examine patterns of neural activation during verb processing in 11 AD patients compared with 16 healthy seniors. Subjects judged the pleasantness of verbs, including MOTION verbs and COGNITION verbs. Healthy seniors and AD patients both activated posterolateral temporal and inferior frontal regions during judgments of verbs. These activations were relatively reduced and somewhat changed in their anatomic distribution in AD patients compared with healthy seniors, particularly for the subcategory of MOTION verbs, but AD patients showed minimal activation in association with COGNITION verbs. These findings imply that poor performance with verbs in AD is due in part to altered activation of the large-scale neural network that supports verb processing.
Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca(2+)](i) events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca(2+)](i) events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca(2+)](i) activity in individual cells and a robust coordination of [Ca(2+)](i) signals in neighboring astrocytes. These findings indicate potential neuron-glia communication in the intact brain.
The structural and functional integrity of the brain depends on the delicate balance between substrate delivery through blood flow and energy demands imposed by neural activity. Complex cerebrovascular control mechanisms ensure that active brain regions receive an adequate amount of blood, but the nature of these mechanisms remains elusive. Recent findings implicate perivascular neurons, gliovascular interactions and intramural vascular signalling in the control of the cerebral microcirculation. Neurons, astrocytes and vascular cells seem to constitute a functional unit, the primary purpose of which is to maintain the homeostasis of the brain's microenvironment. Alterations of these vascular regulatory mechanisms lead to brain dysfunction and disease. The emerging view is that cerebrovascular dysregulation is a feature not only of cerebrovascular pathologies, such as stroke, but also of neurodegenerative conditions, such as Alzheimer's disease.
Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.
Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.
The cerebral vascular supply is constructed to protect the cerebral hemispheres and brainstem from the consequences of blood flow cessation. Reversal of blood flow around local obstructions is a feature of the microvascular beds of the striatum and cerebral cortex. Cerebral capillaries of these beds consist of endothelial cells, basal lamina, and astrocyte end-feet that sit in close apposition. The interaction of astrocytes with neurons indicates the close relationship, of microvessels to neurons, These relationships are altered when blood flow ceases in the supplying artery. Increased endothelial cell permeability and endocytoses load to edema formation, and matrix degradation is associated with hemorrhage, Autoregulation is lost, Ischemia initiates leukocyte adhesion receptor expression, which is promoted by cytokine generation from the neuropil and activated monocytes. "Preactivation" may further augment the inflammatory responses to ischemia, The activation of cerebral microvessels by ischemia is heterogeneous, involving alterations in integrin-matrix interactions, leukocyte-endothelial cell adhesion, permeability changes, and the "no-reflow'' phenomenon due to platelet activation, fibrin formation, and leukocyte adhesion. Ischemia produces swelling of the microvascular endothelium, and rapid detachment and swelling of the astrocyte end-feet. Ischemic injury targets the microvasculature, where the inflammatory responses are initiated and contribute to tissue injury.
We investigated the cerebral blood flow (CBF) response to somatosensory stimulation. Stimulation of neuronal activity was performed by deflection (2-3/s) of the mystacial vibrissae in rats over a period of 60 s, and regional cortical CBF was measured continuously in the contralateral somatosensory cortex with laser-Droppler flowmetry. CBF within the somatosensory cortex wag studied through the parietal bone thinned to translucency (n = 7) or through a closed cranial window with the dura mater removed (n = 7). In addition, the differential effect of anesthetics (halothane-N2O, n = 5; thiobutabarbiturate, n = 5; and alpha-chloralose, n = 7) on the CBF response to stimulation was investigated. After a rapid increase after stimulation onset (maximum reached within 2-3 s), CBF remained above baseline with a slight tendency to decrease despite continued stimulation. On termination of stimulation, CBF fell to near prestimulation values within 2-3 s. The following mean CBF responses above baseline during the 60-s stimulation period were obtained: halothane-N2O anesthesia, 25.4 +/- 5.9%; thiobutabarbiturate anesthesia, 10.6 +/- 2.4%; and alpha-chloralose anesthesia, 16.9 +/- 2.3 (through the translucent bone) and 16.2 +/- 2.9% (closed cranial window, dura removed). We conclude that coupling of CBF to neuronal function has a very high temporal resolution (<3 s) and that whisker deflection in rats provides a physiological stimulus to study coupling with laser-Doppler flowmetry.
