Search

The outgrowth and stabilization of nascent dendritic spines are crucial processes underlying learning and memory. Most new spines retract shortly after growth; only a small subset is stabilized and integrated into the new circuit connections that support learning. New spine stabilization has been shown to rely upon activity-dependent molecular mechanisms that also contribute to long-term potentiation (LTP) of synaptic strength. Indeed, disruption of the activity-dependent targeting of the kinase CaMKIIα to the GluN2B subunit of the NMDA-type glutamate receptor disrupts both LTP and activity-dependent stabilization of new spines. Yet it is not known which of CaMKIIα's many enzymatic and structural functions are important for new spine stabilization. Here, we used two-photon imaging and photolysis of caged glutamate to monitor the activity-dependent stabilization of new dendritic spines on hippocampal CA1 neurons from mice of both sexes in conditions where CaMKIIα functional and structural interactions were altered. Surprisingly, we found that inhibiting CaMKIIα kinase activity either genetically or pharmacologically did not impair activity-dependent new spine stabilization. In contrast, shRNA knockdown of CaMKIIα abolished activity-dependent new spine stabilization, which was rescued by co-expressing shRNA-resistant full-length CaMKIIα, but not by a truncated monomeric CaMKIIα. Notably, overexpression of phospho-mimetic CaMKIIα-T286D, which exhibits activity-independent targeting to GluN2B, enhanced basal new spine survivorship in the absence of additional glutamatergic stimulation, even when kinase activity was disrupted. Together, our results support a model in which nascent dendritic spine stabilization requires structural and scaffolding interactions mediated by dodecameric CaMKIIα that are independent of its enzymatic activities.

Cerebral microbleeds (CMBs) lead to cognitive decline, linked to the axonal structure composed of phospholipid bilayers. Current methods are difficult to obtain in situ changes of biochemical component concentration during CMB. In this study, by Raman spectrum and two‐photon imaging, we achieve in situ changes in the information of biochemical components concentration during CMB. The overall concentration of phospholipids in the damaged tissue significantly decreases after CMB, forming a large region of low concentration, but the relative concentration of phosphatidylinositol (PI) increases, reflecting the inhibition role of the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway. Accordingly, two‐photon images of neurons show a clear decrease in the number of axons, indicating a close correlation between phospholipid hydrolysis and axon damage, as well as cognitive impairment. Therefore, the decrease in phospholipid concentration and the increase in the PI concentration might serve as a pair of indicators for characterizing CMB and its relationship with cognitive decline. [ABSTRACT FROM AUTHOR]

Feedback at the photoreceptor synapse is the first neuronal circuit computation in vision, which influences downstream activity patterns within the visual system. Yet, the identity of the feedback signal and the mechanism of synaptic transmission are still not well understood. Here, we combined perturbations of cell-type-specific genes of mouse horizontal cells with two-photon imaging of the result of light-induced feedback in cones and showed that the electrogenic bicarbonate transporter Slc4a5, but not the electroneutral bicarbonate transporter Slc4a3, both expressed specifically in horizontal cells, is necessary for horizontal cell-to-cone feedback. Pharmacological blockage of bicarbonate transporters and buffering pH also abolished the feedback but blocking sodium-proton exchangers and GABA receptors did not. Our work suggests an unconventional mechanism of feedback at the first visual synapse: changes in horizontal cell voltage modulate bicarbonate transport to the cell, via Slc4a5, which leads to the modulation of feedback to cones. [Display omitted] • Feedback from horizontal cells to cones was measured by imaging calcium in cones • Slc4a5 is an electrogenic Na+-HCO 3 − cotransporter specific to horizontal cells • Knocking out or down Slc4a5 abolished horizontal cell-to-cone feedback • Blocking HCO 3 − transporters and buffering pH also abolished feedback Molecular mechanisms of horizontal cell-to-cone feedback in the retina have been under debate. Morikawa et al. show that Slc4a5, an electrogenic sodium-bicarbonate cotransporter specific to horizontal cells, is crucial for feedback. They suggest that Slc4a5, through its voltage-dependent transport of bicarbonate, regulates pH in the synaptic cleft that mediates feedback. [ABSTRACT FROM AUTHOR]

