Calcium Imaging in Brain Tissue Slices.

Calcium imaging is a method that was first developed in the mid-1970s yet kept developing until current days to allow accurate measurement of free calcium ions in tissues. This widely used method has provided significant advances to our understanding of cellular signal transduction, including the discovery of subcellular compartmentalization of neurons and astrocytes, the identification of multiple signaling pathways, and mapping the functional connectivity between astrocytes and neuronal networks. Here we describe a method for the loading and imaging of cell-permeable AM ester calcium-sensitive dyes for the in vitro measurement of free intracellular Ca2+ ions in acute brain slices.

Environmental enrichment and sex, but not n-acetylcysteine, alter extended-access amphetamine self-administration and cue-seeking.

There are no approved therapeutics for psychostimulant use and recurrence of psychostimulant use. However, in preclinical rodent models environmental enrichment can decrease psychostimulant self-administration of low unit doses and cue-induced amphetamine seeking. We have previously demonstrated that glutamate-dependent therapeutics are able to alter amphetamine seeking to amphetamine-associated cues only in enriched rats. In the current experiment, we will determine if enrichment can attenuate responding and cue-induced amphetamine seeking during extended access to a high dose of intravenous amphetamine. We will also determine if N-acetylcysteine (NAC), a glutamate dependent therapeutic, can attenuate amphetamine seeking in differentially reared rats. Female and male Sprague-Dawley rats were reared in enriched, isolated, or standard conditions from postnatal day 21-51. Rats were trained to self-administer intravenous amphetamine (0.1 mg/kg/infusion) during twelve 6-hour sessions. During the abstinence period, NAC (100 mg/kg) or saline was administered daily. Following a cue-induced amphetamine-seeking test, astrocyte densities within regions of the medial prefrontal cortex (mPFC) and nucleus accumbens (ACb) were quantified using immunohistochemistry. Environmental enrichment decreased responding for amphetamine and during the cue-induced amphetamine-seeking test. NAC did not attenuate cue-induced amphetamine seeking or alter astrocyte density. Across all groups, female rats self-administered less amphetamine but responded more during cue-induced amphetamine seeking than male rats. While amphetamine increased astrocyte densities within the ACb and mPFC, it did not alter mPFC astrocyte densities in female rats. The results suggest that enrichment can attenuate responding during extended access to a high dose of amphetamine and the associated cues. Sex alters amphetamine-induced changes to astrocyte densities in a regionally specific matter.

Microglia are essential for tissue contraction in wound closure after brain injury in zebrafish larvae.

Wound closure after brain injury is crucial for tissue restoration but remains poorly understood at the tissue level. We investigated this process using in vivo observations of larval zebrafish brain injury. Our findings show that wound closure occurs within the first 24 h through global tissue contraction, as evidenced by live-imaging and drug inhibition studies. Microglia accumulate at the wound site before closure, and computational models suggest that their physical traction could drive this process. Depleting microglia genetically or pharmacologically impairs tissue repair. At the cellular level, live imaging reveals centripetal deformation of astrocytic processes contacted by migrating microglia. Laser severing of these contacts causes rapid retraction of microglial processes and slower retraction of astrocytic processes, indicating tension. Disrupting the lcp1 gene, which encodes the F-actin-stabilising protein L-plastin, in microglia results in failed wound closure. These findings support a mechanical role of microglia in wound contraction and suggest that targeting microglial mechanics could offer new strategies for treating traumatic brain injury.

Astroglial membrane camouflaged Ptbp1 siRNA delivery hinders glutamate homeostasis via SDH/Nrf2 pathway.

