Activity-driven synaptic translocation of LGI1 controls excitatory neurotransmission.

The fine control of synaptic function requires robust trans-synaptic molecular interactions. However, it remains poorly understood how trans-synaptic bridges change to reflect the functional states of the synapse. Here, we develop optical tools to visualize in firing synapses the molecular behavior of two trans-synaptic proteins, LGI1 and ADAM23, and find that neuronal activity acutely rearranges their abundance at the synaptic cleft. Surprisingly, synaptic LGI1 is primarily not secreted, as described elsewhere, but exo- and endocytosed through its interaction with ADAM23. Activity-driven translocation of LGI1 facilitates the formation of trans-synaptic connections proportionally to the history of activity of the synapse, adjusting excitatory transmission to synaptic firing rates. Accordingly, we find that patient-derived autoantibodies against LGI1 reduce its surface fraction and cause increased glutamate release. Our findings suggest that LGI1 abundance at the synaptic cleft can be acutely remodeled and serves as a critical control point for synaptic function.

Destroy the old to build the new: Activity-dependent lysosomal exocytosis in neurons.

Lysosomes are organelles that support diverse cellular functions such as terminal degradation of macromolecules and nutrient recycling. Additionally, lysosomes can fuse with the plasma membrane, a phenomenon referred to as lysosomal exocytosis, to release their contents, including hydrolytic enzymes and cargo proteins. Recently, neuronal activity has been shown to induce lysosomal exocytosis in dendrites and axons. Secreted lysosomal enzyme cathepsin B induces and stabilizes synaptic structural changes by degrading the local extracellular matrix. Extracellular matrix reorganization could also enhance the lateral diffusion of the co-released synaptic organizer Cbln1 along the surface of axons to facilitate new synapse formation. Similarly, lateral diffusion of dendritic AMPA-type glutamate receptors could be facilitated to enhance functional synaptic plasticity. Therefore, lysosomal exocytosis is a powerful way of building new cellular structures through the coordinated destruction of the old environment. Understanding the mechanisms by which lysosomal exocytosis is regulated in neurons is expected to lead to the development of new therapeutics for neuronal plasticity following spinal cord injury or neurodegenerative disease.

Resilience to capsaicin-induced mitochondrial damage in trigeminal ganglion neurons.

Capsaicin is an agonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). Strong TRPV1 stimulation with capsaicin causes mitochondrial damage in primary sensory neurons. However, the effect of repetitive and moderate exposure to capsaicin on the integrity of neuronal mitochondria remains largely unknown. Our electron microscopic analysis revealed that repetitive stimulation of the facial skin of mice with 10 mM capsaicin induced short-term damage to the mitochondria in small-sized trigeminal ganglion neurons. Further, capsaicin-treated mice exhibited decreased sensitivity to noxious heat stimulation, indicating TRPV1 dysfunction, in parallel with the mitochondrial damage in the trigeminal ganglion neurons. To analyze the capsaicin-induced mitochondrial damage and its relevant cellular events in detail, we performed cell-based assays using TRPV1-expressing PC12 cells. Dose-dependent capsaicin-mediated mitochondrial toxicity was observed. High doses of capsaicin caused rapid destruction of mitochondrial internal structure, while low doses induced mitochondrial swelling. Further, capsaicin induced a dose-dependent loss of mitochondria and autophagy-mediated degradation of mitochondria (mitophagy). Concomitantly, transcriptional upregulation of mitochondrial proteins, cytochrome c oxidase subunit IV, Mic60/Mitofilin, and voltage-dependent anion channel 1 was observed, which implied induction of mitochondrial biogenesis to compensate for the loss of mitochondria. Collectively, although trigeminal ganglion neurons transiently exhibit mitochondrial damage and TRPV1 dysfunction following moderate capsaicin exposure, they appear to be resilient to such a challenge. Our in vitro data show a dose-response relationship in capsaicin-mediated mitochondrial toxicity. We postulate that induction of mitophagy and mitochondrial biogenesis in response to capsaicin stimulation play important roles in repairing the damaged mitochondrial system.

Novel luciferase-opsin combinations for improved luminopsins.

Previous work has demonstrated that fusion of a luciferase to an opsin, to create a luminescent opsin or luminopsin, provides a genetically encoded means of manipulating neuronal activity via both chemogenetic and optogenetic approaches. Here we have expanded and refined the versatility of luminopsin tools by fusing an alternative luciferase variant with high light emission, Gaussia luciferase mutant GLucM23, to depolarizing and hyperpolarizing channelrhodopsins with increased light sensitivity. The combination of GLucM23 with Volvox channelrhodopsin-1 produced LMO4, while combining GLucM23 with the anion channelrhodopsin iChloC yielded iLMO4. We found efficient activation of these channelrhodopsins in the presence of the luciferase substrate, as indicated by responses measured in both single neurons and in neuronal populations of mice and rats, as well as by changes in male rat behavior during amphetamine-induced rotations. We conclude that these new luminopsins will be useful for bimodal opto- and chemogenetic analyses of brain function.

