Integrated Control of Predatory Hunting by the Central Nucleus of the Amygdala.

Superior predatory skills led to the evolutionary triumph of jawed vertebrates. However, the mechanisms by which the vertebrate brain controls predation remain largely unknown. Here, we reveal a critical role for the central nucleus of the amygdala in predatory hunting. Both optogenetic and chemogenetic stimulation of central amygdala of mice elicited predatory-like attacks upon both insect and artificial prey. Coordinated control of cervical and mandibular musculatures, which is necessary for accurately positioning lethal bites on prey, was mediated by a central amygdala projection to the reticular formation in the brainstem. In contrast, prey pursuit was mediated by projections to the midbrain periaqueductal gray matter. Targeted lesions to these two pathways separately disrupted biting attacks upon prey versus the initiation of prey pursuit. Our findings delineate a neural network that integrates distinct behavioral modules and suggest that central amygdala neurons instruct predatory hunting across jawed vertebrates.

Enhancing Human Treg Cell Induction through Engineered Dendritic Cells and Zinc Supplementation.

Regulatory T (Treg) cells hold promise for the ultimate cure of immune-mediated diseases. However, how to effectively restore Treg function in patients remains unknown. Previous reports suggest that activated dendritic cells (DCs) de novo synthesize locally high concentrations of 1,25-dihydroxy vitamin D, i.e., the active vitamin D or 1,25(OH)2D by upregulating the expression of 25-hydroxy vitamin D 1α-hydroxylase. Although 1,25(OH)2D has been shown to induce Treg cells, DC-derived 1,25(OH)2D only serves as a checkpoint to ensure well-balanced immune responses. Our animal studies have shown that 1,25(OH)2D requires high concentrations to generate Treg cells, which can cause severe side effects. In addition, our animal studies have also demonstrated that dendritic cells (DCs) overexpressing the 1α-hydroxylase de novo synthesize the effective Treg-inducing 1,25(OH)2D concentrations without causing the primary side effect of hypercalcemia (i.e., high blood calcium levels). This study furthers our previous animal studies and explores the efficacy of the la-hydroxylase-overexpressing DCs in inducing human CD4+FOXP3+regulatory T (Treg) cells. We discovered that the effective Treg-inducing doses of 1,25(OH)2D were within a range. Additionally, our data corroborated that the 1α-hydroxylase-overexpressing DCs synthesized 1,25(OH)2D within this concentration range in vivo, thus facilitating effective Treg cell induction. Moreover, this study demonstrated that 1α-hydroxylase expression levels were pivotal for DCs to induce Treg cells because physiological 25(OH)D levels were sufficient for the engineered but not parental DCs to enhance Treg cell induction. Interestingly, adding non-toxic zinc concentrations significantly augmented the Treg-inducing capacity of the engineered DCs. Our new findings offer a novel therapeutic avenue for immune-mediated human diseases, such as inflammatory bowel disease, type 1 diabetes, and multiple sclerosis, by integrating zinc with the 1α-hydroxylase-overexpressing DCs.

Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Rational Design of NIR-II G-Quadruplex Fluorescent Probes for Accurate In Vivo Tumor Metastasis Imaging.

Accurate in vivo imaging of G-quadruplexes (G4) is critical for understanding the emergence and progression of G4-associated diseases like cancer. However, existing in vivo G4 fluorescent probes primarily operate within the near-infrared region (NIR-I), which limits their application accuracy due to the short emission wavelength. The transition to second near-infrared (NIR-II) fluorescent imaging has been of significant interest, as it offers reduced autofluorescence and deeper tissue penetration, thereby facilitating more accurate in vivo imaging. Nonetheless, the advancement of NIR-II G4 probes has been impeded by the absence of effective probe design strategies. Herein, through a "step-by-step" rational design approach, we have successfully developed NIRG-2, the first small-molecule fluorescent probe with NIR-II emission tailored for in vivo G4 detection. Molecular docking calculations reveal that NIRG-2 forms stable hydrogen bonds and strong π-π interactions with G4 structures, which effectively inhibit twisted intramolecular charge transfer (TICT) and, thereby, selectively illuminate G4 structures. Due to its NIR-II emission (940 nm), large Stokes shift (90 nm), and high selectivity, NIRG-2 offers up to 47-fold fluorescence enhancement and a tissue imaging depth of 5 mm for in vivo G4 detection, significantly outperforming existing G4 probes. Utilizing NIRG-2, we have, for the first time, achieved high-contrast visualization of tumor metastasis through lymph nodes and precise tumor resection. Furthermore, NIRG-2 proves to be highly effective and reliable in evaluating surgical and drug treatment efficacy in cancer lymphatic metastasis models. We are optimistic that this study not only provides a crucial molecular tool for an in-depth understanding of G4-related diseases in vivo but also marks a promising strategy for the development of clinical NIR-II G4-activated probes.