INTRODUCTION: It is unclear how early neuronal deficits occur in tauopathies, if these are associated with changes in neuronal network activity, and if they can be alleviated with therapies. METHODS: To address this, we performed in vivo two‐photon Ca2+ imaging in tauopathy mice at 6 versus 12 months, compared to controls, and treated the younger animals with a tau antibody. RESULTS: Neuronal function was impaired at 6 months but did not deteriorate further at 12 months, presumably because cortical tau burden was comparable at these ages. At 6 months, neurons were mostly hypoactive, with enhanced neuronal synchrony, and had dysregulated responses to stimulus. Ex vivo, electrophysiology revealed altered synaptic transmission and enhanced excitability of motor cortical neurons, which likely explains the altered network activity. Acute tau antibody treatment reduced pathological tau and gliosis and partially restored neuronal function. DISCUSSION: Tauopathies are associated with early neuronal deficits that can be attenuated with tau antibody therapy. Highlights: Neuronal hypofunction in awake and behaving mice in early stages of tauopathy.Altered network activity disrupted local circuitry engagement in tauopathy mice.Enhanced neuronal excitability and altered synaptic transmission in tauopathy mice.Tau antibody acutely reduced soluble phospho‐tau and improved neuronal function. [ABSTRACT FROM AUTHOR]

The role of experience in the organization of cortical feedback (FB) remains unknown. We measured the effects of manipulating visual experience on the retinotopic specificity of supragranular and infragranular projections from the lateromedial (LM) visual area to layer (L)1 of the mouse primary visual cortex (V1). LM inputs were, on average, retinotopically matched with V1 neurons in normally and dark-reared mice, but visual exposure reduced the fraction of spatially overlapping inputs to V1. FB inputs from L5 conveyed more surround information to V1 than those from L2/3. The organization of LM inputs from L5 depended on their orientation preference and was disrupted by dark rearing. These observations were recapitulated by a model where visual experience minimizes receptive field overlap between LM inputs and V1 neurons. Our results provide a mechanism for the dependency of surround modulations on visual experience and suggest how expected interarea coactivation patterns are learned in cortical circuits. • Visual experience reduces the receptive field overlap between LM inputs and V1 neurons • LM inputs from L5 convey more surround information to V1 neurons than those from L2/3 • The tuning-dependent organization of LM inputs from L5 requires visual experience • Spatial redundancy minimization explains visual experience effects on LM inputs Dias and Rajan et al. compared the functional organization of cortical feedback inputs in the primary visual cortex of normally and dark-reared mice. They found that visual experience reduces the representational redundancy between feedback inputs and primary visual cortex neurons. [ABSTRACT FROM AUTHOR]

Background: Cerebrospinal fluid (CSF) flow is crucial for brain homeostasis and its dysfunction is highly associated with neurodegenerative diseases. Restoring CSF circulation is proposed as a key strategy for the treatment of the diseases. Among the methods to improve CSF circulation, focused ultrasound (FUS) stimulation has emerged as a promising non-invasive brain stimulation technique, with effectiveness evidenced by ex vivo studies. However, due to technical disturbances in in vivo imaging combined with FUS, direct evidence of real-time in vivo CSF flow enhancement by FUS remains elusive. Objective: To investigate whether FUS administered through the skull base can enhance CSF influx in living animals with various real-time imaging techniques. Methods: We demonstrate a novel method of applying FUS through the skull base, facilitating cortical CSF influx, evidenced by diverse in vivo imaging techniques. Acoustic simulation confirmed effective sonication of our approach through the skull base. After injecting fluorescent CSF tracers into cisterna magna, FUS was administered at the midline of the jaw through the skull base for 30 min, during which imaging was performed concurrently. Results: Enhanced CSF influx was observed in macroscopic imaging, demonstrated by the influx area and intensity of the fluorescent dyes after FUS. In two-photon imaging, increased fluorescence was observed in the perivascular space (PVS) after stimulation. Moreover, particle tracking of microspheres showed more microspheres entering the imaging field, with increased mean speed after FUS. Conclusion: Our findings provide direct real-time in vivo imaging evidence that FUS promotes CSF influx and flow in the PVS.

In the brain, a microvascular sensory web coordinates oxygen delivery to regions of neuronal activity. This involves a dense network of capillaries that send conductive signals upstream to feeding arterioles to promote vasodilation and blood flow. Although this process is critical to the metabolic supply of healthy brain tissue, it may also be a point of vulnerability in disease. Deterioration of capillary networks is a feature of many neurological disorders and injuries and how this web is engaged during vascular damage remains unknown. We performed in vivo two-photon microscopy on young adult mural cell reporter mice and induced focal capillary injuries using precise two-photon laser irradiation of single capillaries. We found that ~59% of the injuries resulted in regression of the capillary segment 7 to 14 d following injury, and the remaining repaired to reestablish blood flow within 7 d. Injuries that resulted in capillary regression induced sustained vasoconstriction in the upstream arteriole-capillary transition (ACT) zone at least 21 days postinjury in both awake and anesthetized mice. The degree of vasomotor dynamics was chronically attenuated in the ACT zone consequently reducing blood flow in the ACT zone and in secondary, uninjured downstream capillaries. These findings demonstrate how focal capillary injury and regression can impair the microvascular sensory web and contribute to cerebral hypoperfusion. [ABSTRACT FROM AUTHOR]

