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.

DNA-AgNC Loaded Liposomes for Measuring Cerebral Blood Flow Using Two-Photon Fluorescence Correlation Spectroscopy.

Unraveling the transport of drugs and nanocarriers in cerebrovascular networks is important for pharmacokinetic and hemodynamic studies but is challenging due to the complexity of sensing individual particles within the circulatory system of a live animal. Here, we demonstrate that a DNA-stabilized silver nanocluster (DNA-Ag16NC) that emits in the first near-infrared window upon two-photon excitation in the second NIR window can be used for multiphoton in vivo fluorescence correlation spectroscopy for the measurement of cerebral blood flow rates in live mice with high spatial and temporal resolution. To ensure bright and stable emission during in vivo experiments, we loaded DNA-Ag16NCs into liposomes, which served the dual purposes of concentrating the fluorescent label and protecting it from degradation. DNA-Ag16NC-loaded liposomes enabled the quantification of cerebral blood flow velocities within individual vessels of a living mouse.

Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy.

BACKGROUND: Skeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy. METHODS: To identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse. RESULTS: In this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest. CONCLUSIONS: Our findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Dendritic Cell Membrane-Derived Nanovesicles for Targeted T Cell Activation.

T cells play an integral role in the generation of an effective immune response and are responsible for clearing foreign microbes that have bypassed innate immune system defenses and possess cognate antigens. The immune response can be directed toward a desired target through the selective priming and activation of T cells. Due to their ability to activate a T cell response, dendritic cells and endogenous vesicles from dendritic cells are being developed for cancer immunotherapy treatment. However, current platforms, such as exosomes and synthetic nanoparticles, are limited by their production methods and application constraints. Here, we engineer nanovesicles derived from dendritic cell membranes with similar properties as dendritic cell exosomes via nitrogen cavitation. These cell-derived nanovesicles are capable of activating antigen-specific T cells through direct and indirect mechanisms. Additionally, these nanovesicles can be produced in large yields, overcoming production constraints that limit clinical application of alternative immunomodulatory vesicle or nanoparticle-based methods. Thus, dendritic cell-derived nanovesicles generated by nitrogen cavitation show potential as an immunotherapy platform to stimulate and direct T cell response.

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.

Gold Ion Beam Milled Gold Zero-Mode Waveguides.

Zero-mode waveguides (ZMWs) are widely used in single molecule fluorescence microscopy for their enhancement of emitted light and the ability to study samples at physiological concentrations. ZMWs are typically produced using photo or electron beam lithography. We report a new method of ZMW production using focused ion beam (FIB) milling with gold ions. We demonstrate that ion-milled gold ZMWs with 200 nm apertures exhibit similar plasmon-enhanced fluorescence seen with ZMWs fabricated with traditional techniques such as electron beam lithography.

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.

Exploring the Role of Surface States in Emissive Carbon Nanodots: Analysis at Single-Particle Level.

Fluorescent carbon nanodots (CDs) have been highlighted as promising semiconducting materials due to their outstanding chemical and optical properties. However, the intrinsic heterogeneity of CDs has impeded a clear understanding of the mechanisms behind their photophysical properties. In this study, as-prepared CDs are fractionated via chromatography to reduce their structural and chemical heterogeneity and analyzed through ensemble and single-particle spectroscopies. Many single particles reveal fluorescence intensity fluctuations between two or more discrete levels with bi-exponential decays. While the intrinsic τ1 components are uniform among single particles, the τ2 components from molecule-like emissions spans a wider range of lifetimes, reflecting the inhomogeneity of the surface states. Furthermore, it is concluded that the relative population and chemical states of surface functional groups in CDs have a significant impact on emissive states, brightness, blinking, stability, and lifetime distribution of photoluminescence.

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.

Neuron-Derived Extracellular Vesicles Modulate Microglia Activation and Function.