Polypyrimidine tract-binding protein 1 (PTBP1) regulates numerous alternative splicing events during tumor progression and neurogenesis. Previously, PTBP1 downregulation was reported to convert astrocytes into functional neurons; however, how PTBP1 regulates astrocytic physiology remains unclear. In this study, we revealed that PTBP1 modulated glutamate uptake via ATP1a2, a member of Na+/K+-ATPases, and glutamate transporters in astrocytes. Ptbp1 knockdown altered mitochondrial function and energy metabolism, which involved PTBP1 regulating mitochondrial redox homeostasis via the succinate dehydrogenase (SDH)/Nrf2 pathway. The malfunction of glutamate transporters following Ptbp1 knockdown resulted in enhanced excitatory synaptic transmission in the cortex. Notably, we developed a biomimetic cationic triblock polypeptide system, i.e., polyethylene glycol44-polylysine30-polyleucine10 (PEG44-PLL30-PLLeu10) with astrocytic membrane coating to deliver Ptbp1 siRNA in vitro and in vivo, which approach allowed Ptbp1 siRNA to efficiently cross the blood-brain barrier and target astrocytes in the brain. Collectively, our findings suggest a framework whereby PTBP1 serves as a modulator in glutamate transport machinery, and indicate that biomimetic methodology is a promising route for in vivo siRNA delivery.

Immunofluorescence Staining of Murine Spinal Cord Sections to Evaluate Microglial Activation and Astrogliosis.

Microglia and astrocytes are the main components of the central nervous system (CNS). Upon activation, microglia is able to phagocyte cell debris, pathogens, and toxins; astrocytes support neuronal functions, blood-brain barrier (BBB) homeostasis, and neurotransmitter uptake and metabolism. Furthermore, both cell types can produce cytokines and chemokines. Aging impacts microglia and astrocytes by promoting the production of pro-inflammatory cytokines, impairing microglial phagocytosis and motility and astrocyte glutamate uptake. During neurodegenerative and neuroinflammatory diseases, the aging process may be accelerated contributing to the alteration of CNS glial cells functions. Multiple sclerosis (MS) is an autoimmune, demyelinating disease in which immunosenescence can promote the conversion from relapsing-remitting form to progressive disease. The murine model of experimental autoimmune encephalomyelitis (EAE) allows to investigate MS pathogenesis. Furthermore, EAE can be developed as acute or progressive, mimicking different forms of human MS. Microglia and astrocytes report morphological and functional changes during neuroinflammation that can be investigated in different ways. We here present a protocol for the study of glial cell activation in the spinal cord tissue of EAE mice.

Adenosine triphosphate release inhibitors targeting pannexin1 improve recovery after spinal cord injury.

Traumatic spinal cord injury is characterized by immediate and irreversible tissue loss at the lesion site and secondary tissue damage. Secondary injuries should, in principle, be preventable, although no effective treatment options currently exist for patients with acute spinal cord injury. Traumatized tissues release excessive amounts of adenosine triphosphate and activate the P2X purinoceptor 7/pannexin1 complex, which is associated with secondary injury. We investigated the neuroprotective effects of the blue dye Brilliant Blue FCF, a selective inhibitor of P2X purinoceptor 7/pannexin1 that is approved for use as a food coloring, by comparing it with Brilliant Blue G, a P2X7 purinoceptor antagonist, and carbenoxolone, which attenuates P2X purinoceptor 7/pannexin1 function, in a rat spinal cord injury model. Brilliant Blue FCF administered early after spinal cord injury reduced spinal cord anatomical damage and improved motor recovery without apparent toxicity. Brilliant Blue G had the highest effect on this neurological recovery, with Brilliant Blue FCF and carbenoxolone having comparable improvement. Furthermore, Brilliant Blue FCF administration reduced local astrocytic and microglial activation and neutrophil infiltration, and no differences in these histological effects were observed between compounds. Thus, Brilliant Blue FCF protects spinal cord neurons after spinal cord injury and suppresses local inflammatory responses as well as Brilliant Blue G and carbenoxolone.

Astrocytic neuroligin 3 regulates social memory and synaptic plasticity through adenosine signaling in male mice.