Hypoxic tumor microenvironments pose significant challenges to the clinical translation of cancer photodynamic therapy (PDT). While heavy atom‐free Type‐I boron dipyrromethenes (BODIPYs) photosensitizers can alleviate this challenge by reducing oxygen dependency, but they remain scarce. Herein, heavy‐atom‐free α,meso‐linked bisBODIPYs were designed and synthesized. These bisBODIPYs exhibit remarkable red‐shifted emission (λemmax ~670 nm), large Stokes shifts (~4480 cm−1) and they are capable of producing both superoxide anion (O2⋅−) and singlet oxygen (1O2) in solution and cells. Additionally, they offer a wide PDT treatment window, ranging from 0.26 to 83.5 μM. Furthermore, these bisBODIPYs also demonstrate superior two‐photon fluorescence, promising for surgical navigation and integrating diagnosis with treatment. [ABSTRACT FROM AUTHOR]

Cerebrospinal fluid (CSF) flow is crucial for brain homeostasis and its dysfunction is highly associated with neurodegenerative diseases. Restoring CSF circulation is proposed as a key strategy for the treatment of the diseases. Among the methods to improve CSF circulation, focused ultrasound (FUS) stimulation has emerged as a promising non-invasive brain stimulation technique, with effectiveness evidenced by ex vivo studies. However, due to technical disturbances in in vivo imaging combined with FUS, direct evidence of real-time in vivo CSF flow enhancement by FUS remains elusive. To investigate whether FUS administered through the skull base can enhance CSF influx in living animals with various real-time imaging techniques. We demonstrate a novel method of applying FUS through the skull base, facilitating cortical CSF influx, evidenced by diverse in vivo imaging techniques. Acoustic simulation confirmed effective sonication of our approach through the skull base. After injecting fluorescent CSF tracers into cisterna magna, FUS was administered at the midline of the jaw through the skull base for 30 min, during which imaging was performed concurrently. Enhanced CSF influx was observed in macroscopic imaging, demonstrated by the influx area and intensity of the fluorescent dyes after FUS. In two-photon imaging, increased fluorescence was observed in the perivascular space (PVS) after stimulation. Moreover, particle tracking of microspheres showed more microspheres entering the imaging field, with increased mean speed after FUS. Our findings provide direct real-time in vivo imaging evidence that FUS promotes CSF influx and flow in the PVS. [Display omitted] • Applying low-intensity FUS through the skull base enhanced cortical CSF influx. • In vivo imaging techniques evidenced real-time CSF dynamics facilitated by FUS. • FUS accelerated CSF dynamics exhibited by microsphere flow in the PVS. [ABSTRACT FROM AUTHOR]

Significance: The initiation of goal-directed actions is a complex process involving the medial prefrontal cortex and dopaminergic inputs through the mesocortical pathway. However, it is unclear what information the mesocortical pathway conveys and how it impacts action initiation. In this study, we unveiled the indispensable role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Aim: To investigate the role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Approach: We designed a lever-press task in which mice internally determine the timing of the press, receiving a larger reward for longer waiting periods. Results: Our study revealed that self-initiated actions depend on dopaminergic signaling mediated by D2 receptors, whereas sensory-triggered lever-press actions do not involve D2 signaling. Microprism-mediated two-photon calcium imaging further demonstrated ramping activity in mesocortical axon terminals approximately 0.5 s before the self-initiated lever press. Remarkably, the ramping patterns remained consistent whether the mice responded to cues immediately for a smaller reward or held their response for a larger reward. Conclusions: We conclude that mesocortical dopamine axon terminals encode the timing of self-initiated actions, shedding light on a crucial aspect of the intricate neural mechanisms governing goal-directed behavior. [ABSTRACT FROM AUTHOR]

Protein kinase A (PKA) plays essential roles in diverse cellular functions. However, the spatiotemporal dynamics of endogenous PKA upon activation remain debated. The classical model predicts that PKA catalytic subunits dissociate from regulatory subunits in the presence of cAMP, whereas a second model proposes that catalytic subunits remain associated with regulatory subunits following physiological activation. Here, we report that different PKA subtypes, as defined by the regulatory subunit, exhibit distinct subcellular localization at rest in CA1 neurons of cultured hippocampal slices. Nevertheless, when all tested PKA subtypes are activated by norepinephrine, presumably via the β-adrenergic receptor, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. These differential spatial dynamics between the subunits indicate that at least a significant fraction of PKA dissociates. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA. These results indicate that endogenous PKA regulatory and catalytic subunits dissociate to achieve PKA function in neurons.