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Phospholipase A2 (PLA2) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA2-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA2 in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA2 has during both physiologic and pathologic states. Several different PLA2 enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA2+-dependent and Ca2+-independent, based on their catalytic dependence on Ca2+. Under physiologic conditions, PLA2 may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA2 activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA2 found in the central nervous system and attempts to define the role of PLA2 during both physiologic and pathologic conditions.
The modulation of blood oxygenation level-dependent (BOLD) cerebral MRI contrast by the vasoconstrictive drug indomethacin (i.v. 0.2 mg/kg b.w.) was investigated in 10 healthy young adults without and with functional challenge (repetitive and sustained visual activation). For comparison, isotonic saline (placebo, 20 mL) and acetylsalicylate (i.v. 500 mg) were investigated as well, each in separate sessions using identical protocols. After indomethacin, dynamic T2*-weighted echo-planar MRI at 2.0 T revealed a rapid decrease in MRI signal intensity by 2.1%–2.6% in different gray matter regions (P ≤ 0.001 compared to placebo), which was not observed for acetylsalicylate and the placebo condition. Regional signal differences were not significant within gray matter, but all gray matter regions differed significantly from the signal decrease of only 1.2% ± 0.7% observed in white matter (P = 0.001). For the experimental parameters used, a 1% MRI signal decrease in response to indomethacin was estimated to correlate with a decrease of the cerebral blood flow by about 12 ml/100 g/minute, and an increase of the oxygen extraction fraction by about 15%. Responses to visual activation were not affected by saline or acetylsalicylate, and yielded 5.0%–5.5% BOLD MRI signal increases both before and after drug application. In contrast, indomethacin reduced the initial response strength to 82%–85% of that obtained without the drug. The steady-state response during sustained activation reached only 47% of the corresponding pre-drug level (P < 0.01). During repetitive activation the BOLD contrast was reduced to 66% of that observed for control conditions (P < 0.001). In conclusion, indomethacin attenuates the vasodilatory force at functional brain activation, indicating different mechanisms governing neurovascular coupling. J. Magn. Reson. Imaging 2001;13:325–334. © 2001 Wiley-Liss, Inc.
To determine the possible role that endogenously produced prostaglandins may play in the regulation of cerebral blood flow, the responses of cerebral precapillary vessels to prostaglandins (PG) D2, E2, G2, and I2 (8.1 X 10(-8) to 2.7 X 10(-5) M) were studied in cats equipped with cranial windows for direct observation of the microvasculature. Local application of PGs induced a dose-dependent dilation of large (greater than or equal to 100 microns) and small (less than 100 microns) arterioles with no effect on arterial blood pressure. The relative vasodilator potency was PGG2 greater than PGE2 greater than PGI2 greater than PGD2. With all PGs, except D2, the percent dilation of small arterioles was greater than the dilation of large arterioles. After application of prostaglandins in a concentration of 2.7 X 10(-5) M, the mean +/- standard error of the percent dilation of large and small arterioles was, respectively, 47.6 +/- 2.7 and 65.3 +/- 6.1 for G2, 34.1 +/- 2.0, and 53.6 +/- 5.5 for E2, 25.4 +/- 1.8, and 40.2 +/- 4.6 for I2, and 20.3 +/- 2.5 and 11.0 +/- 2.2 for D2. Because brain arterioles are strongly responsive to prostaglandins and the brain can synthesize prostaglandins from its large endogenous pool of prostaglandin precursor, prostaglandins may be important mediators of changes in cerebral blood flow under normal and abnormal conditions.
The effect of various anesthetics on the functional-metabolic coupling of cerebral cortex was studied in rats submitted to unilateral somatosensory stimulation. The regional cerebral metabolic rate of glucose (CMRglc) was measured autoradiographically using the 2-deoxyglucose method, and somatosensory activation was carried out by electrical stimulation of the left forepaw. In animals treated with 70% nitrous oxide, 0.5% halothane/70% nitrous oxide or 40 mg/kg pentobarbital, CMRglc of somatosensory cortex did not change despite generation of primary evoked cortical potentials. Anesthesia with 80 mg/kg alpha-chloralose, in contrast, led to a focal increase of CMRglc in the primary somatosensory cortex from 52.1 +/- 18.3 to 73.1 +/- 18.9 mumol/100 g/min (means +/- s.d.). Metabolic activation was strictly confined to the forelimb (FL) area of somatosensory cortex, and it exhibited a laminar pattern with maximal activation in layers I, II and IV. The preservation of functional-metabolic coupling under a surgical dose of chloralose renders this anesthetic particularly suited for the investigation of coupling processes under conditions where the experimental requirements preclude the use of unanaesthetized animals.