Social memory impairment is a key symptom of many brain disorders, but its underlying mechanisms remain unclear. Neuroligins (NLGs) are a family of cell adhesion molecules essential for synapse development and function and their dysfunctions are linked to neurodevelopmental and neuropsychiatric disorders, including autism and schizophrenia. Although NLGs are extensively studied in neurons, their role in glial cells is poorly understood. Here we show that astrocytic deletion of NLG3 in the ventral hippocampus of adult male mice impairs social memory, attenuates astrocytic Ca2+ signals, enhances the expression of EAAT2 and prevents long-term potentiation, and these impairments are rescued by increasing astrocyte activity, reducing EAAT2 function or enhancing adenosine/A2a receptor signaling. This study has revealed an important role of NLG3 in astrocyte function, glutamate homeostasis and social memory and identified the glutamate transporter and adenosine signaling pathway as potential therapeutic strategies to treat brain disorders.

[Mechanism of analgesic effect of electroacupuncture on rats with cervical spondylosis radiculopathy based on activation of astrocytes and HMGB1/TLR4/MyD88 signaling pathway]. / 基于星形胶质细胞活化与HMGB1/TLR4/MyD88ä¿¡å·é€šè·¯æŽ¢è®¨ç”µé’ˆå¯¹ç¥žç»æ ¹åž‹é¢ˆæ¤Žç— å¤§é¼ çš„é•‡ç—›æœºåˆ¶.

OBJECTIVES: To observe effects of electroacupuncture (EA) on the activation of astrocytes and high mobility group protein B1(HMGB1)/Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway, as well as related cytokines in rats with cervical spondylosis radiculopathy(CSR), so as to explore the analgesic mechanism of EA in treating CSR. METHODS: Twenty-four male SD rats were randomly divided into blank, sham surgery, model, and EA groups, with 6 rats in each group. CSR rat model was established by using cervical spinal cord canal puncture method. On the 7th day after successful modeling, EA was applied to rats in the EA group at bilateral "Hegu"(LI4) and "Taichong"(LR3) for 20 minutes(1.5 Hz, 1 mA), once daily for 7 consecutive days. Before and after intervention, gait impairment scores and mechanical pain thresholds were assessed. HE staining was used to observe pathological changes in spinal cord tissue. Western blot was used to detect the expression of HMGB1, TLR4, MyD88, and glial fibrillary acidic protein (GFAP) in the spinal cord. ELISA was used to measure the contents of CXC chemokine ligand 1 (CXCL1), chemokine ligand 2 (CCL2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß in spinal cord. Immunofluorescence staining was used to observe GFAP protein positive expression in spinal cord tissue. RESULTS: There was no significant difference of all indexes between the blank group and the sham surgery group. Compared with the sham surgery group, mechanical pain threshold of rats in the model group was decreased(P<0.01), while gait impairment score, the contents of CXCL1, CCL2, TNF-α, IL-1ß, protein expressions of HMGB1, TLR4, MyD88 and GFAP, and positive expression of GFAP in spinal cord tissue were increased (P<0.01)；HE staining indicated severe overall morphological damage in the spinal cord of rats in the model group, with significant shrinkage of gray matter neurons, reduced number of Nissl bodies, and increased inflammatory cell infiltration. Compared with the model group, mechanical pain threshold in the EA group was increased (P<0.01), while gait impairment score, the contents of CXCL1, CCL2, TNF-α, IL-1ß, protein expressions of HMGB1, TLR4, MyD88 and GFAP, and positive expression of GFAP in spinal cord were reduced (P<0.01)；HE staining showed more intact neuronal cell bodies, increased number of Nissl bodies, and reduced shrinkage of gray matter neurons, inflammatory cell infiltration, and microvascular dilation in the spinal cord of rats in the EA group. CONCLUSIONS: EA can effectively alleviate pain in CSR rats, which is possibly by inhibiting astrocyte activation, HMGB1/TLR4/MyD88 signaling pathway, and reducing the release of related inflammatory cytokines, thus alleviating central sensitization in spinal segments.

IGFBP7+ subpopulation and IGFBP7 risk score in astrocytoma: insights from scRNA-Seq and bulk RNA-Seq.