The positive hemodynamic response (PHR) during stimulation often co-occurs with a strong, sustained negative hemodynamic response (NHR). However, the characteristics and neurophysiological mechanisms of the NHR, especially in regions distal to the PHR, remain incompletely understood. Using intrinsic optical imaging (OI) and two-photon imaging, we observed that forelimb electrical stimulation evoked strong PHR signals in the forelimb region of the primary somatosensory cortex (S1FL). Meanwhile, NHR signals primarily appeared in the primary visual cortex (V1), with a delayed onset and lower amplitude relative to the PHR signals. Additionally, stimulation led to a reduction in cerebral blood flow (CBF) in the NHR region. Notably, there was an overall suppression of the calcium response in the NHR region, although a small proportion (14 %) of neurons exhibited concurrent activation. Axon tracing revealed cortico-cortical projections from S1FL to V1. These findings suggest that neuronal deactivation significantly contributes to the origin of the NHR, offering additional insights into the specific inhibitory mechanisms underlying the NHR.

The superior colliculus (SC) is a prominent and conserved visual center in all vertebrates. In mice, the most superficial lamina of the SC is enriched with neurons that are selective for the moving direction of visual stimuli. Here, we study how these direction selective neurons respond to complex motion patterns known as plaids, using two-photon calcium imaging in awake male and female mice. The plaid pattern consists of two superimposed sinusoidal gratings moving in different directions, giving an apparent pattern direction that lies between the directions of the two component gratings. Most direction selective neurons in the mouse SC respond robustly to the plaids and show a high selectivity for the moving direction of the plaid pattern but not of its components. Pattern motion selectivity is seen in both excitatory and inhibitory SC neurons and is especially prevalent in response to plaids with large cross angles between the two component gratings. However, retinal inputs to the SC are ambiguous in their selectivity to pattern versus component motion. Modeling suggests that pattern motion selectivity in the SC can arise from a nonlinear transformation of converging retinal inputs. In contrast, the prevalence of pattern motion selective neurons is not seen in the primary visual cortex (V1). These results demonstrate an interesting difference between the SC and V1 in motion processing and reveal the SC as an important site for encoding pattern motion. [ABSTRACT FROM AUTHOR]

[Display omitted] The efficacy of imaging-guided photodynamic therapy (PDT) is compromised by the attenuation of fluorescence and decline in reactive oxygen species (ROS) generation efficiency in the physiological environment of conventional photosensitizers, limited near-infrared (NIR) absorption, and high systemic cytotoxicity. This paper presents the synthesis of two cyclometalated Ir (III) complexes (Ir-thpy and Ir-ppy) by using a triphenylamine derivative (DPTPA) as the primary ligand and their encapsulation into an amphiphilic phospholipid to form nanoparticles (NPs). These complexes exhibit aggregation-induced emission features and remarkably enhanced ROS generation compared to Chlorin e6 (Ce6). Moreover, Ir-thpy NPs possess the unique ability to selectively target mitochondria, leading to depolarization of the mitochondrial membrane potential and ultimately triggering apoptosis. Notably, Ir-thpy NPs exhibit exceptional photocytotoxicity even towards cisplatin-resistant A549/DDP tumor cells. In vivo two-photon imaging verified the robust tumor-targeting efficacy of Ir-thpy NPs. The in vivo results unequivocally demonstrate that Ir-thpy NPs exhibit excellent tumor ablation along with remarkable biocompatibility. This study presents a promising approach for the development of multifunctional Ir-NPs for two-photon imaging-guided PDT and provides novel insights for potential clinical applications in oncology. [ABSTRACT FROM AUTHOR]

The prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness to locomotion. We find that PFC astrocytes express receptors for dopamine but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time locked to dopamine release and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in the PFC, contributing to PFC function though neuromodulator receptor crosstalk.

The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2′-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging. [ABSTRACT FROM AUTHOR]

Fetal alcohol spectrum disorders (FASD) caused by developmental ethanol exposure lead to cerebellar impairments, including motor problems, decreased cerebellar weight, and cell death. Alterations in the sole output of the cerebellar cortex, Purkinje cells, and central nervous system immune cells, microglia, have been reported in animal models of FASD. To determine how developmental ethanol exposure affects adult cerebellar microglia and Purkinje cells, we used a human third-trimester binge exposure model in which mice received ethanol or saline from postnatal (P) days 4–9. In adolescence, cerebellar cranial windows were implanted and mice were aged to young adulthood for examination of microglia and Purkinje cells in vivo with two-photon imaging or in fixed tissue. Ethanol had no effect on microglia density, morphology, dynamics, or injury response. However, Purkinje cell linear frequency was reduced by ethanol. Microglia–Purkinje cell interactions in the Purkinje Cell Layer were altered in females compared to males. Overall, developmental ethanol exposure had few effects on cerebellar microglia in young adulthood and Purkinje cells appeared to be more susceptible to its effects. [ABSTRACT FROM AUTHOR]

