The Role of Microglia in Sex- and Region-Specific Blood-Brain Barrier Integrity During Nicotine Withdrawal.

Background: Smoking is the largest preventable cause of death and disease in the United States, with <5% of quit attempts being successful. Microglia activation and proinflammatory neuroimmune signaling in reward neurocircuitry are implicated in nicotine withdrawal symptomology. Microglia are integral regulators of blood-brain barrier (BBB) functionality as well; however, whether the effects of nicotine withdrawal on microglia function impact BBB integrity is unknown. Methods: Mice were treated with chronic nicotine (12 mg/kg/day) and subjected to 48 hours nicotine withdrawal. Regional BBB permeability, together with messenger RNA and protein expression of tight junction proteins, were assessed. PLX5622 chow was used to deplete microglia to evaluate the role of microglia in regulating BBB integrity and nicotine withdrawal symptomology. Results: Female mice had higher baseline BBB permeability in the prefrontal cortex and hippocampus than males. Nicotine withdrawal further exacerbated the BBB permeability selectively in the prefrontal cortex of females. These effects were concurrent with prefrontal cortex alterations in a subset of tight junction proteins with increased proinflammatory responses following nicotine withdrawal in females. Depletion of microglia via PLX5622 treatment prevented all these molecular effects and attenuated withdrawal-induced anxiety-like behavior in female mice. Conclusions: These results are the first to show sex differences in regional BBB permeability during nicotine withdrawal. This represents a possible link to both the reduced smoking cessation success seen in women and women's increased risk for smoking-related neurovascular disorders. Furthermore, these findings open an avenue for sex-specific therapeutics that target microglia and BBB dysfunction during nicotine withdrawal in women.

Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria.

OBJECTIVES: This study aimed to elucidate mechanistic explanation(s) for compositional changes to enteric microbiota by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. METHODS: In vitro cultures of the gastrointestinal bacteria (Bacteroides fragilis 25285, Prevotella bryantii B14, and Acetoanaerobium sticklandii SR) were continuously exposed to nicotine or cotinine. Supernatant samples were collected for fermentation acid analysis. Vesicles were collected and analyzed for physiological changes in number, size, and total protein cargo. Cultured macrophages were stimulated to a tolerogenic phenotype, exposed to control or altered (nicotine or cotinine - exposed) vesicles, and inflammatory plasticity assessed via inflammatory cytokine production. RESULTS: Nicotine/cotinine exposure differentially affected metabolism of all bacteria tested in a Gram (nicotine) and concentration-dependent (cotinine) manner. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Continuous exposure to 1 µM nicotine and 10 µM cotinine concentrations reduced total protein cargo of Gram (-) - 25285 and B14 vesicles, while cotinine generally increased total protein in Gram (+) - SR vesicles. We found that theses physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Tolerogenic macrophages exposed to vesicles from 1 µM nicotine, and 5 or 10 µΜ cotinine cultures produced significantly less IL-12p70, TNFα, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. CONCLUSIONS: Nicotine/cotinine exposure differentially alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.

Programming Cell-Derived Vesicles with Enhanced Immunomodulatory Properties.

Tumor-associated macrophages are the predominant immune cells present in the tumor microenvironment and mostly exhibit a pro-tumoral M2-like phenotype. However, macrophage biology is reversible allowing them to acquire an anti-tumoral M1-like phenotype in response to external stimuli. A potential therapeutic strategy for treating cancer may be achieved by modulating macrophages from an M2 to an M1-like phenotype with the tumor microenvironment. Here, programmed nanovesicles are generated as an immunomodulatory therapeutic platform with the capability to re-polarize M2 macrophages toward a proinflammatory phenotype. Programmed nanovesicles are engineered from cellular membranes to have specific immunomodulatory properties including the capability to bidirectionally modulate immune cell polarization. These programmed nanovesicles decorated with specific membrane-bound ligands can be targeted toward specific cell types including immune cells. Macrophage-derived vesicles are engineered to enhance immune cell reprogramming toward a proinflammatory phenotype.

Study of Calcium Signaling in Astrocytes with a Novel Endoplasmic Reticulum-Targeted GCaMP Sensor.