Anesthetic agents are often administered in the presence of ethyl alcohol, both in research and in the clinical setting. The authors tested the hypothesis that anesthetic agents may affect cerebrovascular responses to ethanol. A closed cranial window preparation in the rat was used to compare the response of pial arterioles to topically applied ethanol (0.01% to 1% vol/vol) in the presence of alpha-chloralose/urethane (50 and 600 mg/kg, respectively) or halothane (0.5% to 1%) anesthesia. Heart rate, mean arterial blood pressure, and blood gas levels were maintained stable and within the physiological range throughout each experiment. Ethanol induced significant vasoconstriction in alpha-chloralose/urethane-anesthetized animals (multivariate analysis of variance (MANOVA), p = 0.039); conversely, ethanol induced significant vasodilation of the pial arterioles in halothane-anesthetized animals (MANOVA, p = 0.017). These responses were significantly different from one another (MANOVA, p = 0.001). Thus, the choice of anesthetic agent alters the cerebrovascular response to ethanol, and care should be taken to ascertain the influence of anesthesia in both research and clinical settings.
The metabolism of arachidonic acid (AA) into vasoactive products by cyclooxygenase and lipoxygenase enzymes has been well described, as has their biological relevance. Recently, a number of studies have demonstrated the ability of cytochrome P-450 (P450) enzymes to metabolize AA into biologically important regulators of vascular tone. There are two categories of vasoactive P450 metabolites, namely those catalyzed by epoxygenase enzymes which generate epoxyeicosatrienoic acids (EETs) and those enzymes which generate hydroxyeicosatetraenoic acids (HETEs). Except for 20-HETE, P450 metabolites of AA occur as stereo- and regioisomers which determine, to some extent, their biological activity. 5,6-, 8,9-, 11,12- and 14,15-EETs are generally potent dilators in a number of vascular beds with a sensitivity which appears to increase as the vasculature decreases in size toward capillaries. HETEs, such as 12R- and 20-HETE, can be potent activators of vascular tissue with 20-HETE contracting cerebral and renal microvessels at concentrations of < 10(-10) M. Both EETs and HETEs can be made by vascular and extravascular tissue. Available data suggests that EETs are formed by endothelial and parenchymal tissue while HETEs can be endogenously formed in arterial muscle where they appear to act as second messengers. This review will discuss the molecular biology, stereochemistry, biological activity and importance of P450 metabolites of AA as para- and autocrine controllers of organ blood flow. We will also discuss the large diversity of P450 enzyme isoforms and how such diversity can provide for precise physiological control of vascular tone.
Calcium-sensitive cytosolic phospholipase A2 (cPLA2) is responsible for receptor-mediated liberation of arachidonic acid, and thus plays an important role in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. In this study we have investigated the cellular distribution of cPLA2 in brain using a monoclonal antibody raised against cPLA2 to immunostain tissue sections of human cerebral cortex. We have localized cPLA2 in astrocytes of the gray matter. Colocalization with glial fibrillary acidic protein (GFAP) confirmed that cPLA2 is associated predominantly with protoplasmic astrocytes. Astrocytes of the white matter, on the other hand, were not immunoreactive. In experiments using different human astrocytoma cell lines we found that cPLA2 can be immunochemically localized in UC-11 MG cells, but cannot be detected in U-373 MG cells. This finding is consistent with the observation that cPLA2 mRNA as well as cPLA2 enzymatic activity can be readily measured in UC-11 MG astrocytoma cells, yet cannot be detected in U-373 MG cells. Our data suggest that the astrocyte is a primary source of cPLA2 in the brain and provide further evidence for the importance of this cell type in inflammatory processes in the brain.