Background: Glioma is the predominant malignant brain tumor that lacks effective treatment options due to its shielding by the blood-brain barrier (BBB). Astrocytes play a role in the development of glioma, yet the diverse cellular composition of astrocytoma has not been thoroughly researched. Methods: We examined the internal diversity of seven distinct astrocytoma subgroups through single-cell RNA sequencing (scRNA-seq), pinpointed crucial subgroups using CytoTRACE, monocle2 pseudotime analysis, and slingshot pseudotime analysis, employed various techniques to identify critical subgroups, and delved into cellular communication analysis. Then, we combined the clinical information of GBM patients and used bulk RNA sequencing (bulk RNA-seq) to analyze the prognostic impact of the relevant molecules on GBM patients, and we performed in vitro experiments for validation. Results: The analysis of the current study revealed that C0 IGFBP7+ Glioma cells were a noteworthy subpopulation of astrocytoma, influencing the differentiation and progression of astrocytoma. A predictive model was developed to categorize patients into high- and low-scoring groups based on the IGFBP7 Risk Score (IGRS), with survival analysis revealing a poorer prognosis for the high-IGRS group. Analysis of immune cell infiltration, identification of genes with differential expression, various enrichment analyses, assessment of copy number variations, and evaluation of drug susceptibility were conducted, all of which highlighted their significant influence on the prognosis of astrocytoma. Conclusion: This research enhances comprehension of the diverse cell composition of astrocytoma, delves into the various factors impacting the prognosis of astrocytoma, and offers fresh perspectives on treating glioma.

Reprogramming astroglia into neurons with hallmarks of fast-spiking parvalbumin-positive interneurons by phospho-site-deficient Ascl1.

Cellular reprogramming of mammalian glia to an induced neuronal fate holds the potential for restoring diseased brain circuits. While the proneural factor achaete-scute complex-like 1 (Ascl1) is widely used for neuronal reprogramming, in the early postnatal mouse cortex, Ascl1 fails to induce the glia-to-neuron conversion, instead promoting the proliferation of oligodendrocyte progenitor cells (OPC). Since Ascl1 activity is posttranslationally regulated, here, we investigated the consequences of mutating six serine phospho-acceptor sites to alanine (Ascl1SA6) on lineage reprogramming in vivo. Ascl1SA6 exhibited increased neurogenic activity in the glia of the early postnatal mouse cortex, an effect enhanced by coexpression of B cell lymphoma 2 (Bcl2). Genetic fate-mapping revealed that most induced neurons originated from astrocytes, while only a few derived from OPCs. Many Ascl1SA6/Bcl2-induced neurons expressed parvalbumin and were capable of high-frequency action potential firing. Our study demonstrates the authentic conversion of astroglia into neurons featuring subclass hallmarks of cortical interneurons, advancing our scope of engineering neuronal fates in the brain.

Neuroprotective Role of AQP4 Knockdown in Astrocytes After Oxygen-Glucose Deprivation.

BACKGROUND: Aquaporin-4 (AQP4), predominantly expressed in astrocytes, has been implicated in the development of brain edema following ischemic events. However, its role in post-stroke neuroinflammation is not fully understood. METHODS: Using a middle cerebral artery occlusion (MCAO) mouse model, we assessed AQP4's role in post-stroke inflammation. Brain tissue slices from male C57BL/6 mice were subjected to immunohistochemistry and western blot post-MCAO. Additionally, primary astrocytes were isolated for quantitative real-time PCR and immunofluorescence assays to evaluate the expression of inflammatory markers glial fibrillary acidic protein (GFAP) and AQP4. AQP4 modulation was achieved using viral knockdown and overexpression methods. Neuronal damage was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests in co-culture studies. RESULTS: MCAO mice exhibited a significant upregulation in GFAP. This reactive astrogliosis corresponded with an elevation in inflammatory markers. AQP4 expression responded to this inflammatory trend, peaking at 6 h after OGD and returning to baseline levels at 24 and 48 h. Co-culture experiments revealed that AQP4(+) astrocytes exacerbated injury in OGD-treated neurons, as evidenced by increased TUNEL positivity and apoptotic events. Conversely, AQP4(-) astrocytes appeared to have a protective effect. Knockdown of AQP4 resulted in reduced post-OGD inflammatory response, whereas AQP4 overexpression intensified the injury to neurons post-OGD. In vivo experiments also confirmed that AQP4 inhibitor TGN-020 reduced and overexpression of AQP4 increased behavioral abnormalities and brain infarcts. CONCLUSION: Our findings underscore AQP4's pivotal role in modulating post-stroke neuroinflammation. Targeting AQP4 may present a novel therapeutic avenue for mitigating ischemia-induced neuronal damage.