Fluorescence microscopy remains one of the single most widely applied experimental approaches in neuroscience and beyond and is continuously evolving to make it easier and more versatile. The success of the approach is based on synergistic developments in imaging technologies and fluorophore labeling strategies that have allowed it to greatly diversify and be used across preparations for addressing structure as well as function. Yet, while targeted labeling strategies are a key strength of fluorescence microscopy, they reciprocally impose general limitations on the possible types of experiments and analyses. One recent development that overcomes some of these limitations is fluorescence microscopy shadow imaging, where membranebound cellular structures remain unlabeled while the surrounding extracellular space is made to fluoresce to provide a negative contrast shadow image. When based on super-resolution STED microscopy, the technique in effect provides a positive image of the extracellular space geometry and entire neuropil in the field of view. Other noteworthy advantages include the near elimination of the adverse effects of photobleaching and toxicity in live imaging, exhaustive and homogeneous labeling across the preparation, and the ability to apply and adjust the label intensity on the fly. Shadow imaging is gaining popularity and has been applied on its own or combined with conventional positive labeling to visualize cells and synaptic proteins in their parenchymal context. Here, we highlight the inherent limitations of fluorescence microscopy and conventional labeling and contrast these against the pros and cons of recent shadow imaging approaches. Our aim is to describe the brief history and current trajectory of the shadow imaging technique in the neuroscience field, and to draw attention to its ease of application and versatility. [ABSTRACT FROM AUTHOR]

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2 , an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD. [ABSTRACT FROM AUTHOR]

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

Background: The treatment of patellar tendon injury has always been an unsolved problem, and mechanical characterization is very important for its repair and reconstruction. Elastin is a contributor to mechanics, but it is not clear how it affects the elasticity, viscoelastic properties, and structure of patellar tendon.Methods: The patellar tendons from six fresh adult experimental pigs were used in this study and they were made into 77 samples. The patellar tendon was specifically degraded by elastase, and the regional mechanical response and structural changes were investigated by: (1) Based on the previous study of elastase treatment conditions, the biochemical quantification of collagen, glycosaminoglycan and total protein was carried out; (2) The patellar tendon was divided into the proximal, central, and distal regions, and then the axial tensile test and stress relaxation test were performed before and after phosphate-buffered saline (PBS) or elastase treatment; (3) The dynamic constitutive model was established by the obtained mechanical data; (4) The structural relationship between elastin and collagen fibers was analyzed by two-photon microscopy and histology.Results: There was no statistical difference in mechanics between patellar tendon regions. Compared with those before elastase treatment, the low tensile modulus decreased by 75%–80%, the high tensile modulus decreased by 38%–47%, and the transition strain was prolonged after treatment. For viscoelastic behavior, the stress relaxation increased, the initial slope increased by 55%, the saturation slope increased by 44%, and the transition time increased by 25% after enzyme treatment. Elastin degradation made the collagen fibers of patellar tendon become disordered and looser, and the fiber wavelength increased significantly.Conclusion: The results of this study show that elastin plays an important role in the mechanical properties and fiber structure stability of patellar tendon, which supplements the structure-function relationship information of patellar tendon. The established constitutive model is of great significance to the prediction, repair and replacement of patellar tendon injury. In addition, human patellar tendon has a higher elastin content, so the results of this study can provide supporting information on the natural properties of tendon elastin degradation and guide the development of artificial patellar tendon biomaterials.

Primates rely on two eyes to perceive depth, while maintaining stable vision when either one eye or both eyes are open. Although psychophysical and modeling studies have investigated how monocular signals are combined to form binocular vision, the underlying neuronal mechanisms, particularly in V1 where most neurons exhibit binocularity with varying eye preferences, remain poorly understood. Here, we used two-photon calcium imaging to compare the monocular and binocular responses of thousands of simultaneously recorded V1 superficial-layer neurons in three awake macaques. During monocular stimulation, neurons preferring the stimulated eye exhibited significantly stronger responses compared to those preferring both eyes. However, during binocular stimulation, the responses of neurons preferring either eye were suppressed on the average, while those preferring both eyes were enhanced, resulting in similar neuronal responses irrespective of their eye preferences, and an overall response level similar to that with monocular viewing. A neuronally realistic model of binocular combination, which incorporates ocular dominance-dependent divisive interocular inhibition and binocular summation, is proposed to account for these findings.

