Quantum dot/antibody conjugates for in vivo cytometric imaging in mice

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (∼15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine–norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1⁺c-Kit⁺ cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues.Studying migration of individual endogenous cells and their interactions with the surrounding microenvironment in vivo would greatly help expand our knowledge on how cells behave in their complex native biological network. However, single-cell imaging of a rare population in vivo places highly stringent constraints on targeting fluorophores. First, identifying specific cell populations among various types of cells expressing overlapping surface markers demands simultaneous labeling of multiple markers, and therefore requires fluorophores with narrow emission features. Second, the size of targeting fluorophore conjugates needs to be optimized to simultaneously achieve both long blood circulation times and high diffusion in dense in vivo environments. Third, because only a small fraction of systemically administered fluorophores is delivered to the targeting sites, resulting in low signal, targeting fluorophore conjugates must emit bright signals and exhibit minimal nonspecific binding to serum proteins and cells. Fourth, targeting fluorophore conjugate samples must be free of unbound targeting molecules, because unbound targeting molecules can block target sites and yield decreased signal.Because of the lack of such a technology, direct labeling of single cells from an endogenous rare cell population in live and nonmanipulated animal models has not been possible. Instead, researchers have used (i) immunohistochemistry (1), (ii) ex vivo (2) or intravital imaging (3) of injected target cells, which are fluorescently labeled ex vivo, or (iii) ex vivo (4) or intravital imaging (5, 6) of target cells that are genetically modified to express fluorescent markers. However, none of these methods reproduce the native microenvironment of target cells. Tissue immunohistochemistry and ex vivo imaging only provide a snapshot image of a perturbed state. Intravital imaging allows for real-time imaging but requires either genetically engineered mouse models to induce endogenous expression of fluorescence proteins in cells or irradiation of mice for marrow depletion and repopulation with systemically infused cells.Quantum dots (QDs) possess unique optical properties that are ideal for in vivo imaging in live animals. Specifically, QDs have a tunable band gap ranging from the visible to the IR, high quantum yields (QYs), narrow and symmetric emission features, broad absorption above the band gap, large multiphoton absorption cross-sections, and high photostability (7–10). These properties make QDs amenable to optical multiplexing for simultaneous study of various targets, long-term tracking, and deep-tissue imaging using multiphoton microscopy. Despite these exciting capabilities, most imaging studies using QDs have involved either in vitro targeting or ensemble measurements of QD signals over large volumes of tissue in vivo (11–15). This restriction is caused by a lack of technology for synthesizing QD conjugates that are optimal for single-cell imaging in vivo.Here, we report the development of QD-Ab conjugates that satisfy all of the constraints described above. Moreover, we used these QD-Ab conjugates for in vivo cytometry of endogenous bone marrow cells (BMCs) in their unperturbed microenvironment. Our technique provides opportunities to study the movements of single cells and the interactions between cells and between a cell and its environment in their native states.     

High-resolution,noninvasive longitudinal live imaging of immune responses

Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.     

Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R² = 0.998) or i.v. (R² = 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.     

In vivo visualization of butterfly scale cell morphogenesis in Vanessa cardui

During metamorphosis, the wings of a butterfly sprout hundreds of thousands of scales with intricate microstructures and nano-structures that determine the wings’ optical appearance, wetting characteristics, thermodynamic properties, and aerodynamic behavior. Although the functional characteristics of scales are well known and prove desirable in various applications, the dynamic processes and temporal coordination required to sculpt the scales’ many structural features remain poorly understood. Current knowledge of scale growth is primarily gained from ex vivo studies of fixed scale cells at discrete time points; to fully understand scale formation, it is critical to characterize the time-dependent morphological changes throughout their development. Here, we report the continuous, in vivo, label-free imaging of growing scale cells of Vanessa cardui using speckle-correlation reflection phase microscopy. By capturing time-resolved volumetric tissue data together with nanoscale surface height information, we establish a morphological timeline of wing scale formation and gain quantitative insights into the underlying processes involved in scale cell patterning and growth. We identify early differences in the patterning of cover and ground scales on the young wing and quantify geometrical parameters of growing scale features, which suggest that surface growth is critical to structure formation. Our quantitative, time-resolved in vivo imaging of butterfly scale development provides the foundation for decoding the processes and biomechanical principles involved in the formation of functional structures in biological materials.