The endoplasmic reticulum (ER), the major organelle for the storage of Ca2+ , maintains a concentration of Ca2+ much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca2+ activity in neuronal and glial cells, but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important for measuring Ca2+ transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca2+ exchange between the ER and other organelles, such as the mitochondria. We recently developed an ER-targeted GCaMP6f anchored to the cytosolic side of the ER that can measure minute calcium exchange occurring in this region. In this article, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and assess its expression and calcium imaging in astrocytes in rodent brains. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression and characterization of ER-GCaMP6f Support Protocol 1: ER-GCaMP6f-expressing stable cell line generation Basic Protocol 2: In vitro calcium imaging with ER-GCaMP6f Support Protocol 2: Imaging of drug-induced calcium activity Alternate Protocol 1: Transduction of astrocytes with ER-GCaMP6f AAV Alternate Protocol 2: Calcium imaging of astrocytes with Fluo-4 AM Basic Protocol 3: In vivo ER-GCaMP6f expression and slice calcium imaging Support Protocol 3: Pharmacological studies with 2-APB in brain slices.

ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes.

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.

Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia: Roles of P2 receptors and CD39 in ATP-stimulated microglia.

Adenosine triphosphate (ATP) and its metabolites drive microglia migration and cytokine production by activating P2X- and P2Y- class purinergic receptors. Purinergic receptor activation gives rise to diverse intracellular calcium (Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these characteristics of diverse waveforms influence microglia function is not well-established. We developed a computational model trained with data from published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration, as well as, how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced Ca2+ waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. Namely, it was necessary to account for CD39 metabolism of ATP to align the model's predicted purinoreceptor responses with published experimental data. In addition, our modeling results indicate that small Ca2+ transients accompany migration, while large and sustained transients are needed for cytokine responses. Lastly, as a proof-of-principal, we predict Ca2+ transients and cell membrane displacements in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model may be extrapolated to other microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence microglial responses to ATP.

Tinospora cordifolia (Willd.) Miers: phytochemical composition, cytotoxicity, proximate analysis and their biological activities.

The present research work has been performed to evaluate the phenolic content, flavonoids content, and cytotoxicity of a multidimensional medicinal plant; Tinospora cordifolia and as well as to determine nutritive value by proximate analysis. The total phenolic and flavonoids contents of Tinospora cordifolia were found to be significantly greater in methanol extract as compared to corresponding hexane extract. Brine shrimp bioassay indicated Tinospora cordifolia is pharmacologically active. The percentage composition of different nutrition parameters namely moisture, total ash, crude fat, protein, fibre, carbohydrate, and vitamin C were assessed. The nutritive values of fresh and dried stem samples were evaluated as 156.44 Kcal/100g and 232.61 Kcal/100g respectively. From Gas column mass spectrometry analysis, it can be reported that inositol, 1-deoxy-, trans-sinapyl alcohol, n-hexadecanoic acid were present in the major amount in methanol stem extract. The findings from this study reveal Tinospora cordifolia contains an adequate amount of phenolic and flavonoids content, vital bioactive antioxidant compounds, and a good source of carbohydrates and fibers which potentially adds to the overall value of the plant.

Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique.

Astrocytic processes interact with synapses throughout the brain modulating neurotransmitter signaling and synaptic communication. During conditions such as exposure to drugs of abuse and neurological diseases, astrocytes respond by altering their morphological and functional properties. Reactive astrocyte phenotypes exhibit a bushy morphology with altered soma volume and an increased number of processes compared to resting astrocytes. The reactive astrocytic phenotype also overexpresses proteins one of which can be glial fibrillary acidic protein (GFAP). Fluorescence microscopy on thin tissue sections (<20 µm) requires reconstruction, often through multiple sections, to delineate the full astrocytic morphology. In contrast, tissue clearing methods have been developed that enable imaging of larger sections including the whole brain, providing an opportunity to see in-depth changes in single cell structure. In this article, a detailed protocol for studying astrocyte morphology using tissue clearing and subsequent imaging of whole brains as well as region-specific slices is provided. This method is ideal for understanding the effect of different physiological conditions on astrocyte morphology. A standard biochemistry laboratory has the resources to accomplish tissue clearing using this protocol and most universities have the required imaging facilities. Protocols to study brains from both genetically modified mice that contain an astrocyte-specific marker and from wild-type mice using antibody labeling steps after tissue clearing are provided. We also describe general protocols to conduct fluorescence imaging of astrocytes in cleared tissue to characterize their morphology. This protocol could be useful for researchers working in the rapidly growing field of astrocyte biology. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Brain perfusion, fixation, and tissue clearing Alternate Protocol: Clearing brain tissue with passive clarity Basic Protocol 2: Antibody labeling and refractive index matching Basic Protocol 3: Fluorescence imaging and characterization of astrocyte morphology.