We investigated the cerebral blood flow (CBF) response to somatosensory stimulation. Stimulation of neuronal activity was performed by deflection (2-3/s) of the mystacial vibrissae in rats over a period of 60 s, and regional cortical CBF was measured continuously in the contralateral somatosensory cortex with laser-Doppler flowmetry. CBF within the somatosensory cortex was studied through the parietal bone thinned to translucency (n = 7) or through a closed cranial window with the dura mater removed (n = 7). In addition, the differential effect of anesthetics (halothane-N2O, n = 5; thiobutabarbiturate, n = 5; and alpha-chloralose, n = 7) on the CBF response to stimulation was investigated. After a rapid increase after stimulation onset (maximum reached within 2-3 s), CBF remained above baseline with a slight tendency to decrease despite continued stimulation. On termination of stimulation, CBF fell to near prestimulation values within 2-3 s. The following mean CBF responses above baseline during the 60-s stimulation period were obtained: halothane-N2O anesthesia, 25.4 +/- 5.9%; thiobutabarbiturate anesthesia, 10.6 +/- 2.4%; and alpha-chloralose anesthesia, 16.9 +/- 2.3 (through the translucent bone) and 16.2 +/- 2.9% (closed cranial window, dura removed). We conclude that coupling of CBF to neuronal function has a very high temporal resolution (< 3 s) and that whisker deflection in rats provides a physiological stimulus to study coupling with laser-Doppler flowmetry.
Hemodynamics play a significant role in the propensity of intracranial arteriovenous malformations (AVMs) to hemorrhage and in influencing both therapeutic strategies and their complications. AVM hemodynamics are difficult to quantitate, particularly within or in close proximity to the nidus. Biomathematical models represent a theoretical method of investigating AVM hemodynamics but currently provide limited information because of the simplicity of simulated anatomic and physiological characteristics in available models. Our purpose was to develop a new detailed biomathematical model in which the morphological, biophysical, and hemodynamic characteristics of an intracranial AVM are replicated more faithfully. The technique of electrical network analysis was used to construct the biomathematical AVM model to provide an accurate rendering of transnidal and intranidal hemodynamics. The model represented a complex, noncompartmentalized AVM with 4 arterial feeders (with simulated pial and transdural supply), 2 draining veins, and a nidus consisting of 28 interconnecting plexiform and fistulous components. Simulated vessel radii were defined as observed in human AVMs. Common values were assigned for normal systemic arterial pressure, arterial feeder pressures, draining vein pressures, and central venous pressure. Using an electrical analogy of Ohm's law, flow was determined based on Poiseuille's law given the aforementioned pressures and resistances of each nidus vessel. Circuit analysis of the AVM vasculature based on the conservation of flow and voltage revealed the flow rate through each vessel in the AVM network. Once the flow rate was established, the velocity, the intravascular pressure gradient, and the wall shear stress were determined. Total volumetric flow through the AVM was 814 ml/min. Hemodynamic analysis of the AVM showed increased flow rate, flow velocity, and wall shear stress through the fistulous component. The intranidal flow rate varied from 5.5 to 57.0 ml/min with and average of 31.3 ml/min for the plexiform vessels and from 595.1 to 640.1 ml/min with an average of 617.6 ml/min for the fistulous component. The blood flow velocity through the AVM nidus ranged from 11.7 to 121.1 cm/s with an average of 66.4 cm/s for the plexiform vessels and from 446.9 to 480 dyne/cm2 with an average of 463.5 dyne/cm2 for the fistulous component. The wall shear stress ranged in magnitude from 33.2 to 342.1 dyne/cm2 with an average of 187.7 dyne/cm2 for the plexiform vessels and from 315.9 to 339.7 cm/s with an average of 327.8 cm/s for the fistulous component. The described novel biomathematical model characterizes the transnidal and intranidal hemodynamics of an intracranial AVM more accurately than was possible previously. This model should serve as a useful research tool for further theoretical investigations of intracranial AVMs and their hemodynamic sequelae.
We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, respectively. The olefinic compounds N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS) and 12, 12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.
To determine whether brain function is altered in cognitively normal individuals at high risk for AD several years before the typical age at onset for this illness.
Neuropathologic alterations in AD precede cognitive impairment by several years. It is unknown whether functional alterations in neural circuitry accompany these neuropathologic changes, and if so, whether they may be detectable before onset of symptoms.
We used functional MRI to compare cortical activation between two groups of cognitively normal women differing only in their risk for developing AD. Visual naming and letter fluency tasks were used to activate brain areas subserving object and face recognition, previously described sites of hypometabolism and neuropathologic alteration in AD. The risk groups differed in family history of AD and apolipoprotein E allele status, but were matched in age, education, and measures of cognitive performance. Average age of the study participants was 52 years.
The regional patterns of brain activation were similar between groups. However, the high risk group showed areas of significantly reduced activation in the mid- and posterior inferotemporal regions bilaterally during both tasks despite identical naming and letter fluency performance.