Multiple cholinergic receptor subtypes coordinate dual modulation of acetylcholine on anterior and posterior paraventricular thalamic neurons.

Paraventricular thalamus (PVT) plays important roles in the regulation of emotion and motivation through connecting many brain structures including the midbrain and the limbic system. Although acetylcholine (ACh) neurons of the midbrain were reported to send projections to PVT, little is known about how cholinergic signaling regulates PVT neurons. Here, we used both RNAscope and slice patch-clamp recordings to characterize cholinergic receptor expression and ACh modulation of PVT neurons in mice. We found ACh excited a majority of anterior PVT (aPVT) neurons but predominantly inhibited posterior PVT (pPVT) neurons. Compared to pPVT with more inhibitory M2 receptors, aPVT expressed higher levels of all excitatory receptor subtypes including nicotinic α4, α7, and muscarinic M1 and M3. The ACh-induced excitation was mimicked by nicotine and antagonized by selective blockers for α4ß2 and α7 nicotinic ACh receptor (nAChR) subtypes as well as selective antagonists for M1 and M3 muscarinic ACh receptors (mAChR). The ACh-induced inhibition was attenuated by selective M2 and M4 mAChR receptor antagonists. Furthermore, we found ACh increased the frequency of excitatory postsynaptic currents (EPSCs) on a majority of aPVT neurons but decreased EPSC frequency on a larger number of pPVT neurons. In addition, ACh caused an acute increase followed by a lasting reduction in inhibitory postsynaptic currents (IPSCs) on PVT neurons of both subregions. Together, these data suggest that multiple AChR subtypes coordinate a differential modulation of ACh on aPVT and pPVT neurons.

Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells.

Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.

Plasmonic Imaging of Multivalent NTD-Nucleic Acid Interactions for Broad-Spectrum Antiviral Drug Analysis.

The discovery and identification of broad-spectrum antiviral drugs are of great significance for blocking the spread of pathogenic viruses and corresponding variants of concern. Herein, we proposed a plasmonic imaging-based strategy for assessing the efficacy of potential broad-spectrum antiviral drugs targeting the N-terminal domain of a nucleocapsid protein (NTD) and nucleic acid (NA) interactions. With NTD and NA conjugated gold nanoparticles as core and satellite nanoprobes, respectively, we found that the multivalent binding interactions could drive the formation of core-satellite nanostructures with enhanced scattering brightness due to the plasmonic coupling effect. The core-satellite assembly can be suppressed in the presence of antiviral drugs targeting the NTD-NA interactions, allowing the drug efficacy analysis by detecting the dose-dependent changes in the scattering brightness by plasmonic imaging. By quantifying the changes in the scattering brightness of plasmonic nanoprobes, we uncovered that the constructed multivalent weak interactions displayed a 500-fold enhancement in affinity as compared with the monovalent NTD-NA interactions. We demonstrated the plasmonic imaging-based strategy for evaluating the efficacy of a potential broad-spectrum drug, PJ34, that can target the NTD-NA interactions, with the IC50 as 24.35 and 14.64 µM for SARS-CoV-2 and SARS-CoV, respectively. Moreover, we discovered that ceftazidime holds the potential as a candidate drug to inhibit the NTD-NA interactions with an IC50 of 22.08 µM from molecular docking and plasmonic imaging-based drug analysis. Finally, we validated that the potential antiviral drug, 5-benzyloxygramine, which can induce the abnormal dimerization of nucleocapsid proteins, is effective for SARS-CoV-2, but not effective against SARS-CoV. All these demonstrations indicated that the plasmonic imaging-based strategy is robust and can be used as a powerful strategy for the discovery and identification of broad-spectrum drugs targeting the evolutionarily conserved viral proteins.