The functional structures of butterfly wing scales form during pupal development: scale cells grow protrusions that serve as templates for finely sculpted nanoscale cuticle morphologies (1–3). By tailoring these scale morphologies, butterflies produce unique visual appearances (4–7), ensure thermal regulation (8) and water repellency (9), or generate beneficial acoustic (10) and aerodynamic effects (11). Interdisciplinary interest in these material functionalities has led to scientific advances in the comprehensive assessment of the scales’ multifunctional material properties (12), design of next-generation bioinspired functional materials (13, 14), identification of key genes in patterning and structural color (15–19), and evaluation of the impact of ecological factors on biodiversity (20, 21). Although the enviable functionality of butterfly wings depends heavily on the precise structural architecture of the wing scales, little is known about the dynamics, processes, and phenomena involved in scale development (22).Each scale on a butterfly’s wing is formed by an individual cell, which secretes a chitinous cuticle that forms a single-cell exo- skeleton. In many butterflies, these scales are further organized in rows of alternating cover and ground scale morphologies (1). The mature scales of the painted lady butterfly (Vanessa cardui) exemplify a skeletal scale blueprint, which is widely reflected in both the simple and the sophisticated wing scale morphologies found across Lepidoptera (Fig. 1A–D). In general, the upper surface of a scale consists of ridges running down its length; these ridges are composed of overlapping lamellae and are connected by crossribs (23). Supporting trabeculae bridge the upper features and lower scale surface, which is essentially a thin lamina on the order of 100 nm in thickness. The rich diversity of scale morphologies in other butterflies and moths may be thought of as modulations of the basic structures found in this generic scale architecture. Consequently, the easy-to-rear V. cardui is a favorable model system for gaining insights into the processes and mechanical phenomena underlying biological formation of functional micro- and nanostructures (15, 24, 25).Open in a separate windowFig. 1.Imaging the structure of fully formed butterfly scales. (A) The painted lady butterfly, V. cardui. (B) Optical micrograph of orange and black wing scales. (C) Scanning electron micrograph of individual adult scales, with ridges running down the length of the scale. (D) Magnification of scale finger showing that ridges consist of stacked lamellae and are connected by crossribs. (E) Volumetric image of an adult scale acquired by speckle-correlation reflection phase microscopy (red, top of the volumetric data stack; green, bottom). (F) A single slice of phase data for the same scale. (Scale bars: B, 200 µm; C, 20 µm; D, 5 µm; and E and F, 20 µm.)Key insights into the formation of these structures have resulted from the analysis of dissected and stained wing tissues at discrete developmental time points. Almost a century ago, the sequence of cellular division, scale protrusion, growth, and ridge formation was documented in flour moths (26). Since then, electron microscopy served to elucidate nanoscale structures in the wing tissue and provided glimpses of cuticle growth on the scale cell (27–29). Seminal studies emphasized the optical function of the scale and offered hypotheses for lamella formation on the scale ridges via mechanical wrinkling (30) and for three-dimensional (3D) structure formation via internal membrane templates (23). This rationale has been used to explain how gyroids and other cubic structures form in scales (31). More recently, confocal imaging has allowed closer examination of material distribution, albeit in fixed wing tissues due to a lack of endogenous labeling methods in Lepidoptera. These studies described the signaling factors involved in patterning scale positions (32), explored the role of actin in the formation of scale fingers and ridges (24), and quantified actin bundle spacing and chitin distribution during and after development (25). Together with discrete snapshots from wings fixed at different developmental stages (33), the cuticle structures of mature scales may hint at their formation: in some adult scales, internal gyroids are ordered in increasing size up the length of the scale, possibly illustrating the timing in onset and growth (34).While these time-discrete imaging efforts provide glimpses into scale development, a comprehensive understanding of the processes underlying scale structure formation can be gained only by continuous observation of the spatiotemporal progression of living scale cells (22). Recent exogenous fluorescent imaging of live lepidopteran pupae captured the young scale cell and the initial protrusion of scales (35, 36). Despite this progress, visualizing subcellular features of live scale cells throughout development remains an unsolved challenge, due to complications inherently associated with the imaging of tissues that feature heterogenous and pronounced micro- and nanoscale refractive index variations. Additionally, imaging over long durations with fluorescent techniques is susceptible to photobleaching and photodamage; moreover, genetic constructs for fluorescent labeling in live organisms are still limited for butterflies.Here, we report the continuous, in vivo, label-free imaging of developing scale cells in the living V. cardui butterfly using speckle-correlation reflection phase microscopy (see Fig. 1 for comparison with scanning electron microscopy data). This quantitative phase imaging technique offers a versatile strategy for observing the growth of functional materials in vivo with high temporal and spatial resolution. We capture critical moments of lepidopteran scale structure formation in living organisms on a continuous timeline. In particular, we identified a two-step process of tissue patterning in the early epithelial sheet and quantified the morphological changes occurring across various length scales as scale cells grow. Insights from continuous imaging of scale formation form the foundation for understanding the biomechanical processes involved in the genesis of functional cuticle morphologies.     