Cognitively normal individuals at high risk for AD demonstrate decreased brain activation in key areas engaged during naming and fluency tasks. Decreased activation in the high risk group may be a consequence of the presence of subclinical neuropathology in the inferotemporal region or in the inputs to that region. If so, these findings provide evidence of a window of opportunity for disease-modifying treatment before the onset of symptomatic AD.
Epidemiological and clinical studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit cyclooxygenase (COX) slow the progression and delay the onset of Alzheimer disease (AD). Two isoforms of cyclooxygenase have been identified. Although much effort has recently been focused on the inducible COX-2 isoform, little is known about COX-1 expression in human brain. We report that COX-1 message and immunoreactivity are localized to human hippocampal CA3 and CA4 neurons, granular neurons in neocortical layer IV, and occasional cortical pyramidal neurons. Quantitative in situ hybridization showed no differences between COX-1 mRNA levels in control and AD CA3 hippocampal neurons. COX-1 immunoreactivity was also present in microglial cells in gray and white matter in all brain regions examined. COX-1 appeared to be expressed in microglial cells regardless of their activation state as determined by HLA-DR immunostaining. However, COX-1 immunopositive microglia were found in association with Abeta plaques, and the density of COX-1 immunopositive microglia in AD fusiform cortex was increased. This pattern suggests an overall increase of COX-1 expression in AD. Currently used NSAIDs inhibit both isoforms of cyclooxygenase. The present study shows that COX-1 is widely expressed in human brain, and raises the possibility that COX-1 may contribute to CNS pathology.
Crus II is an area of the cerebellar cortex that receives trigeminal afferents from the perioral region. We investigated the mechanisms of functional hyperemia in cerebellum using activation of crus II by somatosensory stimuli as a model. In particular, we sought to determine whether stimulation of the perioral region increases cerebellar blood flow (BFcrb) in crus II and, if so, whether the response depends on activation of 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-kainate receptors and nitric oxide (NO) production. Crus II was exposed in anesthetized rats, and the site was superfused with Ringer. Field potentials were recorded, and BFcrb was measured by laser-Doppler flowmetry. Crus II was activated by electrical stimulation of the perioral region (upper lip). Perioral stimulation evoked the characteristic field potentials in crus II and increased BFcrb (34 +/- 6%; 10 Hz-25 V; n = 6) without changing arterial pressure. The BFcrb increases were associated with a local increase in glucose utilization (74 +/- 8%; P < 0.05; n = 5) and were attenuated by the AMPA-kainate receptor antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline (-71 +/- 3%; 100 microM; P < 0.01; n = 5). The neuronal NO synthase inhibitor 7-nitroindazole (7-NI, 50 mg/kg; n = 5) virtually abolished the increases in BFcrb (-90 +/- 2%; P < 0.01) but did not affect the amplitude of the field potentials. In contrast, 7-NI attenuated the increase in neocortical cerebral blood flow produced by perioral stimulation by 52 +/- 6% (P < 0.05; n = 5). We conclude that crus II activation by somatosensory stimuli produces localized increases in local neural activity and BFcrb that are mediated by activation of glutamate receptors and NO. Unlike in neocortex, in cerebellum the vasodilation depends almost exclusively on NO. The findings underscore the unique role of NO in the mechanisms of synaptic function and blood flow regulation in cerebellum.
The cerebral vascular supply is constructed to protect the cerebral hemispheres and brainstem from the consequences of blood flow cessation. Reversal of blood flow around local obstructions is a feature of the microvascular beds of the striatum and cerebral cortex. Cerebral capillaries of these beds consist of endothelial cells, basal lamina, and astrocyte end-feet that sit in close apposition. The interaction of astrocytes with neurons indicates the close relationship of microvessels to neurons. These relationships are altered when blood flow ceases in the supplying artery. Increased endothelial cell permeability and endocytoses lead to edema formation, and matrix degradation is associated with hemorrhage. Autoregulation is lost. Ischemia initiates leukocyte adhesion receptor expression, which is promoted by cytokine generation from the neuropil and activated monocytes. "Preactivation" may further augment the inflammatory responses to ischemia. The activation of cerebral microvessels by ischemia is heterogeneous, involving alterations in integrin-matrix interactions, leukocyte-endothelial cell adhesion, permeability changes, and the "no-reflow" phenomenon due to platelet activation, fibrin formation, and leukocyte adhesion. Ischemia produces swelling of the microvascular endothelium, and rapid detachment and swelling of the astrocyte end-feet. Ischemic injury targets the microvasculature, where the inflammatory responses are initiated and contribute to tissue injury.