Activity-Dependent Secretion of Synaptic Organizer Cbln1 from Lysosomes in Granule Cell Axons.

Synapse formation is achieved by various synaptic organizers. Although this process is highly regulated by neuronal activity, the underlying molecular mechanisms remain largely unclear. Here we show that Cbln1, a synaptic organizer of the C1q family, is released from lysosomes in axons but not dendrites of cerebellar granule cells in an activity- and Ca2+-dependent manner. Exocytosed Cbln1 was retained on axonal surfaces by binding to its presynaptic receptor neurexin. Cbln1 further diffused laterally along the axonal surface and accumulated at boutons by binding postsynaptic δ2 glutamate receptors. Cbln1 exocytosis was insensitive to tetanus neurotoxin, accompanied by cathepsin B release, and decreased by disrupting lysosomes. Furthermore, overexpression of lysosomal sialidase Neu1 not only inhibited Cbln1 and cathepsin B exocytosis in vitro but also reduced axonal bouton formation in vivo. Our findings imply that co-release of Cbln1 and cathepsin B from lysosomes serves as a new mechanism of activity-dependent coordinated synapse modification.

Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology.

Background Recent genome-wide association studies have identified transient receptor potential M8 ( TRPM8) as a migraine susceptibility gene. TRPM8 is a nonselective cation channel that mediates cool perception. However, its precise role in migraine pathophysiology is elusive. Transient receptor potential V1 (TRPV1) is a nonselective cation channel activated by noxious heat. Both TRPM8 and TRPV1 are expressed in trigeminal ganglion (TG) neurons. Methods We investigated the functional roles of TRPM8 and TRPV1 in a meningeal inflammation-based migraine model by measuring the effects of facial TRPM8 activation on thermal allodynia and assessing receptor coexpression changes in TG neurons. We performed retrograde tracer labeling to identify TG neurons innervating the face and dura. Results We found that pharmacological TRPM8 activation reversed the meningeal inflammation-induced lowering of the facial heat pain threshold, an effect abolished by genetic ablation of TRPM8. No significant changes in the heat pain threshold were seen in sham-operated animals. Meningeal inflammation caused dynamic alterations in TRPM8/TRPV1 coexpression patterns in TG neurons, and colocalization was most pronounced when the ameliorating effect of TRPM8 activation on thermal allodynia was maximal. Our tracer assay disclosed the presence of dura-innervating TG neurons sending collaterals to the face. Approximately half of them were TRPV1-positive. We also demonstrated functional inhibition of TRPV1 by TRPM8 in a cell-based assay using c-Jun N-terminal kinase phosphorylation as a surrogate marker. Conclusions Our findings provide a plausible mechanism to explain how facial TRPM8 activation can relieve migraine by suppressing TRPV1 activity. Facial TRPM8 appears to be a promising therapeutic target for migraine.

Signaling Pathways Relevant to Nerve Growth Factor-induced Upregulation of Transient Receptor Potential M8 Expression.

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that primarily detects the innocuous cold. In pathological conditions, TRPM8 plays a role in the development of cold hyperalgesia/allodynia. Nerve growth factor (NGF) is an important mediator involved in various pain disorders. In the present study, the NGF-TrkA pathway increased TRPM8 expression by stabilizing TRPM8 mRNA through the actions of phosphatidylinositol 3-kinase and p38 MAP kinase. Moreover, c-Jun N-terminal kinase and Src tyrosine kinase were identified as a positive and negative regulator of TRPM8 expression, respectively, via post-transcriptional mechanisms independent of mRNA stabilization. PTEN activity was found to increase protein TRPM8 expression. Calcium imaging confirmed that NGF induced TRPM8 functional upregulation. Time-lapse fluorescence microscopic analysis and a cell fractionation assay revealed that NGF promoted the trafficking of TRPM8 to the plasma membrane. In the presence of NGF, lysosome-associated membrane protein-2 (LAMP-2) was localized to TRPM8-positive dot-like and linear structures, the latter of which were observed in the periphery of the cytoplasm. It was inferred that LAMP-2 was involved in the vesicular transport of TRPM8. Pharmacological blockade of the proteasome with MG132 led to a further increase in NGF-induced TRPM8 expression, indicating that the proteasome system played a pivotal role in the degradation of TRPM8. Our findings provide novel insight into the signaling pathways involved in NGF-mediated TRPM8 upregulation and its reversion to the normal state.

Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons.

Chemical labelling for visualizing native AMPA receptors in live neurons.

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors.

Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.

Controlling the Regional Identity of hPSC-Derived Neurons to Uncover Neuronal Subtype Specificity of Neurological Disease Phenotypes.

The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs.

Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors.

BACKGROUND: The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets. RESULTS: Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state. CONCLUSIONS: Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.

Development of luminescent coelenterazine derivatives activatable by ß-galactosidase for monitoring dual gene expression.

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect ß-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing ß-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain ß-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for ß-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect ß-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by ß-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations.

Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

Analysis of aquaporin-mediated diffusional water permeability by coherent anti-stokes Raman scattering microscopy.

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P(f)), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P(d)). Here, we report an accurate and instantaneous method for measuring the P(d) of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H(2)O/D(2)O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ(CARS)) for the external solution H(2)O/D(2)O exchange was 16.1 ms, whereas the intracellular H(2)O/D(2)O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ(CARS) for the intracellular H(2)O/D(2)O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P(d). The average P(d) values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10(-3) and 8.3 ± 2.6 × 10(-4) cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.

Rapid synaptic scaling induced by changes in postsynaptic firing.

Homeostatic synaptic scaling adjusts a neuron's excitatory synaptic strengths up or down to compensate for perturbations in activity. Little is known about the molecular pathway(s) involved, nor is it clear which aspect of "activity"-local synaptic signaling, postsynaptic firing, or large-scale changes in network activity-is required to induce synaptic scaling. Here, we selectively block either postsynaptic firing in individual neurons or a fraction of presynaptic inputs, while optically monitoring changes in synaptic strength. We find that synaptic scaling is rapidly induced by block of postsynaptic firing, but not by local synaptic blockade, and is mediated through a drop in somatic calcium influx, reduced activation of CaMKIV, and an increase in transcription. Cortical neurons thus homeostatically adjust synaptic strengths in response to changes in their own firing rate, a mechanism with the computational advantage of efficiently normalizing synaptic strengths without interfering with synapse-specific mechanisms of information storage.

Postsynaptic expression of homeostatic plasticity at neocortical synapses.

Synaptic scaling is a form of homeostatic plasticity that scales synaptic strengths up or down to compensate for prolonged changes in activity. It has been controversial whether this plasticity is expressed presynaptically, postsynaptically, or both. Here we describe in detail the homeostatic changes that take place at excitatory synapses in visual cortical cultures after 1 or 2 d of activity blockade. After 7-10 d in vitro, activity blockade significantly increased postsynaptic accumulation of synaptic AMPA receptors via proportional increases in glutamate receptor 1 (GluR1) and GluR2. Time-lapse imaging of enhanced green fluorescent protein-tagged AMPA receptors revealed that receptor accumulation increased progressively over 2 d of activity blockade and affected the entire population of imaged synapses. The strength of synaptic connections between pyramidal neurons was more than doubled after activity blockade without affecting short-term depression or the coefficient of variation of the postsynaptic responses. Furthermore, uptake of the fluorescent styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl] pyridinium dibromide) by presynaptic terminals was not different at control and activity-blocked synapses. In addition to the increased accumulation of postsynaptic AMPA receptors, boosting of dendritic AMPA currents by sodium channels was increased by activity blockade. These data indicate that, at young neocortical synapses, synaptic scaling has a predominantly postsynaptic locus and functions as a gain control mechanism to regulate neuronal activity without affecting the dynamics of synaptic transmission.

Non-polarized distribution of synaptotagmin IV in neurons: evidence that synaptotagmin IV is not a synaptic vesicle protein.

Synaptotagmin IV (Syt IV) expression is regulated by neuronal development and by depolarization in the brain and in neuronal cell cultures. In cultures, immunocytochemical analysis has shown that Syt IV is localized at the Golgi and at the tips of growing neurites, but little was known about associations between Syt IV and vesicles or organelles [J. Neurochem. 74 (2000) 518]. In this study we performed an electron microscopic (EM) analysis of developing mouse neocortex to determine the exact localization of Syt IV in native mouse tissues. In neurons of layer II/III, Syt IV was found to be localized in the dendrites and axons, and at the Golgi in the cell body. Some Syt IV signals were clearly associated with vesicles and/or organelles, but EM and cell fractionation studies showed no Syt IV signals at synaptic vesicles. Detection of fluorescence protein-tagged Syt IV (Syt IV-EGFP) in hippocampal neurons also showed the presence of Syt IV-EGFP vesicles or organelles in the axons and dendrites. These results suggest that Syt IV regulates non-polarized membrane trafficking in neurons, which may be involved in synaptic plasticity or neuronal development.

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.