Lanthanide Inorganic Nanoparticles Enhance Semiconducting Polymer Nanoparticles Afterglow Luminescence for In Vivo Afterglow/Magnetic Resonance Imaging.

Dual/multimodal imaging strategies are increasingly recognized for their potential to provide comprehensive diagnostic insights in cancer imaging by harnessing complementary data. This study presents an innovative probe that capitalizes on the synergistic benefits of afterglow luminescence and magnetic resonance imaging (MRI), effectively eliminating autofluorescence interference and delivering a superior signal-to-noise ratio. Additionally, it facilitates deep tissue penetration and enables noninvasive imaging. Despite the advantages, only a limited number of probes have demonstrated the capability to simultaneously enhance afterglow luminescence and achieve high-resolution MRI and afterglow imaging. Herein, we introduce a cutting-edge imaging platform based on semiconducting polymer nanoparticles (PFODBT) integrated with NaYF4@NaGdF4 (Y@Gd@PFO-SPNs), which can directly amplify afterglow luminescence and generate MRI and afterglow signals in tumor tissues. The proposed mechanism involves lanthanide nanoparticles producing singlet oxygen (1O2) upon white light irradiation, which subsequently oxidizes PFODBT, thereby intensifying afterglow luminescence. This innovative platform paves the way for the development of high signal-to-background ratio imaging modalities, promising noninvasive diagnostics for cancer.

Achieving Low-Dose Rate X-Ray Imaging Based on 2D/3D-Mixed Perovskite Films.

X-ray detection and imaging are widely used in medical diagnosis, product inspection, security monitoring, etc. Large-scale polycrystalline perovskite thick films possess high potential for direct X-ray imaging. However, the notorious problems of baseline drift and high detection limit caused by ions migration are still remained. Here, ion migration is reduced by incorporating 2D perovskite into 3D perovskite, thereby increasing the ion activation energy. This approach hinders ion migration within the perovskite film, consequently suppressing baseline drift and reducing the lowest detection limit(LOD) of the device. As a result, the baseline drifting declines by 20 times and the LOD reduces to 21.1 nGy s-1, while the device maintains a satisfactory sensitivity of 5.6 × 103 µC Gy-1 cm-2. This work provides a new strategy to achieve low ion migration in large-scale X-ray detectors and may provide new thoughts for the application of mixed-dimension perovskite.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Chemical Design of Activatable Photoacoustic Probes for Precise Biomedical Applications.

Photoacoustic (PA) imaging technology, a three-dimensional hybrid imaging modality that integrates the advantage of optical and acoustic imaging, has great application prospects in molecular imaging due to its high imaging depth and resolution. To endow PA imaging with the ability for real-time molecular visualization and precise biomedical diagnosis, numerous activatable molecular PA probes which can specifically alter their PA intensities upon reacting with the targets or biological events of interest have been developed. This review highlights the recent developments of activatable PA probes for precise biomedical applications including molecular detection of the biotargets and imaging of the biological events. First, the generation mechanism of PA signals will be given, followed by a brief introduction to contrast agents used for PA probe design. Then we will particularly summarize the general design principles for the alteration of PA signals and activatable strategies for developing precise PA probes. Furthermore, we will give a detailed discussion of activatable PA probes in molecular detection and biomedical imaging applications in living systems. At last, the current challenges and outlooks of future PA probes will be discussed. We hope that this review will stimulate new ideas to explore the potentials of activatable PA probes for precise biomedical applications in the future.

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

DNA-Templated Anchoring of Proteins for Programmable Cell Functionalization and Immunological Response.

Membrane protein engineering exhibits great potential for cell functionalization. Although genetic strategies are sophisticated for membrane protein engineering, there still exist some issues, including transgene insertional mutagenesis, laborious, complicated procedures, and low tunability. Herein, we report a DNA-templated anchoring of exogenous proteins on living cell membranes to realize programmable functionalization of living cells. Using DNA as a scaffold, the model cell membranes are readily modified with proteins, on which the density and ratio of proteins as well as their interactions can be precisely controlled through predictable DNA hybridization. Then, the natural killer (NK) cells were engineered to gain the ability to eliminate the immune checkpoint signaling at the NK-tumor synapse, which remarkably promoted NK cell activation in immunotherapy. Given the versatile functions of exogenous proteins and flexible designs of programmable DNA, this method has the potential to facilitate membrane-protein-based cell engineering and therapy.