Adenosine is a modulator that has a pervasive and generally inhibitory effect on neuronal activity. Tonic activation of adenosine receptors by adenosine that is normally present in the extracellular space in brain tissue leads to inhibitory effects that appear to be mediated by both adenosine A1 and A2A receptors. Relief from this tonic inhibition by receptor antagonists such as caffeine accounts for the excitatory actions of these agents. Characterization of the effects of adenosine receptor agonists and antagonists has led to numerous hypotheses concerning the role of this nucleoside. Previous work has established a role for adenosine in a diverse array of neural phenomena, which include regulation of sleep and the level of arousal, neuroprotection, regulation of seizure susceptibility, locomotor effects, analgesia, mediation of the effects of ethanol, and chronic drug use.
Cyclooxygenase (COX) is a prostanoid-synthesizing enzyme present in 2 isoforms: COX-1 and COX-2. Although it has long been hypothesized that prostanoids participate in cerebrovascular regulation, the lack of adequate pharmacological tools has led to conflicting results and has not permitted investigators to define the relative contribution of COX-1 and COX-2. We used the COX-1 inhibitor SC-560 and COX-1-null (COX-1(-/-)) mice to investigate whether COX-1 plays a role in cerebrovascular regulation. Mice were anesthetized (urethane and chloralose) and equipped with a cranial window. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry or by the (14)C-iodoantipyrine technique with quantitative autoradiography. In wild-type mice, SC-560 (25 micromol/L) reduced resting CBF by 21+/-4% and attenuated the CBF increase produced by topical application of bradykinin (-59%) or calcium ionophore A23187 (-49%) and by systemic hypercapnia (-58%) (P<0.05 to 0.01). However, SC-560 did not reduce responses to acetylcholine or the increase in somatosensory cortex blood flow produced by vibrissal stimulation. In COX-1(-/-) mice, resting CBF assessed by (14)C-iodoantipyrine was reduced (-13% to -20%) in cerebral cortex and other telencephalic regions (P<0.05). The CBF increase produced by bradykinin, A23187, and hypercapnia, but not acetylcholine or vibrissal stimulation, were attenuated (P<0.05 to 0.01). The free radical scavenger superoxide dismutase attenuated responses to bradykinin and A23187 in wild-type mice but not in COX-1(-/-) mice, suggesting that COX-1 is the source of the reactive oxygen species known to mediate these responses. The data provide evidence for a critical role of COX-1 in maintaining resting vascular tone and in selected vasodilator responses of the cerebral microcirculation.
Cyclooxygenase (COX) is the obligate, rate-limiting enzyme for the conversion of arachidonic acid into prostaglandins. Two COX enzymes have been identified: a constitutively expressed COX-1 and an inducible, highly regulated COX-2. Widely used to treat chronic inflammatory disorders, COX inhibitors have shown promise in attenuating inflammation associated with brain injury. However, the use of COX inhibition in the treatment of brain injury has met with mixed success. This review summarizes our current understanding of COX expression in the central nervous system and the effects of COX inhibitors on brain injury. Three major targets for COX inhibition in the treatment brain injury have been identified. These are the cerebrovasculature, COX-2 expression by vulnerable neurons, and the neuroinflammatory response. Evidence suggests that given the right treatment paradigm, COX inhibition can influence each of these three targets. Drug interactions and general considerations for administrative paradigms are also discussed. Although therapies targeted to specific prostaglandin species, such as PGE2, might prove more ameliorative for brain injury, at the present time non-specific COX inhibitors and COX-2 specific inhibitors are readily available to researchers and clinicians. We believe that COX inhibition will be a useful, ameliorative adjunct in the treatment of most forms of brain injury.
The cerebral circulation is tightly regulated to meet the brain's metabolic demands. Although the mechanism is not fully understood, the major physiologic influences on cerebral blood flow have been well documented. In this chapter the basic vascular anatomy, and physiologic control of the cerebral circulation are reviewed. Clinical implications are emphasized.