Brain Metabolism in Health and Neurodegeneration: The Interplay Among Neurons and Astrocytes.

The regulation of energy in the brain has garnered substantial attention in recent years due to its significant implications in various disorders and aging. The brain's energy metabolism is a dynamic and tightly regulated network that balances energy demand and supply by engaging complementary molecular pathways. The crosstalk among these pathways enables the system to switch its preferred fuel source based on substrate availability, activity levels, and cell state-related factors such as redox balance. Brain energy production relies on multi-cellular cooperation and is continuously supplied by fuel from the blood due to limited internal energy stores. Astrocytes, which interface with neurons and blood vessels, play a crucial role in coordinating the brain's metabolic activity, and their dysfunction can have detrimental effects on brain health. This review characterizes the major energy substrates (glucose, lactate, glycogen, ketones and lipids) in astrocyte metabolism and their role in brain health, focusing on recent developments in the field.

Sex Differences in Astrocyte Activity.

Astrocytes are essential for maintaining brain homeostasis. Alterations in their activity have been associated with various brain pathologies. Sex differences were reported to affect astrocyte development and activity, and even susceptibility to different neurodegenerative diseases. This review aims to summarize the current knowledge on the effects of sex on astrocyte activity in health and disease.

Glia dysfunction in schizophrenia: evidence of possible therapeutic effects of nervonic acid in a preclinical model.

RATIONALE: Neuroinflammation may inhibit oligodendrocyte and astrocyte differentiation, which causes demyelination and synaptic degeneration. The myelin component nervonic acid (NA) may improve demyelinating and neurodegenerative diseases. OBJECTIVES: This study firstly explored relationships between glial cell dysfunction and demyelination or synaptic degeneration in schizophrenia patients, and secondly determined nervonic acid therapeutic effects in a preclinical schizophrenia model of mice. METHODS: Plasma samples were collected from 18 male healthy controls and 18 male schizophrenic patients (diagnosed by DSM-V) at aged 18-55. Mouse brain samples were collected from a maternal immune activation (MIA) model of schizophrenia via injecting 5 mg/kg polyinosinic-polycytidylic acid. Male mouse offspring (age 2.5 months, n = 12) were treated by clozapine (15 mg/kg/day) or fed 0.5% NA for 6 weeks. Cytokine and dopamine (DA) concentrations, and glial phenotypes and myelin markers were measured in both human plasma and mouse brain samples. RESULTS: In patient plasma, increased proinflammatory cytokines were associated with reactive microglia (Iba-1) up-regulation, while decreased anti-inflammatory cytokines were related to microglia (CD206) downregulation. Decreased astrocyte marker (p11) concentrations were accompanied by reduced concentrations of oligodendrocyte and synaptic markers. However, NA and DA contents were increased. Compared with control mice, SZ-like behaviors appeared in MIA male mice. Changes in microglia and astrocytes markers, and cytokine concentrations in the frontal cortex were consistent with those observed in patients' plasma. Hippocampal oligodendrocyte and synaptic marker expression were also decreased. DA content and DA/metabolite (DAPOC) were increased in MIA mouse brains. Most of these changes were normalized by both clozapine and NA. Even though some NA effects were more pronounced than clozapine, only clozapine restored cytokine function. CONCLUSION: The data suggest a possible therapeutic route for schizophrenia patients.

Astrocyte-Mediated Neuroinflammation in Neurological Conditions.