Zinc-Carnosine Metallodrug Network as Dual Metabolism Inhibitor Overcoming Metabolic Reprogramming for Efficient Cancer Therapy.

The targeting of tumor metabolism as a novel strategy for cancer therapy has attracted tremendous attention. Herein, we develop a dual metabolism inhibitor, Zn-carnosine metallodrug network nanoparticles (Zn-Car MNs), which exhibits good Cu-depletion and Cu-responsive drug release, causing potent inhibition of both OXPHOS and glycolysis. Notably, Zn-Car MNs can decrease the activity of cytochrome c oxidase and the content of NAD+, so as to reduce ATP production in cancer cells. Thereby, energy deprivation, together with the depolarized mitochondrial membrane potential and increased oxidative stress, results in apoptosis of cancer cells. In result, Zn-Car MNs exerted more efficient metabolism-targeted therapy than the classic copper chelator, tetrathiomolybdate (TM), in both breast cancer (sensitive to copper depletion) and colon cancer (less sensitive to copper depletion) models. The efficacy and therapy of Zn-Car MNs suggest the possibility to overcome the drug resistance caused by metabolic reprogramming in tumors and has potential clinical relevance.

In vitro evaluation of the toxicological effects of cooking oil fumes using a self-designed microfluidic chip.

Cooking oil fumes (COFs) are widely acknowledged as substantial contributors to indoor air pollution, having detrimental effects on human health. Despite the existence of commercialized in vitro aerosol exposure platforms, assessment risks of aerosol pollutants are primarily evaluated based on multiwell plate experiments by trapping and redissolving aerosols to conduct comprehensive in vitro immersion exposure manner. Therefore, an innovative real-time exposure system for COF aerosol was constructed, featuring a self-designed microfluidic chip as its focal component. The chip was used to assess toxicological effects of in vitro exposure to COF aerosol on cells cultured at the gas-liquid interface. Meanwhile, we used transcriptomics to analyze genes that exhibited differential expression in cells induced by COF aerosol. The findings indicated that the MAPK signaling pathway, known for its involvement in inflammatory response and oxidative stress, played a crucial role in the biological effects induced by COF aerosol. Biomarkers associated with inflammatory response and oxidative stress exhibited corresponding alterations. Furthermore, the concentration of COF aerosol exposure and post-exposure duration exert decisive effects on these biomarkers. Thus, the study suggests that COF can induce oxidative stress and inflammatory response in BEAS-2B cells, potentially exerting a discernible impact on human health.

Chemical Approaches to Optimize the Properties of Organic Fluorophores for Imaging and Sensing.

Organic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high-performance fluorophores.

Dual-Locked Fluorescent Probes Activated by Aminopeptidase N and the Tumor Redox Environment for High-Precision Imaging of Tumor Boundaries.

Clear delineation of tumor margins is essential for accurate resection and decreased recurrence rate in the clinic. Fluorescence imaging is emerging as a promising alternative to traditional visual inspection by surgeons for intraoperative imaging. However, traditional probes lack accuracy in tumor diagnosis, making it difficult to depict tumor boundaries accurately. Herein, we proposed an offensive and defensive integration (ODI) strategy based on the "attack systems (invasive peptidase) and defense systems (reductive microenvironment)" of multi-dimensional tumor characteristics to design activatable fluorescent probes for imaging tumor boundaries precisely. Screened out from a series of ODI strategy-based probes, ANQ performed better than traditional probes based on tumor unilateral correlation by distinguishing between tumor cells and normal cells and minimizing false-positive signals from living metabolic organs. To further improve the signal-to-background ratio in vivo, derivatized FANQ, was prepared and successfully applied to distinguish orthotopic hepatocellular carcinoma tissues from adjacent tissues in mice models and clinical samples. This work highlights an innovative strategy to develop activatable probes for rapid diagnosis of tumors and high-precision imaging of tumor boundaries, providing more efficient tools for future clinical applications in intraoperative assisted resection.

"Zero" Intrinsic Fluorescence Sensing-Platforms Enable Ultrasensitive Whole Blood Diagnosis and In Vivo Imaging.

