Protein nanobarcodes enable single-step multiplexed fluorescence imaging.

Multiplexed cellular imaging typically relies on the sequential application of detection probes, as antibodies or DNA barcodes, which is complex and time-consuming. To address this, we developed here protein nanobarcodes, composed of combinations of epitopes recognized by specific sets of nanobodies. The nanobarcodes are read in a single imaging step, relying on nanobodies conjugated to distinct fluorophores, which enables a precise analysis of large numbers of protein combinations. Fluorescence images from nanobarcodes were used as input images for a deep neural network, which was able to identify proteins with high precision. We thus present an efficient and straightforward protein identification method, which is applicable to relatively complex biological assays. We demonstrate this by a multicell competition assay, in which we successfully used our nanobarcoded proteins together with neurexin and neuroligin isoforms, thereby testing the preferred binding combinations of multiple isoforms, in parallel.

Using different geometries to modulate the cardiac fibroblast phenotype and the biomechanical properties of engineered connective tissues.

Tissue engineering with human cardiac fibroblasts (CF) allows identifying novel mechanisms and anti-fibrotic drugs in the context of cardiac fibrosis. However, substantial knowledge on the influences of the used materials and tissue geometries on tissue properties and cell phenotypes is necessary to be able to choose an appropriate model for a specific research question. As there is a clear lack of information on how CF react to the mold architecture in engineered connective tissues (ECT), we first compared the effect of two mold geometries and materials with different hardnesses on the biomechanical properties of ECT. We could show that ECT, which formed around two distant poles (non-uniform model) were less stiff and more strain-resistant than ECT, which formed around a central rod (uniform model), independent of the materials used for poles and rods. Next, we investigated the cell state and could demonstrate that in the uniform versus non-uniform model, the embedded cells have a higher cell cycle activity and display a more pronounced myofibroblast phenotype. Differential gene expression analysis revealed that uniform ECT displayed a fibrosis-associated gene signature similar to the diseased heart. Furthermore, we were able to identify important relationships between cell and tissue characteristics, as well as between biomechanical tissue parameters by implementing cells from normal heart and end-stage heart failure explants from patients with ischemic or dilated cardiomyopathy. Finally, we show that the application of pro- and anti-fibrotic factors in the non-uniform and uniform model, respectively, is not sufficient to mimic the effect of the other geometry. Taken together, we demonstrate that modifying the mold geometry in tissue engineering with CF offers the possibility to compare different cellular phenotypes and biomechanical tissue properties.

Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R.

The brain extracellular matrix (ECM) consists of extremely long-lived proteins that assemble around neurons and synapses, to stabilize them. The ECM is thought to change only rarely, in relation to neuronal plasticity, through ECM proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules. Using multiple ECM labeling and imaging assays, from super-resolution optical imaging to nanoscale secondary ion mass spectrometry, both in culture and in brain slices, we find that a key ECM protein, Tenascin-R, is frequently endocytosed, and later resurfaces, preferentially near synapses. The TNR molecules complete this cycle within ~3 days, in an activity-dependent fashion. Interfering with the recycling process perturbs severely neuronal function, strongly reducing synaptic vesicle exo- and endocytosis. We conclude that the neuronal ECM can be remodeled frequently through mechanisms that involve endocytosis and recycling of ECM proteins.

Human brain organoids assemble functionally integrated bilateral optic vesicles.

During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day 30, brain organoids attempt to assemble optic vesicles, which develop progressively as visible structures within 60 days. These optic vesicle-containing brain organoids (OVB-organoids) constitute a developing optic vesicle's cellular components, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. OVB-organoids also display synapsin-1, CTIP-positive myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photosensitive activity of OVB-organoids, and light sensitivities could be reset after transient photobleaching. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow interorgan interaction studies within a single organoid.

Developmental GABA polarity switch and neuronal plasticity in Bioengineered Neuronal Organoids.