Astrocytes are one of the key glial types of the central nervous system (CNS), accounting for over 20% of total glial cells in the brain. Extensive evidence has established their indispensable functions in the maintenance of CNS homeostasis, as well as their broad involvement in neurological conditions. In particular, astrocytes can participate in various neuroinflammatory processes, e.g., releasing a repertoire of cytokines and chemokines or specific neurotrophic factors, which result in both beneficial and detrimental effects. It has become increasingly clear that such astrocyte-mediated neuroinflammation, together with its complex crosstalk with other glial cells or immune cells, designates neuronal survival and the functional integrity of neurocircuits, thus critically contributing to disease onset and progression. In this review, we focus on the current knowledge of the neuroinflammatory responses of astrocytes, summarizing their common features in neurological conditions. Moreover, we highlight several vital questions for future research that promise novel insights into diagnostic or therapeutic strategies against those debilitating CNS diseases.

Cellular Profile of Subfornical Organ Insulin Receptors in Mice.

Brain insulin receptor signaling is strongly implicated in cardiovascular and metabolic physiological regulation. In particular, we recently demonstrated that insulin receptors within the subfornical organ (SFO) play a tonic role in cardiovascular and metabolic regulation in mice. The SFO is a forebrain sensory circumventricular organ that regulates cardiometabolic homeostasis due to its direct exposure to the circulation and thus its ability to sense circulating factors, such as insulin. Previous work has demonstrated broad distribution of insulin receptor-expressing cells throughout the entire SFO, indirectly indicating insulin receptor expression in multiple cell types. Based on this, we sought to determine the cellular phenotypes that express insulin receptors within the SFO by combining immunohistochemistry with genetically modified reporter mouse models. Interestingly, SFO neurons, including both excitatory and inhibitory types, were the dominant cell site for insulin receptor expression, although a weak degree of insulin receptor expression was also detected in astrocytes. Moreover, SFO angiotensin type 1a receptor neurons also expressed insulin receptors. Collectively, these anatomical findings indicate the existence of potentially complex cellular networks within the SFO through which insulin signaling can influence physiology and further point to the SFO as a possible brain site for crosstalk between angiotensin-II and insulin.

Comprehensive Analysis of the 5xFAD Mouse Model of Alzheimer's Disease Using dMRI, Immunohistochemistry, and Neuronal and Glial Functional Metabolic Mapping.

Alzheimer's disease (AD) is characterized by complex interactions between neuropathological markers, metabolic dysregulation, and structural brain changes. In this study, we utilized a multimodal approach, combining immunohistochemistry, functional metabolic mapping, and microstructure sensitive diffusion MRI (dMRI) to progressively investigate these interactions in the 5xFAD mouse model of AD. Our analysis revealed age-dependent and region-specific accumulation of key AD markers, including amyloid-beta (Aß), GFAP, and IBA1, with significant differences observed between the hippocampal formation and upper and lower regions of the cortex by 6 months of age. Functional metabolic mapping validated localized disruptions in energy metabolism, with glucose hypometabolism in the hippocampus and impaired astrocytic metabolism in the cortex. Notably, increased cortical glutaminolysis suggested a shift in microglial metabolism, reflecting an adaptive response to neuroinflammatory processes. While dMRI showed no significant microstructural differences between 5xFAD and wild-type controls, the study highlights the importance of metabolic alterations as critical events in AD pathology. These findings emphasize the need for targeted therapeutic strategies addressing specific metabolic disturbances and underscore the potential of integrating advanced imaging with metabolic and molecular analyses to advance our understanding of AD progression.

Changes in Cx43 and AQP4 Proteins, and the Capture of 3 kDa Dextran in Subpial Astrocytes of the Rat Medial Prefrontal Cortex after Both Sham Surgery and Sciatic Nerve Injury.