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re-engineered far-red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing-platforms were created by systematically regulating the open-loop/spirocyclic forms. Leveraging these advancements, we devised various ultra-sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300-fold). Among these indicators, 8-LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8-LAP with an endoplasmic reticulum-targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases.

Oxytocin Receptor-Expressing Neurons in the Paraventricular Thalamus Regulate Feeding Motivation through Excitatory Projections to the Nucleus Accumbens Core.

Oxytocin receptors (OTR) have been found in the paraventricular thalamus (PVT) for the regulation of feeding and maternal behaviors. However, the functional projections of OTR-expressing PVT neurons remain largely unknown. Here, we used chemogenetic and optogenetic tools to test the role of OTR-expressing PVT neurons and their projections in the regulation of food intake in both male and female OTR-Cre mice. We found chemogenetic activation of OTR-expressing PVT neurons promoted food seeking under trials with a progressive ratio schedule of reinforcement. Using Feeding Experimentation Devices for real-time meal measurements, we found chemogenetic activation of OTR-expressing PVT neurons increased meal frequency but not cumulative food intake because of a compensatory decrease in meal sizes. In combination with anterograde neural tracing and slice patch-clamp recordings, we found optogenetic stimulation of PVT OTR terminals excited neurons in the posterior basolateral amygdala (pBLA) and nucleus accumbens core (NAcC) as well as local PVT neurons through monosynaptic glutamatergic transmissions. Photostimulation of OTR-expressing PVT-NAcC projections promoted food-seeking, whereas selective activation of PVT-pBLA projections produced little effect on feeding. In contrast to selective activation of OTR terminals, photostimulation of a broader population of glutamatergic PVT terminals exerted direct excitation followed by indirect lateral inhibition on neurons in both NAcC and anterior basolateral amygdala. Together, these results suggest that OTR-expressing PVT neurons are a distinct population of PVT glutamate neurons that regulate feeding motivation through projections to NAcC.SIGNIFICANCE STATEMENT The paraventricular thalamus plays an important role in the regulation of feeding motivation. However, because of the diversity of paraventricular thalamic neurons, the specific neuron types promoting food motivation remain elusive. In this study, we provide evidence that oxytocin receptor-expressing neurons are a specific group of glutamate neurons that primarily project to the nucleus accumbens core and posterior amygdala. We found that activation of these neurons promotes the motivation for food reward and increases meal frequency through projections to the nucleus accumbens core but not the posterior amygdala. As a result, we postulate that oxytocin receptor-expressing neurons in the paraventricular thalamus and their projections to the nucleus accumbens core mainly regulate feeding motivation but not food consumption.

"Four-In-One" Design of a Hemicyanine-Based Modular Scaffold for High-Contrast Activatable Molecular Afterglow Imaging.

Afterglow luminescence (long persistent luminescence) holds great potential for nonbackground molecular imaging. However, current afterglow probes are mainly nanoparticles, and afterglow imaging systems based on organic small molecules are still lacking and have rarely been reported. Moreover, the lack of reactive sites and a universal molecular scaffold makes it difficult to design activatable afterglow probes. To address these issues, this study reports a novel kind of hemicyanine-based molecule scaffolds with stimuli-responsive afterglow luminescence, which is dependent on an intramolecular cascade photoreaction between 1O2 and the afterglow molecule to store the photoenergy for delayed luminescence after light cessation. As a proof of concept, three modular activatable molecular afterglow probes (MAPs) with a "four-in-one" molecular design by integrating a stimuli-responsive unit, 1O2-generating unit, 1O2-capturing unit, and luminescent unit into one probe are customized for quantification and imaging of targets including pH, superoxide anions, and aminopeptidase. Notably, MAPs show higher sensitivity in afterglow imaging than in fluorescence imaging because the responsive unit simultaneously controls the initiation of fluorescence (S1 to S0) and 1O2 generation (S1 to T1). Finally, MAPs are applied for high-contrast afterglow imaging of drug-induced hepatotoxicity, which is poorly evaluated in clinics and drug discovery. By reporting the sequential occurrence of oxidative stress and upregulation of aminopeptidase, such activatable afterglow probes allow noninvasive imaging of hepatotoxicity earlier than the serological and histology manifestation, indicating their promise for early diagnosis of hepatotoxicity.