Thermodynamics of protein destabilization in live cells

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.Unlike their static impression in X-ray structures and textbook illustrations, some proteins are tuned to work at marginal structural stability. The advantage of such tuning is that it enables the protein to easily switch from one conformation to another, providing sensitive functional control. A well-known example is the tumor suppressor P53 whose function in gene regulation relies on a complex interplay of local folding–unfolding transitions (1). Likewise, the maturation pathway of the radical scavenger Cu/Zn superoxide dismutase (SOD1) involves a marginally stable apo species that seems required for interorganelle trafficking (2) and effective chaperone-assisted metal loading (3). As an inevitable consequence of such near-equilibrium action, however, the proteins become critically sensitive to perturbations (1): Mutation of SOD1 triggers with full penetrance late-onset neurodegenerative disease even though the causative mutations shift the structural equilibrium only by less than a factor of 3 (4). In the latter case, it is not the loss of native function that poses the acute problem, but rather the promotion of competing disordered SOD1 conformations that eventually exhaust the cellular proteostasis system and end up in pathologic deposits (5–8). Uncovering the rules, capacity and limitations of this delicate interplay between individual proteins and the cellular components (9, 10) requires not only information about the in vivo response to molecular perturbations, but also precise quantification of the structural equilibria at play. The question is then, to what extent are existing data obtained under simplified conditions in vitro transferable to the complex environment in live cells (11)? The answer is not clear cut. Defying predictions from steric crowding effects (11–13), experimental data have shown that cells in some cases stabilize (14–19) and in other cases destabilize (20–25) the native protein structures. In this study, we shed light on these seemingly conflicting results by mapping out the thermodynamic behavior of a marginally stable β-barrel protein (SOD1^barrel), using in-cell NMR. Our results show that mammalian and bacterial cells not only destabilize SOD1^barrel, but also render its structure essentially disordered at 37 °C. The effect is assigned to transient interactions with the cellular interior, which counterbalance the crowding pressure, narrow the width of the thermal unfolding transitions, and move both cold and heat unfolding into the physiological regime. Moreover, these transient interactions are seen to be sequence and context dependent, reconciling the previous observations that different proteins yield different results. The emerging picture is thus that proteins are optimized not only for structure and function but also for their interplay with the host-cell environment, raising interesting questions about the physiological manifestation of marginal stability, as well as the constraints on protein behavior across evolutionary diverse organisms.     

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery.     