The hippocampus consists of a stereotyped neuronal circuit repeated along the septal-temporal axis. This transverse circuit contains distinct subfields with stereotyped connectivity that support crucial cognitive processes, including episodic and spatial memory. However, comprehensive measurements across the transverse hippocampal circuit in vivo are intractable with existing techniques. Here, we developed an approach for two-photon imaging of the transverse hippocampal plane in awake mice via implanted glass microperiscopes, allowing optical access to the major hippocampal subfields and to the dendritic arbor of pyramidal neurons. Using this approach, we tracked dendritic morphological dynamics on CA1 apical dendrites and characterized spine turnover. We then used calcium imaging to quantify the prevalence of place and speed cells across subfields. Finally, we measured the anatomical distribution of spatial information, finding a non-uniform distribution of spatial selectivity along the DG-to-CA1 axis. This approach extends the existing toolbox for structural and functional measurements of hippocampal circuitry.

The primary motor cortex (M1) and the dorsal striatum play a critical role in motor learning and the retention of learned behaviors. Motor representations of corticostriatal ensembles emerge during motor learning. In the coordinated reorganization of M1 and the dorsal striatum for motor learning, layer 5a (L5a) which connects M1 to the ipsilateral and contralateral dorsal striatum, should be a key layer. Although M1 L5a neurons represent movement-related activity in the late stage of learning, it is unclear whether the activity is retained as a memory engram. Here, using Tlx3-Cre male transgenic mice, we conducted two-photon calcium imaging of striatum-projecting L5a intratelencephalic (IT) neurons in forelimb M1 during late sessions of a self-initiated lever-pull task and in sessions after 6 d of nontraining following the late sessions. We found that trained male animals exhibited stable motor performance before and after the nontraining days. At the same time, we found that M1 L5a IT neurons strongly represented the well-learned forelimb movement but not uninstructed orofacial movements. A subset of M1 L5a IT neurons consistently coded the well-learned forelimb movement before and after the nontraining days. Inactivation of M1 IT neurons after learning impaired task performance when the lever was made heavier or when the target range of the pull distance was narrowed. These results suggest that a subset of M1 L5a IT neurons continuously represent skilled movement after learning and serve to fine-tune the kinematics of well-learned movement. [ABSTRACT FROM AUTHOR]

Abnormal expression of monoamine oxidase A (MAO−A) has been implicated in the development of human glioma, making MAO−A a promising target for therapy. Therefore, a rapid determination of MAO−A is critical for diagnosis. Through in silico screening of two‐photon fluorophores, we discovered that a derivative of N,N‐dimethyl‐naphthalenamine (pre‐mito) can effectively fit into the entrance of the MAO−A cavity. Substitutions on the N‐pyridine not only further explore the MAO−A cavity, but also enable mitochondrial targeting ability. The aminopropyl substituted molecule, CD1, showed the fastest MAO−A detection (within 20 s), high MAO−A affinity and selectivity. It was also used for in situ imaging of MAO−A in living cells, enabling a comparison of the MAO−A content in human glioma and paracancerous tissues. Our results demonstrate that optimizing the affinity binding‐based fluorogenic probes significantly improves their detection rate, providing a general approach for rapid detection probe design and optimization. [ABSTRACT FROM AUTHOR]

Introduction: The visual cortex is a key region in the mouse brain, responsible for processing visual information. Comprised of six distinct layers, each with unique neuronal types and connections, the visual cortex exhibits diverse decoding properties across its layers. This study aimed to investigate the relationship between visual stimulus decoding properties and the cortical layers of the visual cortex while considering howthis relationship varies across different decoders and brain regions. Methods: This study reached the above conclusions by analyzing two publicly available datasets obtained through two-photon microscopy of visual cortex neuronal responses. Various types of decoders were tested for visual cortex decoding. Results: Our findings indicate that the decoding accuracy of neuronal populations with consistent sizes varies among visual cortical layers for visual stimuli such as drift gratings and natural images. In particular, layer 4 neurons in VISp exhibited significantly higher decoding accuracy for visual stimulus identity compared to other layers. However, in VISm, the decoding accuracy of neuronal populations with the same size in layer 2/3 was higher than that in layer 4, despite the overall accuracy being lower than that in VISp and VISl. Furthermore, SVM surpassed other decoders in terms of accuracy, with the variation in decoding performance across layers being consistent among decoders. Additionally, we found that the difference in decoding accuracy across different imaging depths was not associated with the mean orientation selectivity index (OSI) and the mean direction selectivity index (DSI) neurons, but showed a significant positive correlation with the mean reliability and mean signal-to-noise ratio (SNR) of each layer's neuron population. Discussion: These findings lend new insights into the decoding properties of the visual cortex, highlighting the role of different cortical layers and decoders in determining decoding accuracy. The correlations identified between decoding accuracy and factors such as reliability and SNR pave the way for more nuanced understandings of visual cortex functioning. [ABSTRACT FROM AUTHOR]

Non-rapid eye movement (NREM) sleep, characterized by slow-wave electrophysiological activity, underlies several critical functions, including learning and memory. However, NREM sleep is heterogeneous, varying in duration, depth, and spatially across the cortex. While these NREM sleep features are thought to be largely independently regulated, there is also evidence that they are mechanistically coupled. To investigate how cortical NREM sleep features are controlled, we examined the astrocytic network, comprising a cortex-wide syncytium that influences population-level neuronal activity. We quantified endogenous astrocyte activity in mice over natural sleep and wake, then manipulated specific astrocytic G-protein-coupled receptor (GPCR) signaling pathways in vivo. We find that astrocytic Gi- and Gq-coupled GPCR signaling separately control NREM sleep depth and duration, respectively, and that astrocytic signaling causes differential changes in local and remote cortex. These data support a model in which the cortical astrocyte network serves as a hub for regulating distinct NREM sleep features.