Brain organoids are promising tools for disease modeling and drug development. For proper neuronal network formation excitatory and inhibitory neurons as well as glia need to co-develop. Here, we report the directed self-organization of human induced pluripotent stem cells in a collagen hydrogel towards a highly interconnected neuronal network at a macroscale tissue format. Bioengineered Neuronal Organoids (BENOs) comprise interconnected excitatory and inhibitory neurons with supportive astrocytes and oligodendrocytes. Giant depolarizing potential (GDP)-like events observed in early BENO cultures mimic early network activity of the fetal brain. The observed GABA polarity switch and reduced GDPs in >40 day BENO indicate progressive neuronal network maturation. BENOs demonstrate expedited complex network burst development after two months and evidence for long-term potentiation. The similarity of structural and functional properties to the fetal brain may allow for the application of BENOs in studies of neuronal plasticity and modeling of disease.

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology.

AIMS: Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. RESULTS: Our redox indicator mice widely express Thy1-driven roGFP1 (reduction-oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. INNOVATION AND CONCLUSION: Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell- and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding of cellular redox signals in their full complexity. Antioxid. Redox Signal. 25, 41-58.

Ca(2+)-BK channel clusters in olfactory receptor neurons and their role in odour coding.

Olfactory receptor neurons (ORNs) have high-voltage-gated Ca(2+) channels whose physiological impact has remained enigmatic since the voltage-gated conductances in this cell type were first described in the 1980s. Here we show that in ORN somata of Xenopus laevis tadpoles these channels are clustered and co-expressed with large-conductance potassium (BK) channels. We found approximately five clusters per ORN and twelve Ca(2+) channels per cluster. The action potential-triggered activation of BK channels accelerates the repolarization of action potentials and shortens interspike intervals during odour responses. This increases the sensitivity of individual ORNs to odorants. At the level of mitral cells of the olfactory bulb, odour qualities have been shown to be coded by first-spike-latency patterns. The system of Ca(2+) and BK channels in ORNs appears to be important for correct odour coding because the blockage of BK channels not only affects ORN spiking patterns but also changes the latency pattern representation of odours in the olfactory bulb.

Response properties of the genetically encoded optical H2O2 sensor HyPer.

Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H2O2 reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H2O2 treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H2O2 was still present. Although HyPer should be H2O2 specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Cl(-) exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H2O2-albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H2O2 increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H2O2 decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.

Fast and accurate fitting and filtering of noisy exponentials in Legendre space.

The parameters of experimentally obtained exponentials are usually found by least-squares fitting methods. Essentially, this is done by minimizing the mean squares sum of the differences between the data, most often a function of time, and a parameter-defined model function. Here we delineate a novel method where the noisy data are represented and analyzed in the space of Legendre polynomials. This is advantageous in several respects. First, parameter retrieval in the Legendre domain is typically two orders of magnitude faster than direct fitting in the time domain. Second, data fitting in a low-dimensional Legendre space yields estimates for amplitudes and time constants which are, on the average, more precise compared to least-squares-fitting with equal weights in the time domain. Third, the Legendre analysis of two exponentials gives satisfactory estimates in parameter ranges where least-squares-fitting in the time domain typically fails. Finally, filtering exponentials in the domain of Legendre polynomials leads to marked noise removal without the phase shift characteristic for conventional lowpass filters.

An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions.

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.

Regulation of amygdalar PKA by beta-arrestin-2/phosphodiesterase-4 complex is critical for fear conditioning.

Beta-arrestins, key regulators of receptor signaling, are highly expressed in the central nervous system, but their roles in brain physiology are largely unknown. Here we show that beta-arrestin-2 is critically involved in the formation of associative fear memory and amygdalar synaptic plasticity. In response to fear conditioning, beta-arrestin-2 translocates to amygdalar membrane where it interacts with PDE-4, a cAMP-degrading enzyme, to inhibit PKA activation. Arrb2(-/-) mice exhibit impaired conditioned fear memory and long-term potentiation at the lateral amygdalar synapses. Moreover, expression of the beta-arrestin-2 in the lateral amygdala of Arrb2(-/-) mice, but not its mutant form that is incapable of binding PDE-4, restores basal PKA activity and rescues conditioned fear memory. Taken together, our data demonstrate that the feedback regulation of amygdalar PKA activation by beta-arrestin-2 and PDE-4 complex is critical for the formation of conditioned fear memory.