The present study was designed to investigate whether cyclooxygenase products are involved in the regulation of the regional cerebral blood flow, evoked by somatosensory activation (evoked rCBF) under normo- and hypercapnia. Indomethacin (IMC) was used as cyclooxygenase inhibitor. It was applied intravenously (i.v., 10 mg/kg/h) in two experimental protocols-before hypercapnia (i) and after hypercapnia (ii). Somatosensory activation was induced by electrical hind paw stimulation (5 Hz frequency, 5 s duration, 1.5 mA). The evoked rCBF-response was measured in alpha -chloralose anesthetized rats using laser-Doppler flowmetry. IMC abolished completely the effect of hypercapnia on the baseline level of CBF. The drug reduced significantly evoked rCBF-response also. The inhibitory effect of IMC on evoked rCBF-response is better expressed under normocapnia (approximately 70%) than that under hypercapnia (approximately 40%). After IMC application, the normalized evoked rCBF curves peaked earlier as compared to that before its application (P<0.05), although the rise time of 0.5 s was nearly constant regardless of stimulus frequency. In conclusion, the results suggest a participation of IMC-sensitive and cyclooxygenase-dependent mechanisms in the regulation of evoked rCBF, induced by somatosensory stimulation.
Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H(2)O(2) have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H(2)O(2) and menadione, a compound known to release H(2)O(2) intracellularly, were used to examine the phospholipases A(2) (PLA(2)) responsible for AA release from primary murine astrocytes. Both H(2)O(2) and menadione dose-dependently stimulated AA release, and the release mediated by H(2)O(2) was completely inhibited by catalase. H(2)O(2) also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A(2) (cPLA(2)). However, complete inhibition of cPLA(2) phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H(2)O(2)-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA(2) and the Ca(2+)-independent iPLA(2), nearly completely inhibited H(2)O(2)-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA(2), only inhibited H(2)O(2)-mediated AA release by 40%. Along with the observation that H(2)O(2)-mediated AA release was only partially inhibited upon chelating intracellular Ca(2+) by BAPTA, these results indicate the involvement of both cPLA(2) and iPLA(2) in H(2)O(2)-mediated AA release in murine astrocytes.
Characterization of astrocyte Ca2+ dynamics has been a topic of considerable emphasis for more than a decade. Only recently, however, has the physiological significance of astrocyte Ca2+ signaling started to become clear. Several studies have shown that astrocyte Ca2+ levels become elevated in response to neuronal input and that this, in turn, influences synaptic activity. A novel function of astrocyte Ca2+ signaling has been described by Zonta et al., whereby neuron-induced astrocyte Ca2+ elevations can lead to secretion of vasodilatory substances from perivascular astrocyte endfeet, resulting in improved local blood flow. This finding represents a breakthrough in our knowledge both of astrocyte function and of the mechanism of activity-dependent cerebral blood flow regulation.
Upregulation and activation of phospholipases A2 (PLA2) and cyclooxygenases (COX) leading to prostaglandin E2(PGE2) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE2 production in primary rat astrocytes in response to agents that activate PLA2 including pro-inflammatory cytokines (IL-1beta, TNFalpha and IFNgamma), the P2 nucleotide receptor agonist ATP, and oxidants (H2O2 and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE2 production that was marked by increased expression of secretory sPLA2 and COX-2, but not COX-1 and cytosolic cPLA2. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA2 phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE2. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H2O2 or menadione, markedly increased PGE2 production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE2 production and may contribute to chronic inflammation seen in Alzheimer's disease.
Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequency spiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade in ameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury. We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumatic zone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results in excitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent and functional sequelae of acute SCI.
Cerebral blood flow (CBF) is coupled to neuronal activity and is imaged in vivo to map brain activation. CBF is also modified by afferent projection fibres that release vasoactive neurotransmitters in the perivascular region, principally on the astrocyte endfeet that outline cerebral blood vessels. However, the role of astrocytes in the regulation of cerebrovascular tone remains uncertain. Here we determine the impact of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in astrocytes on the diameter of small arterioles by using two-photon Ca(2+) uncaging to increase [Ca(2+)](i). Vascular constrictions occurred when Ca(2+) waves evoked by uncaging propagated into the astrocyte endfeet and caused large increases in [Ca(2+)](i). The vasoactive neurotransmitter noradrenaline increased [Ca(2+)](i) in the astrocyte endfeet, the peak of which preceded the onset of arteriole constriction. Depressing increases in astrocyte [Ca(2+)](i) with BAPTA inhibited the vascular constrictions in noradrenaline. We find that constrictions induced in the cerebrovasculature by increased [Ca(2+)](i) in astrocyte endfeet are generated through the phospholipase A(2)-arachidonic acid pathway and 20-hydroxyeicosatetraenoic acid production. Vasoconstriction by astrocytes is a previously unknown mechanism for the regulation of CBF.