Microglia act as the immune cells of the central nervous system (CNS). They play an important role in maintaining brain homeostasis but also in mediating neuroimmune responses to insult. The interactions between neurons and microglia represent a key process for neuroimmune regulation and subsequent effects on CNS integrity. However, the molecular mechanisms of neuron-glia communication in regulating microglia function are not fully understood. One recently described means of this intercellular communication is via nano-sized extracellular vesicles (EVs) that transfer a large diversity of molecules between neurons and microglia, such as proteins, lipids, and nucleic acids. To determine the effects of neuron-derived EVs (NDEVs) on microglia, NDEVs were isolated from the culture supernatant of rat cortical neurons. When NDEVs were added to primary cultured rat microglia, we found significantly improved microglia viability via inhibition of apoptosis. Additionally, application of NDEVs to cultured microglia also inhibited the expression of activation surface markers on microglia. Furthermore, NDEVs reduced the LPS-induced proinflammatory response in microglia according to reduced gene expression of proinflammatory cytokines (TNF-α, IL-6, MCP-1) and iNOS, but increased expression of the anti-inflammatory cytokine, IL-10. These findings support that neurons critically regulate microglia activity and control inflammation via EV-mediated neuron-glia communication. (Supported by R21AA025563 and R01AA025591).

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.

Early satellite cell communication creates a permissive environment for long-term muscle growth.

Using in vivo muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, in vitro cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses Wisp1 expression required for appropriate ECM remodeling. Late-stage communication from myogenic cells during loading is widespread but may be targeted toward endothelial cells. Satellite cells coordinate adaptation by influencing the phenotype of recipient cells, which extends our understanding of their role in muscle adaptation beyond regeneration and myonuclear donation.

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.

Fusion-Independent Satellite Cell Communication to Muscle Fibers During Load-Induced Hypertrophy.

The "canonical" function of Pax7+ muscle stem cells (satellite cells) during hypertrophic growth of adult muscle fibers is myonuclear donation via fusion to support increased transcriptional output. In recent years, however, emerging evidence suggests that satellite cells play an important secretory role in promoting load-mediated growth. Utilizing genetically modified mouse models of delayed satellite cell fusion and in vivo extracellular vesicle (EV) tracking, we provide evidence for satellite cell communication to muscle fibers during hypertrophy. Myogenic progenitor cell-EV-mediated communication to myotubes in vitro influences extracellular matrix (ECM)-related gene expression, which is congruent with in vivo overload experiments involving satellite cell depletion, as well as in silico analyses. Satellite cell-derived EVs can transfer a Cre-induced, cytoplasmic-localized fluorescent reporter to muscle cells as well as microRNAs that regulate ECM genes such as matrix metalloproteinase 9 (Mmp9), which may facilitate growth. Delayed satellite cell fusion did not limit long-term load-induced muscle hypertrophy indicating that early fusion-independent communication from satellite cells to muscle fibers is an underappreciated aspect of satellite cell biology. We cannot exclude the possibility that satellite cell-mediated myonuclear accretion is necessary to maintain prolonged growth, specifically in the later phases of adaptation, but these data collectively highlight how EV delivery from satellite cells can directly contribute to mechanical load-induced muscle fiber hypertrophy, independent of cell fusion to the fiber.

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.

In Vivo Single-Molecule Detection of Nanoparticles for Multiphoton Fluorescence Correlation Spectroscopy to Quantify Cerebral Blood Flow.

We present the application of multiphoton in vivo fluorescence correlation spectroscopy (FCS) of fluorescent nanoparticles for the measurement of cerebral blood flow with excellent spatial and temporal resolution. Through the detection of single nanoparticles within the complex vessel architecture of a live mouse, this new approach enables the quantification of nanoparticle dynamics occurring within the vasculature along with simultaneous measurements of blood flow properties in the brain. In addition to providing high resolution blood flow measurements, this approach enables real-time quantification of nanoparticle concentration, degradation, and transport. This method is capable of quantifying flow rates at each pixel with submicron resolution to enable monitoring of dynamic changes in flow rates in response to changes in the animal's physiological condition. Scanning the excitation beam using FCS provides pixel by pixel mapping of flow rates with subvessel resolution across capillaries 300 µm deep in the brains of mice.