Interactions of Na+,K+-ATPase and co-expressed delta-opioid receptor.

To investigate interference of delta-opioid receptor with the Na(+),K(+)-ATPase in a simple model system, we used the Xenopus oocytes as an expression system. Our results indicate that expression of the delta-opioid receptor (DOR) results in reduction of endogenous sodium-pump activity. Stimulation of DOR by the DOR agonist [(D)-Pen(2,5)]-enkephalin (DPDPE) had no pronounced additional effect on pump activity. Qualitatively similar results were obtained in experiments with a variety of co-expressed exogenous sodium pumps. We suggest that reduced pump activity with DOR expression is brought about by an interaction of the pump with DOR. Direct interaction is also supported by co-immunoprecipitation, not only in the Xenopus oocytes but also in rat hippocampal neurons. The interaction may be responsible for altered agonist sensitivity of DOR; activation of the sodium pump led to an increase of the K(m) value for DOR activation by DPDPE from about 0.17 to 0.27muM. In conclusion, pump activity not only affects neural activity directly but our results also suggest that pump activity is affected through functional interaction with DOR that will modulate pain sensation.

Stimulation- and palmitoylation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptors.

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP- and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.

Morphine and heroin differentially modulate in vivo hippocampal LTP in opiate-dependent rat.

Addictive drugs have been shown to severely influence many neuronal functions, which are considered as the underlying mechanisms for physiological and psychological dependences. We previously showed that in vivo LTP in rat hippocampal CA1 region is significantly reduced during withdrawal following chronic opiates treatment, and the reduced LTP can be restored by re-exposure of animals to corresponding drugs. Here, we further demonstrated that during opiates withdrawal, the re-exposure of morphine either systemically (subcutaneously) or locally (intracerebroventricularly) could restore the reduced LTP in heroin-dependent rats, but heroin could not restore the reduced LTP, in morphine-dependent rats, indicating differential modulations of hippocampal functions by those two opiates. In contrast, DAMGO, a mu-opioid receptor (MOR) agonist, could restore the reduced LTP, and CTOP, a MOR antagonist, could block the restoration in rats dependent on both opiates, showing that MOR is functional under such conditions. However, the upregulation of hippocampal PKA activity during morphine withdrawal could be suppressed by re-exposure of morphine but not that of heroin, suggesting a likely underlying mechanism of the differential modulation of LTP by two opiates. Taken together, our study clearly demonstrates that chronic abuse of opiates inevitably leads to severe alteration of hippocampal LTP, and reveals the interesting differences between morphine and heroin in their effects on the differential modulation of hippocampal synaptic plasticity.

Activation of beta2-adrenergic receptor stimulates gamma-secretase activity and accelerates amyloid plaque formation.

Amyloid plaque is the hallmark and primary cause of Alzheimer disease. Mutations of presenilin-1, the gamma-secretase catalytic subunit, can affect amyloid-beta (Abeta) production and Alzheimer disease pathogenesis. However, it is largely unknown whether and how gamma-secretase activity and amyloid plaque formation are regulated by environmental factors such as stress, which is mediated by receptors including beta(2)-adrenergic receptor (beta(2)-AR). Here we report that activation of beta(2)-AR enhanced gamma-secretase activity and thus Abeta production. This enhancement involved the association of beta(2)-AR with presenilin-1 and required agonist-induced endocytosis of beta(2)-AR and subsequent trafficking of gamma-secretase to late endosomes and lysosomes, where Abeta production was elevated. Similar effects were observed after activation of delta-opioid receptor. Furthermore, chronic treatment with beta(2)-AR agonists increased cerebral amyloid plaques in an Alzheimer disease mouse model. Thus, beta(2)-AR activation can stimulate gamma-secretase activity and amyloid plaque formation, which suggests that abnormal activation of beta(2)-AR might contribute to Abeta accumulation in Alzheimer disease pathogenesis.

A nuclear function of beta-arrestin1 in GPCR signaling: regulation of histone acetylation and gene transcription.

Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. Here, we show that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of beta-arrestin 1 (betaarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, betaarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. Our results reveal a novel function of betaarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification.

