Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.DOI: http://dx.doi.org/10.7554/eLife.14315.001

To clarify the role of microglia in brain homeostasis and disease, an understanding of their maintenance, proliferation and turnover is essential. The lifespan of brain microglia, however, remains uncertain, and reflects confounding factors in earlier assessments that were largely indirect. We genetically labeled single resident microglia in living mice and then used multiphoton microscopy to monitor these cells over time. Under homeostatic conditions, we found that neocortical resident microglia were long-lived, with a median lifetime of well over 15 months; thus, approximately half of these cells survive the entire mouse lifespan. While proliferation of resident neocortical microglia under homeostatic conditions was low, microglial proliferation in a mouse model of Alzheimer's β-amyloidosis was increased threefold. The persistence of individual microglia throughout the mouse lifespan provides an explanation for how microglial priming early in life can induce lasting functional changes and how microglial senescence may contribute to age-related neurodegenerative diseases.

Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~ 1 mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations.

Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.cyclic GMP | gap junctions | ovarian follicle | oocyte meiosis | luteinizing hormone M eiosis in mammalian oocytes begins during embryonic development and then arrests in late prophase, for up to 50 y in women and for many months in mice. At the time of ovulation, luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to release the arrest and restart meiosis in preparation for fertilization (1-3). In the mouse preovulatory follicle, inhibition of meiotic progression is dependent upon the cyclic nucleotide cyclic GMP (cGMP), which diffuses from the granulosa cells into the oocyte through gap junctions that connect all cells of the follicle (4-6). The cGMP is produced by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2, also known as guanylyl cyclase B), which is present in all of the granulosa cells, but not in the oocyte (7-11). In the oocyte, cGMP inhibits the degradation of another cyclic nucleotide, cAMP, which depends primarily on the phosphodiesterase PDE3A, an enzyme whose activity is antagonized by cGMP (4, 5). The resulting high level of cAMP, through a series of intermediate steps, inhibits meiotic progression (2, 3, 12).LH signaling is initiated in the outer (mural) layers of granulosa cells; receptors for LH are absent in the oocyte and in the granulosa cells that directly surround it (the cumulus cells) (13-15). Ensuing events cause meiosis to resume by reducing cGMP in the oocyte (4, 5), but how LH receptor activation up to 10 cell layers away lowers oocyt...

Rationale Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. Objective To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer–based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. Methods and Results Mouse lines with smooth muscle–specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase–activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were <100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide–induced cGMP transients with peak concentrations of ≈1 to >3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide–stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. Conclusions These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.

To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119 ؉ erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119 ؉ cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca 2؉ concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.apoptosis ͉ Ca2ϩ channels ͉ phosphatidylserine ͉ spleen N itric oxide (NO) has been shown to be a powerful regulator of cell survival (1, 2). Depending on the source or concentration of NO and the influence of additional regulators, NO may stimulate or inhibit apoptosis (3). NO exerts its effects in part through S-nitrosylation of target proteins. However, the effect of NO donors on Ca 2ϩ -induced phosphatidylserine (PS) exposure, a hallmark of apoptosis, could be mimicked by cGMP analogs (4), suggesting the involvement of soluble guanylyl cyclase, cGMP, and cGMP-dependent protein kinase type I (cGKI), a well known signaling cascade downstream of NO (5, 6).Recent studies indicated that erythroid cells possess a functional NO/cGMP pathway (7-9), which may be involved in the regulation of eryptosis, the suicidal death of erythrocytes (10). Erythrocyte cGMP production might also be stimulated by NO generated in the endothelium. The cGKI can also be activated independent of cGMP in response to oxidative stress (11). Eryptosis may follow osmotic shock, energy depletion, and oxidative stress (10), which activate Ca 2ϩ -permeable cation channels (12). Subsequent Ca 2ϩ entry leads to activation of Ca 2ϩ -sensitive K ϩ channels, exit of KCl with osmotically obliged water, and thus cell shrinkage (13). In addition, Ca 2ϩ entry triggers Ca 2ϩ -sensitive scrambling of the cell membrane (14, 15) with subsequent exposure of PS at the erythrocyte surface (12). Cell membrane scrambling may further be triggered by ceramide (16) and activation of protein kinase C (17). PS-exposing erythrocytes bind to PS receptors (18) and are recognized, ...

Background.-Functional Magnetic Resonance Imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available due to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, while microscopic (e.g., 2-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a

Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.