A subpopulation of astrocytes on the brain's surface, known as subpial astrocytes, constitutes the "glia limitans superficialis" (GLS), which is an interface between the brain parenchyma and the cerebrospinal fluid (CSF) in the subpial space. Changes in connexin-43 (Cx43) and aquaporin-4 (AQP4) proteins in subpial astrocytes were examined in the medial prefrontal cortex at postoperative day 1, 3, 7, 14, and 21 after sham operation and sciatic nerve compression (SNC). In addition, we tested the altered uptake of TRITC-conjugated 3 kDa dextran by reactive subpial astrocytes. Cellular immunofluorescence (IF) detection and image analysis were used to examine changes in Cx43 and AQP4 protein levels, as well as TRITC-conjugated 3 kDa dextran, in subpial astrocytes. The intensity of Cx43-IF was significantly increased, but AQP4-IF decreased in subpial astrocytes of sham- and SNC-operated rats during all survival periods compared to naïve controls. Similarly, the uptake of 3 kDa dextran in the GLS was reduced following both sham and SNC operations. The results suggest that both sciatic nerve injury and peripheral tissue injury alone can induce changes in subpial astrocytes related to the spread of their reactivity across the cortical surface mediated by increased amounts of gap junctions. At the same time, water transport and solute uptake were impaired in subpial astrocytes.

Single Nuclei Transcriptomics Reveals Obesity-Induced Endothelial and Neurovascular Dysfunction: Implications for Cognitive Decline.

Obesity confers risk for cardiovascular disease and vascular dementia. However, genomic alterations modulated by obesity in endothelial cells in the brain and their relationship to other neurovascular unit (NVU) cells are unknown. We performed single nuclei RNA sequencing (snRNAseq) of the NVU (endothelial cells, astrocytes, microglia, and neurons) from the hippocampus of obese (ob/ob) and wild-type (WT) male mice to characterize obesity-induced transcriptomic changes in a key brain memory center and assessed blood-brain barrier permeability (BBB) by gadolinium-enhanced magnetic resonance imaging (MRI). Ob/ob mice displayed obesity, hyperinsulinemia, and impaired glucose tolerance. snRNAseq profiled 14 distinct cell types and 32 clusters within the hippocampus of ob/ob and WT mice and uncovered differentially expressed genes (DEGs) in all NVU cell types, namely, 4462 in neurons, 1386 in astrocytes, 125 in endothelial cells, and 154 in microglia. Gene ontology analysis identified important biological processes such as angiogenesis in endothelial cells and synaptic trafficking in neurons. Cellular pathway analysis included focal adhesion and insulin signaling, which were common to all NVU cell types. Correlation analysis revealed significant positive correlations between endothelial cells and other NVU cell types. Differentially expressed long non-coding RNAs (lncRNAs) were observed in cells of the NVU-affecting pathways such as TNF and mTOR. BBB permeability showed a trend toward increased signal intensity in ob/ob mice. Taken together, our study provides in-depth insight into the molecular mechanisms underlying cognitive dysfunction in obesity and may have implications for therapeutic gene targeting.

Persistence of post-stress blood pressure elevation requires activation of astrocytes.

The reflexive excitation of the sympathetic nervous system in response to psychological stress leads to elevated blood pressure, a condition that persists even after the stress has been alleviated. This sustained increase in blood pressure, which may contribute to the pathophysiology of hypertension, could be linked to neural plasticity in sympathetic nervous activity. Given the critical role of astrocytes in various forms of neural plasticity, we investigated their involvement in maintaining elevated blood pressure during the post-stress phase. Specifically, we examined the effects of arundic acid, an astrocytic inhibitor, on blood pressure and heart rate responses to air-jet stress. First, we confirmed that the inhibitory effect of arundic acid is specific to astrocytes. Using c-Fos immunohistology, we then observed that psychological stress activates neurons in cardiovascular brain regions, and that this stress-induced neuronal activation was suppressed by arundic acid pre-treatment in rats. By evaluating astrocytic process thickness, we also confirmed that astrocytes in the cardiovascular brain regions were activated by stress, and this activation was blocked by arundic acid pre-treatment. Next, we conducted blood pressure measurements on unanesthetized, unrestrained rats. Air-jet stress elevated blood pressure, which remained high for a significant period during the post-stress phase. However, pre-treatment with arundic acid, which inhibited astrocytic activation, suppressed stress-induced blood pressure elevation both during and after stress. In contrast, arundic acid had no significant impact on heart rate. These findings suggest that both neurons and astrocytes play integral roles in stress-induced blood pressure elevation and its persistence after stress, offering new insights into the pathophysiological mechanisms underlying hypertension.