Modification of hippocampal neurogenesis and neuroplasticity by social environments.

Synaptic plasticity and neurogenesis in the brain are affected by environmental stimuli. The present study was designed to investigate the effects of social environments on learning and memory, neurogenesis, and neuroplasticity. Twenty-two-day-old rats were housed in isolation or in groups for 4 or 8 weeks and injected intraperitoneally with bromodeoxyuridine to detect proliferation among progenitor cells. The animals were also tested for learning in a water maze and for hippocampal CA1 long-term potentiation in vivo and in vitro. The results show that the number of newborn neurons in the dentate gyrus and the learning in a water maze decreased significantly in rats reared in isolation for 4 or 8 weeks, as compared with grouped controls. Induction of long-term potentiation in the CA1 area of rat hippocampus in vivo and in vitro was also significantly reduced by isolation. Furthermore, the effects of isolation rearing on spatial learning, hippocampal neurogenesis, and long-term potentiation could be reversed by subsequent group rearing. These findings demonstrated that social environments can modify neurogenesis and synaptic plasticity in adult hippocampal regions, which is associated with alterations in spatial learning and memory.

Morphine withdrawal increases glutamate uptake and surface expression of glutamate transporter GLT1 at hippocampal synapses.

Opiate abuse causes adaptive changes in several processes of synaptic transmission in which the glutamatergic system appears a critical element involved in opiate tolerance and dependence, but the underlying mechanisms remain unclear. In the present study, we found that glutamate uptake in hippocampal synaptosomes was significantly increased (by 70% in chronic morphine-treated rats) during the morphine withdrawal period, likely attributable to an increase in the number of functional glutamate transporters. Immunoblot analysis showed that expression of GLT1 (glutamate transporter subtype 1) was identified to be upregulated in synaptosomes but not in total tissues, suggesting a redistribution of glutamate transporter expression. Moreover, the increase in glutamate uptake was reproduced in cultured neurons during morphine withdrawal, and the increase of uptake in neurons could be blocked by dihydrokainate, a specific inhibitor of GLT1. Cell surface biotinylation and immunoblot analysis showed that morphine withdrawal produced an increase in GLT1 expression rather than EAAC1 (excitatory amino acids carrier 1), a neuronal subtype, at the cultured neuronal cell surface, whereas no significant change was observed in that of cultured astrocytes. Electron microscopy also revealed that GLT1 expression was markedly increased in the nerve terminals of hippocampus and associated with the plasma membrane in vivo. These results suggest that GLT1 in hippocampal neurons can be induced to translocate to the nerve terminals and express on the cell surface during morphine withdrawal. The translocation of GLT1 at synapses during morphine withdrawal provides a neuronal mechanism for modulation of excitatory neurotransmission during opiate abuse.

Hippocampal long-term potentiation is reduced by chronic opiate treatment and can be restored by re-exposure to opiates.

Chronic exposure to opiates eventually leads to drug addiction, which is believed to involve maladaptive changes in brain function, but the underlying neuronal mechanisms remain primarily unknown. Given the known effects of opiates such as morphine and heroin on hippocampal function, we investigated the potential effect of chronic opiate treatment on long-term potentiation (LTP) at CA1 synapses in rat hippocampus, a leading experimental model for studying synaptic plasticity. Our results revealed that chronic exposure of rats to morphine or heroin, which induced severe drug tolerance and dependence, markedly reduced the capacity of hippocampal CA1 LTP during the period of drug withdrawal (from approximately 190% in control to approximately 120%). More interestingly, the capacity of LTP could be restored to the normal level by re-exposure of the animals to opiates, indicating that the synaptic function was already adapted to opiates. Morris water maze test, which measures behavioral consequences of synaptic plasticity, showed parallel learning deficits after chronic exposure to opiates. Moreover, the opiate-reduced LTP could also be restored by inhibitors of cAMP-dependent protein kinase A (PKA), suggesting that upregulation of cAMP pathway was likely one of the underlying mechanisms of the observed phenomena. These findings demonstrated that chronic opiate treatment can significantly modulate synaptic plasticity in the hippocampus, leading to an opiate dependence of the plasticity.