Real-time measurements of aminoglycoside effects on protein synthesis in live cells

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome’s translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Antibiotic resistance has become one of the biggest public health challenges of the 21st century. What used to be easily treatable diseases are becoming deadly as a consequence of commonly used antibiotics increasingly turning ineffective. To aid the development of new strategies to address this challenge, it is necessary to improve our understanding of the mechanism of action of these antibacterial compounds. Many antibiotics currently in use target the bacterial ribosome with high specificity (1). These compounds affect different stages of protein synthesis, depending on their binding sites in the bacterial ribosome or their binding to protein factors involved in protein synthesis.Aminoglycosides are a class of natural and semisynthetic chemical compounds of broad-spectrum therapeutic relevance (2, 3) categorized as critically important by the World Health Organization (4). Aminoglycosides are presently used against multidrug-resistant bacterial infections (5, 6) and, more recently, considered as potential treatments for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy (3, 7, 8). The clinical relevance of aminoglycosides is only shadowed by side effects such as nephrotoxicity and irreversible ototoxicity (5, 6). A subclass of these molecules has a conserved aminocyclitol, a 2-deoxystreptamine, with linked amino sugar groups at different positions. Structural studies showed that these molecules bind at the major groove of the 16S ribosomal RNA (rRNA) in the A-site in close contact with the decoding center of the bacterial 30S ribosomal subunit (9–12). At the decoding center, the adenines A1492 and A1493 take part in monitoring the correct codon–anticodon interaction (13). Aminoglycoside molecules bound to this site have been suggested to interact with A1492/1493 and restrict their mobility (12, 14), which in turn interferes with the selection of cognate transfer RNA (tRNA) (9, 11, 15–18) as well as with the translocation step (11, 16, 19–22).A secondary binding site for 4,5- and 4,6-substituted aminoglycosides has been identified at H69 in the 50S ribosomal subunit, in close contact with A- and P-site tRNAs (23). Based on crystal structures (23) and in vitro kinetics assays (24), it has been suggested that drugs bound to this secondary binding site affect ribosome recycling and also intersubunit rotation—potentially also affecting translocation.The synergistic effect of aminoglycosides binding to multiple sites in the bacterial ribosome contributes to the misreading of codons and defective translocation, which eventually leads to cell death. The mechanism of action of various aminoglycosides on the ribosome has been characterized using diverse structure biology methods (as reviewed in ref. 25), classical in vitro functional biochemical assays (15, 20, 26), and, more recently, in vitro single-molecule approaches (11, 21, 27). Even though the mechanistic steps are described in detail by these complementary in vitro techniques, the reported effects of these drugs on the kinetics of protein synthesis are significantly different. For example, whereas single-molecule Förster resonance energy transfer (FRET) studies report a four- to sixfold inhibition of messenger RNA (mRNA) movement during translocation (21), stopped-flow experiments report a 160-fold inhibition (20). Recent advances in live-cell single-molecule tracking methods have now opened up the possibility to measure the drug’s effects on protein synthesis kinetics directly in live cells (28, 29).In the present study, we measured the effect of three structurally different aminoglycosides, apramycin, gentamicin, and paromomycin, on the kinetics of translation elongation at a single-ribosome level in live Escherichia coli cells. By tracking single dye-labeled tRNAs and analyzing the diffusion trajectories using a Hidden Markov Model-based (HMM) approach, we measured dwell-times of elongator [Cy5]tRNA^Phe and initiator [Cy5]tRNA^fMet on the ribosome, which suggest an overall slower, but ongoing, protein synthesis in intact cells exposed to the aminoglycosides.     

Moderate hypoglycaemia after learning does not affect memory consolidation and brain activation during recognition in non-diabetic adults

INTRODUCTION: Some aspects of memory performance are impaired during acute hypoglycaemia. The hippocampus is critical to formation of long-term memory, and may be particularly sensitive to hypoglycaemia. This study examined whether moderate hypoglycaemia occurring after learning would disrupt the consolidation process, and used functional magnetic resonance imaging (fMRI) to identify accompanying changes in brain activation. METHODS: Sixteen non-diabetic subjects each underwent two glucose clamp studies. During euglycaemia (4.5 mmol/L), subjects tried to memorize a series of words and a series of pictures of faces. Then, either hypoglycaemia (2.5 mmol/L) was induced for one hour, or euglycaemia was maintained. During subsequent uncontrolled euglycaemia, subjects' recognition of the word and face stimuli was tested, with simultaneous fMRI to measure brain activation during recognition. RESULTS: Face identification scores were 67.2% after euglycaemia and 66.9% after hypoglycaemia (p = 0.895). Word identification scores were 78.0 and 77.1% respectively (p = 0.701). Analysis of the fMRI identified two foci where activation was altered after hypoglycaemia compared with euglycaemia, but these were not in regions associated with memory, and were probably statistical artefacts. CONCLUSIONS: One hour of hypoglycaemia at 2.5 mmol/L induced 20-40 min after learning did not disrupt memory consolidation. fMRI did not show evidence of altered brain activation after hypoglycaemia. Consolidation may be relatively resistant to hypoglycaemia, or may have been complete before hypoglycaemia was induced. The study was powered to detect a large effect, and provides some reassurance that moderate hypoglycaemia does not cause major disruption of previously learned memories in people with insulin-treated diabetes.     

Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice

The selection of reward-seeking and aversive behaviors is controlled by two distinct D1 and D2 receptor-expressing striatal medium spiny neurons, namely the direct pathway MSNs (dMSNs) and the indirect pathway MSNs (iMSNs), but the dynamic modulation of signaling cascades of dMSNs and iMSNs in behaving animals remains largely elusive. We developed an in vivo methodology to monitor Förster resonance energy transfer (FRET) of the activities of PKA and ERK in either dMSNs or iMSNs by microendoscopy in freely moving mice. PKA and ERK were coordinately but oppositely regulated between dMSNs and iMSNs by rewarding cocaine administration and aversive electric shocks. Notably, the activities of PKA and ERK rapidly shifted when male mice became active or indifferent toward female mice during mating behavior. Importantly, manipulation of PKA cascades by the Designer Receptor recapitulated active and indifferent mating behaviors, indicating a causal linkage of a dynamic activity shift of PKA and ERK between dMSNs and iMSNs in action selection.In changing environments, animals are forced to choose actions among several alternatives to survive and to keep offspring for the next generation. A brain region critical for initiation and selection of actions is the striatum (1–3), and its dysfunction leads to devastating neurological and psychiatric disorders such as Parkinson’s disease and drug addiction (4–7). The striatum receives convergent glutamatergic inputs from virtually all cortical areas and the thalamus, and dopaminergic inputs from the substantia nigra pars compacta (SNc) and the ventral tegmental area (8, 9). The glutamatergic inputs convey various sensory, motor, and cognitive information and drive striatal medium spiny projection neurons (MSNs) to fire, whereas the dopaminergic inputs strongly influence synaptic transmission and excitability of MSNs (10, 11). MSNs are divided into two subpopulations, the direct pathway MSN (dMSN) expressing Gs-coupled dopamine D1 receptors and sending axons directly to the internal segment of the globus pallidus (GPi) and the substantia nigra pars reticulata (SNr), and the indirect pathway MSN (iMSN) expressing Gi-coupled D2 receptors and sending axons indirectly to the SNr via the external segment of the globus pallidus (GPe) and subthalamic nucleus (12, 13).Intracellular signaling cascades operating in these two types of MSNs are important for plastic modification of synaptic transmission and excitability (14, 15). Among them, protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) have been shown to be the key molecules in these signaling cascades (10, 14, 16). PKA is positively and negatively regulated by Gs-coupled D1 and Gi-coupled D2 receptors, respectively, and contributes to the regulation of a wide range of cellular substrates (10, 11, 16). ERK has been shown to sense coincidental dopaminergic and glutamatergic activations to induce protein synthesis and synaptic modification (17).Although both the ventral and dorsal striatum are essential for naturally occurring reward-seeking and aversive behaviors, such as eating, mating, and escaping from uncomfortable environments (7, 18, 19), the dorsal striatum plays a key role in efficiently choosing suitable outcomes of actions (1, 2). Among naturally occurring rewarding behaviors, male mating behavior is particularly interesting, because it represents innate rapid selection behavior, sometimes actively seeking a female animal but at other times becoming impassive toward it (20, 21). However, little is known about how dMSNs and iMSNs are involved in such action selection. The poor understanding of dynamic intracellular signaling mechanisms in action selection behavior is mainly due to the lack of effective techniques to monitor time-lapsed changes in such activities in behaving animals. In this study, we developed an in vivo methodology in which genetically encoded Förster resonance energy transfer (FRET) biosensors of PKA and ERK (22) were specifically expressed in either dMSNs (D1-PKA and D1-ERK) or iMSNs (D2-PKA and D2-ERK), and cell-specific fluorescence changes in the active and inactive forms of PKA and ERK of the dorsal striatum were monitored by newly designed microendoscopy in freely moving mice (23, 24). We revealed that the activities of PKA and ERK in dMSNs and iMSNs rapidly shifted when male mice became active or impassive toward a female mouse during mating behavior and that the dynamic activity shift of PKA and ERK between dMSNs and iMSNs was causally linked to the selection of alternative actions.     

Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood-brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a "Nissl stain" contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimer's disease using (traditionally) non-BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ～18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.     

桂枝加减汤对MRL小鼠心肌细胞凋亡及Bax mRNA、Caspase3mRNA基因表达的影响

目的:探讨复方中药对MRL小鼠心肌细胞凋亡的作用及机理.方法:将MRL小鼠分为复方中药组、桂枝组、强的松组和空白对照组,检测MRL小鼠心肌组织凋亡细胞,计算心肌细胞凋亡指数(AI),RT-PCR法检测心肌细胞凋亡基因Bax mRNA、Caspase 3 mRNA表达,观察复方中药(组成:丹参、桂枝、西洋参、冰片)对MRL小鼠心肌细胞凋亡的影响.结果:复方中药能抑制Bax mRNA、Caspase 3 mRNA表达,明显抑制心肌细胞凋亡(心肌细胞凋亡指数显著下降).其疗效明显优于桂枝组和空白对照组(P<0.05),与强的松组疗效相当,P>0.05.结论:复方中药可能通过调控凋亡基因Bax mRNA、Caspase 3 mRNA表达而抑制心肌细胞凋亡,从而延缓或部分逆转MRL心肌损害的发生、发展.     

应用实时PCR检测红鹿感染牛型结核杆菌后IFN-γ和IL-4 mRNA的表达

目的：揭示红鹿抗结核杆菌感染的免疫反应途径。方法：共采用20只红鹿，其中一组10只对牛型结核杆菌(Mycobacterium boris，M．bovis)有自然抵抗力，另一组10只对M．bovis敏感。两组各取5只接种活的卡介苗(BCG)，余下5只不接种。接种卡介苗8周后所有的红鹿都注射有毒力的M．bovis，在注射M．bovis 4周前以及6周后分别收集外周血单核细胞(Peripheral blood mononuclear cells，PBMC)，PBMC在与美洲商陆原(Pokeweed Mitogen，PWM)共育后测定细胞因子Interferon-γ(IFN-γ)和Interleukin-4(IL-4)mRNA的表达水平。mRNA的定量测定是在ABI PRISM7700序列检测系统上应用TaqMan Real-timell PCR技术进行的。结果：在红鹿接受M．bovis刺激前后PBMC中IFN-γ和IL-4 mRNA的表达水平有显著性差异。结论：红鹿抵抗结核杆菌感染的免疫反应是复杂的混合细胞反应型，Th1，Th2细胞反应均加强。     

正常老年人数字记忆功能的磁共振成像

目的 探讨正常健康老年人数字工作记忆的脑加工神经机制.方法 实验采用事件相关设计,利用功能磁共振技术(fMRI)对15例正常老年人进行数字工作记忆的数据采集,数据采用AFNI 软件进行数据分析和脑功能区活动图像,分析编码期、保持期、提取期的脑激活情况.结果 进行数字记忆工作任务时,15例正常老年人平均反应时为(1 102.4±99.3)ms,正确率为(95.2±2.5)%.数字工作记忆的编码期激活双侧中央前回、双侧梭状回、双侧枕下回、双侧小脑;保持期脑激活位于左侧中央前回、中央后回、岛叶、额下回及额中回、双侧壳核、双侧梭状回及右额下回也有少量激活;提取期脑激活位于双侧壳核、双侧丘脑、双侧岛叶、双侧顶下小叶、左侧额下回、左侧中央后回、左侧颞中回、左侧颞下回、双侧小脑.结论 左侧额叶在正常老年人脑数字工作记忆中起重要作用,其他脑区也共同参与完成数字工作记忆的信息处理过程.     