Single-Molecule Studies of Membrane Receptors from Brain Region Specific Nanovesicles.

Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with isolating single proteins within a biological system. Here we provide a detailed protocol to conduct ex vivo single molecule imaging where single transmembrane proteins are isolated by rapidly extracting nanovesicles containing receptors of interest from different regions of the brain and subjecting them to single molecule study by using total internal reflection fluorescence (TIRF) microscopy. This protocol discusses the isolation and separation of brain region specific nanovesicles as well as a detailed method to perform TIRF microscopy with those nanovesicles at the single molecule level. This technique can be applied to study trafficking and stoichiometry of various transmembrane proteins from the central nervous system. This approach can be applied to a wide range of animals that are genetically modified to express a membrane protein-fluorescent protein fusion with a wide range of potential applications in many aspects of neurobiology. Graphic abstract: EX vivo single molecue imaging of membrane receptors.

Nicotine induces morphological and functional changes in astrocytes via nicotinic receptor activity.

Nicotine is a highly addictive compound present in tobacco, which causes the release of dopamine in different regions of the brain. Recent studies have shown that astrocytes express nicotinic acetylcholine receptors (nAChRs) and mediate calcium signaling. In this study, we examine the morphological and functional adaptations of astrocytes due to nicotine exposure. Utilizing a combination of fluorescence and atomic force microscopy, we show that nicotine-treated astrocytes exhibit time-dependent remodeling in the number and length of both proximal and fine processes. Blocking nAChR activity with an antagonist completely abolishes nicotine's influence on astrocyte morphology indicating that nicotine's action is mediated by these receptors. Functional studies show that 24-hr nicotine treatment induces higher levels of calcium activity in both the cell soma and the processes with a more substantial change observed in the processes. Nicotine does not induce reactive astrocytosis even at high concentrations (10 µM) as determined by cytokine release and glial fibrillary acidic protein expression. We designed tissue clearing experiments to test whether morphological changes occur in vivo using astrocyte specific Aldh1l1-tdTomato knock in mice. We find that nicotine induces a change in the volume of astrocytes in the prefrontal cortex, CA1 of the hippocampus, and the substantia nigra. These results indicate that nicotine directly alters the functional and morphological properties of astrocytes potentially contributing to the underlying mechanism of nicotine abuse.

Photoluminescence Enhancement, Blinking Suppression, and Improved Biexciton Quantum Yield of Single Quantum Dots in Zero Mode Waveguides.

The capability of quantum dots to generate both single and multiexcitons can be harnessed for a wide variety of applications, including those that require high optical gain. Here, we use time-correlated photoluminescence (PL) spectroscopy to demonstrate that the isolation of single CdSeTe/ZnS core-shell, nanocrystal quantum dots (QDs) in Zero Mode Waveguides (ZMWs) leads to a significant modification in PL intensity, blinking dynamics, and biexciton behavior. QDs in aluminum ZMWs (AlZMWs) exhibited a 15-fold increase in biexciton emission, indicating a preferential enhancement of the biexciton radiative decay rate as compared to the single exciton rate. The increase in biexciton behavior was accompanied by a decrease in blinking events due to a shortening in the dark state residence time. These results indicate that plasmon mediated enhanced decay rates of QDs in AlZMWs lead to substantial changes in the photophysical properties of single quantum dots, including an increase in biexciton behavior.

Macrophage-Engineered Vesicles for Therapeutic Delivery and Bidirectional Reprogramming of Immune Cell Polarization.

Macrophages, one of the most important phagocytic cells of the immune system, are highly plastic and are known to exhibit diverse roles under different pathological conditions. The ability to repolarize macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) or vice versa offers a promising therapeutic approach for treating various diseases such as traumatic injury and cancer. Herein, it is demonstrated that macrophage-engineered vesicles (MEVs) generated by disruption of macrophage cellular membranes can be used as nanocarriers capable of reprogramming macrophages and microglia toward either pro- or anti-inflammatory phenotypes. MEVs can be produced at high yields and easily loaded with diagnostic molecules or chemotherapeutics and delivered to both macrophages and cancer cells in vitro and in vivo. Overall, MEVs show promise as potential delivery vehicles for both therapeutics and their ability to controllably modulate macrophage/microglia inflammatory phenotypes.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe "pokes" the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.