Non‐linear excitation microscopy offers several advantages for in‐vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in‐vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, Fibroblast cell culture plano‐convex microlenses to be used for non‐linear imaging of biological tissue are developed and tested. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. Magnified fluorescence images can be retrieved through the microlenses coupled to commercial confocal and two‐photon excitation scanning microscopes. The signal‐to‐noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro‐optics for optical in‐vivo inspection of biological processes. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) peptide deposition, hyperphosphorylated tau proteins, reactive astrocytes, high levels of metal ions, and upregulated monoamine oxidases are considered to be the primary pathological markers of Alzheimer's disease (AD). Among them, Aβ peptide deposition or Aβ plaques, is regarded as the initial factor in the pathogenesis of AD and a critical pathological hallmark in AD. This review highlights recently Aβ-specific fluorescent probes for two-photon imaging of Aβ plaques in vivo. It includes the synthesis and detection mechanism of probes, as well as their application to two-photon imaging of Aβ plaques in vivo. [ABSTRACT FROM AUTHOR]

Over the past 30 years, the calcium (Ca2+) hypothesis of brain aging has provided clear evidence that hippocampal neuronal Ca2+ dysregulation is a key biomarker of aging. Age‐dependent Ca2+‐mediated changes in intrinsic excitability, synaptic plasticity, and activity have helped identify some of the mechanisms engaged in memory and cognitive decline based on work done mostly at the single‐cell level and in the slice preparation. Recently, our lab identified age‐ and Ca2+‐related neuronal network dysregulation in the cortex of the anesthetized animal. Still, investigations in the awake animal are needed to test the generalizability of the Ca2+ hypothesis of brain aging. Here, we used in vigilo two‐photon imaging in ambulating mice, to image GCaMP8f in the primary somatosensory cortex (S1), during ambulation and at rest. We investigated aging‐ and sex‐related changes in neuronal networks in the C56BL/6J mouse. Following imaging, gait behavior was characterized to test for changes in locomotor stability. During ambulation, in both young adult and aged mice, an increase in network connectivity and synchronicity was noted. An age‐dependent increase in synchronicity was seen in ambulating aged males only. Additionally, females displayed increases in the number of active neurons, Ca2+ transients, and neuronal activity compared to males, particularly during ambulation. These results suggest S1 Ca2+ dynamics and network synchronicity are likely contributors of locomotor stability. We believe this work raises awareness of age‐ and sex‐dependent alterations in S1 neuronal networks, perhaps underlying the increase in falls with age. [ABSTRACT FROM AUTHOR]

The auditory cortex (AC) modulates the activity of upstream pathways in the auditory brainstem via descending (corticofugal) projections. This feedback system plays an important role in the plasticity of the auditory system by shaping response properties of neurons in many subcortical nuclei. The majority of layer (L) 5 corticofugal neurons project to the inferior colliculus (IC). This corticocollicular (CC) pathway is involved in processing of complex sounds, auditory-related learning, and defense behavior. Partly due to their location in deep cortical layers, CC neuron population activity patterns within neuronal AC ensembles remain poorly understood. We employed two-photon imaging to record the activity of hundreds of L5 neurons in anesthetized as well as awake animals. CC neurons are broader tuned than other L5 pyramidal neurons and display weaker topographic order in core AC subfields. Network activity analyses revealed stronger clusters of CC neurons compared to non-CC neurons, which respond more reliable and integrate information over larger distances. However, results obtained from secondary auditory cortex (A2) differed considerably. Here CC neurons displayed similar or higher topography, depending on the subset of neurons analyzed. Furthermore, specifically in A2, CC activity clusters formed in response to complex sounds were spatially more restricted compared to other L5 neurons. Our findings indicate distinct network mechanism of CC neurons in analyzing sound properties with pronounced subfield differences, demonstrating that the topography of sound-evoked responses within AC is neuron-type dependent. [ABSTRACT FROM AUTHOR]