Imaging T-cell movement in the brain during experimental cerebral malaria

T-cells are known to play a role in the pathology associated with experimental cerebral malaria, although it has not previously been possible to examine their behaviour in brain. Using multiphoton laser scanning microscopy, we have examined the migration and movement of these cells in brain tissue. We believe that this approach will help define host–parasite interactions and examine how intervening in these relationships affects the development of cerebral pathology.     

Effect of resveratrol treatment on graft revascularization after islet transplantation in streptozotocin-induced diabetic mice

We evaluated the effect of resveratrol (RSV) on graft survival after islet transplantation (ITx) in diabetic mice. Isolated islets from Balb/c mice (200 IEQ) were transplanted under the kidney capsule of diabetic Balb/c mice. Vehicle or RSV (200 mg/kg/day, orally) was given for 14 days after ITx. Two more control groups [STZ-treated (No-ITx-Control) and STZ+RSV-treated (No-ITx-RSV) mice without ITx] were added. Glucose tolerance tests (GTT) was performed at 14 days after ITx. In vitro, isolated islets pretreated with vehicle or RSV (1 μM) were incubated in a hypoxic chamber (O₂ 1%, 1hr). Some of the ITx was performed in mouse insulin 1 gene promoter-green fluorescent protein (MIP-GFP) transgenic mice and analyzed using an in vivo imaging system. After 14 days of ITx, 2-hr glucose levels on GTT in the RSV-treated group were significantly lower than those of other control groups. But the glucose status was not improved in No-ITx mice with RSV. At day 3, the percentage of Ki-67/insulin co-stained cells in islet graft was significantly increased in the RSV-ITx group. Immunostaining with anti-insulin and anti-BS-1 antibodies revealed significantly higher insulin-stained area and vascular density in RSV-treated islet grafts. The mean vessel volume per islet graft measured by in vivo imaging was significantly higher in the RSV-treated group at day 3. In isolated islets cultured in hypoxic conditions, the cell death rate and oxidative stress were significantly attenuated with RSV pretreatment. Hypoxic treatment for isolated islets decreased the expression of SIRT-1 mRNA, and this attenuation was recovered by RSV pretreatment. Our data suggest that RSV treatment improved glycemic control, beta-cell proliferation, reduced oxidative stress, and enhanced islet revascularization and the outcome of ITx in diabetic mice.     

Imaging rapid redistribution of sensory-evoked depolarization through existing cortical pathways after targeted stroke in mice

Evidence suggests that recovery from stroke damage results from the production of new synaptic pathways within surviving brain regions over weeks. To address whether brain function might redistribute more rapidly through preexisting pathways, we examined patterns of sensory-evoked depolarization in mouse somatosensory cortex within hours after targeted stroke to a subset of the forelimb sensory map. Brain activity was mapped with voltage-sensitive dye imaging allowing millisecond time resolution over 9 mm² of brain. Before targeted stroke, we report rapid activation of the forelimb area within 10 ms of contralateral forelimb stimulation and more delayed activation of related areas of cortex such as the hindlimb sensory and motor cortices. After stroke to a subset of the forelimb somatosensory cortex map, function was lost in ischemic areas within the forelimb map center, but maintained in regions 200–500 μm from blood flow deficits indicating the size of a perfused, but nonfunctional, penumbra. In many cases, stroke led to only partial loss of the forelimb map, indicating that a subset of a somatosensory domain can function on its own. Within the forelimb map spared by stroke, forelimb-stimulated responses became delayed in kinetics, and their center of activity shifted into adjacent hindlimb and posterior-lateral sensory areas. We conclude that the focus of forelimb-specific somatosensory cortex activity can be rapidly redistributed after ischemic damage. Given that redistribution occurs within an hour, the effect is likely to involve surviving accessory pathways and could potentially contribute to rapid behavioral compensation or direct future circuit rewiring.