Neuronal activity in the brain is thought to be coupled to cerebral arterioles (functional hyperemia) through Ca2+ signals in astrocytes. Although functional hyperemia occurs rapidly, within seconds, such rapid signaling has not been demonstrated in situ, and Ca2+ measurements in parenchymal arterioles are still lacking. Using a laser scanning confocal microscope and fluorescence Ca2+ indicators, we provide the first evidence that in a brain slice preparation, increased neuronal activity by electrical stimulation (ES) is rapidly signaled, within seconds, to cerebral arterioles and is associated with astrocytic Ca2+ waves. Smooth muscle cells in parenchymal arterioles exhibited Ca2+ and diameter oscillations ("vasomotion") that were rapidly suppressed by ES. The neuronal-mediated Ca2+ rise in cortical astrocytes was dependent on intracellular (inositol trisphosphate [IP3]) and extracellular voltage-dependent Ca2+ channel sources. The Na+ channel blocker tetrodotoxin prevented the rise in astrocytic [Ca2+]i and the suppression of Ca2+ oscillations in parenchymal arterioles to ES, indicating that neuronal activity was necessary for both events. Activation of metabotropic glutamate receptors in astrocytes significantly decreased the frequency of Ca2+ oscillations in parenchymal arterioles. This study supports the concept that astrocytic Ca2+ changes signal the cerebral microvasculature and indicate the novel concept that this communication occurs through the suppression of arteriolar [Ca2+]i oscillations and corresponding vasomotion. The full text of this article is available online at http://circres.ahajournals.org.
A key goal in functional neuroimaging is to use signals that are related to local changes in metabolism and blood flow to track the neuronal correlates of mental activity. Recent findings indicate that the dendritic processing of excitatory synaptic inputs correlates more closely than the generation of spikes with brain imaging signals. The correlation is often nonlinear and context-sensitive, and cannot be generalized for every condition or brain region. The vascular signals are mainly produced by increases in intracellular calcium in neurons and possibly astrocytes, which activate important enzymes that produce vasodilators to generate increments in flow and the positive blood oxygen level dependent signal. Our understanding of the cellular mechanisms of functional imaging signals places constraints on the interpretation of the data.
In contrast to traditional neuroncentric views of Alzheimer's disease (AD), recent findings indicate that neurovascular dysfunction contributes to cognitive decline and neurodegeneration in AD. Here, I propose the neurovascular hypothesis of AD, suggesting that faulty clearance of amyloid beta peptide (A beta) across the blood-brain barrier (BBB), aberrant angiogenesis and senescence of the cerebrovascular system could initiate neurovascular uncoupling, vessel regression, brain hypoperfusion and neurovascular inflammation. Ultimately, this would lead to BBB compromise, to chemical imbalance in the neuronal environment and to synaptic and neuronal dysfunction, injury and loss. Based on the neurovascular hypothesis, I suggest an array of new potential therapeutic approaches that could be developed for AD, to enhance A beta clearance and neurovascular repair, and to protect the neurovascular unit from divergent inducers of injury and apoptosis.
One of the responses to cerebral ischemia is an increase in the production of nitric oxide, catalyzed by enzymes expressed in both resident and infiltrating cells. The nitric oxide that is generated does contribute to the ensuing pathology, but it can also be beneficial. The effects of nitric oxide depend on the cell site of production, the amount generated, and the chemical nature of the products of further oxidation. Understanding how nitric oxide production from microglia and astrocytes contributes to ischemic pathology is important for the development and application of future therapeutics based on inhibiting or amplifying its production in the injured brain.
The cellular components of the brain are neurons, glia and vascular cells. These three entities form a metabolic network to sustain brain activity. Interactions among these cell types have been studied extensively in vitro, where the cells are easily accessible to physiological and pharmacological manipulations. With the advent of optical tools, it has become possible to investigate the cerebral metabolic network in vitro at the cellular and subcellular levels. However, the metabolic and homeostatic nature of neuronal-glial-vascular interactions must eventually be examined in vivo, and multi-photon imaging now provides a means to monitor neurovascular units in living experimental animals.