Aggregation‐induced emission luminogens (AIEgens) as a new class of optical probes for two‐photon imaging (2PI) have attracted extensive attention in the research community. However, their non‐radiative energy dissipation plays a non‐negligible role in energy consumption, thereby weakening their performance in 2PI. Exploration of a facile and general approach to control the energy relaxation pathways is thus a fascinating yet significantly challenging task. To address this concern, an organic doping method is proposed herein by using TPE‐Br as the doping AIEgen and MeOTTMN as the functional AIEgen. With increased amount of doping molecules, a steadily strengthened brightness (up to 29‐fold of quantum yield) is observed at the expense of reactive oxygen species production. Remarkably, an ultradeep imaging depth of 1000 µm in a mouse brain is realized via 2PI using the heavily organic‐doped AIEgen(ODA) nanoparticles (NPs). Such result is far superior to the imaging depth of 156 µm enabled by MeOTTMN NPs. The safe use of ODA NPs is further evaluated by in vitro and in vivo toxicity assessment. This study thus provides an attractive paradigm to control the energy relaxation of AIEgen NPs and maximize their performance in 2PI while circumventing the difficulties in molecular engineering. [ABSTRACT FROM AUTHOR]

Structural plasticity of dendritic spines is a key component of the refinement of synaptic connections during learning. Recent studies highlight a novel role for the NMDA receptor (NMDAR), independent of ion flow, in driving spine shrinkage and LTD. Yet little is known about the molecular mechanisms that link conformational changes in the NMDAR to changes in spine size and synaptic strength. Here, using two-photon glutamate uncaging to induce plasticity at individual dendritic spines on hippocampal CA1 neurons from mice and rats of both sexes, we demonstrate that p38 MAPK is generally required downstream of non-ionotropic NMDAR signaling to drive both spine shrinkage and LTD. In a series of pharmacological and molecular genetic experiments, we identify key components of the non-ionotropic NMDAR signaling pathway driving dendritic spine shrinkage, including the interaction between NOS1AP (nitric oxide synthase 1 adaptor protein) and neuronal nitric oxide synthase (nNOS), nNOS enzymatic activity, activation of MK2 (MAPK-activated protein kinase 2) and cofilin, and signaling through CaMKII. Our results represent a large step forward in delineating the molecular mechanisms of non-ionotropic NMDAR signaling that can drive shrinkage and elimination of dendritic spines during synaptic plasticity.SIGNIFICANCE STATEMENT Signaling through the NMDA receptor (NMDAR) is vitally important for the synaptic plasticity that underlies learning. Recent studies highlight a novel role for the NMDAR, independent of ion flow, in driving synaptic weakening and dendritic spine shrinkage during synaptic plasticity. Here, we delineate several key components of the molecular pathway that links conformational signaling through the NMDAR to dendritic spine shrinkage during synaptic plasticity.

Modulation of synaptic strength through trafficking of AMPA receptors (AMPARs) is a fundamental mechanism underlying synaptic plasticity, learning, and memory. However, the dynamics of AMPAR trafficking in vivo and its correlation with learning have not been resolved. Here, we used in vivo two-photon microscopy to visualize surface AMPARs in mouse cortex during the acquisition of a forelimb reaching task. Daily training leads to an increase in AMPAR levels at a subset of spatially clustered dendritic spines in the motor cortex. Surprisingly, we also observed increases in spine AMPAR levels in the visual cortex. There, synaptic potentiation depends on the availability of visual input during motor training, and optogenetic inhibition of visual cortex activity impairs task performance. These results indicate that motor learning induces widespread cortical synaptic potentiation by increasing the net trafficking of AMPARs into spines, including in non-motor brain regions.

Abstract Microglia are highly dynamic immune cells of the central nervous system (CNS). Microglial processes interact with neuronal elements constantly on the order of minutes. The functional significance of this acute microglia-neuron interaction and its potential role in the context of pain is still largely unknown. Here, we found that spinal microglia increased their process motility and electrophysiological reactivity within an hour after the insult in a mouse model of formalin-induced acute, sustained, inflammatory pain. Using an ablation strategy to specifically deplete resident microglia in the CNS, we demonstrate that microglia participate in formalin-induced acute sustained pain behaviors by amplifying neuronal activity in the spinal dorsal horn. Moreover, we identified that the P2Y12 receptor, which is specifically expressed in microglia in the CNS, was required for microglial function in formalin-induced pain. Taken together, our study provides a novel insight into the contribution of microglia and the P2Y12 receptor in inflammatory pain that could be used for potential therapeutic strategies.

Exposure to combinations of environmental toxins is growing in prevalence; and therefore, understanding their interactions is of increasing societal importance. Here, we examined the mechanisms by which two environmental toxins, polychlorinated biphenyls (PCBs) and high-amplitude acoustic noise, interact to produce dysfunction in central auditory processing. PCBs are well established to impose negative developmental impacts on hearing. However, it is not known whether developmental exposure to this ototoxin alters the sensitivity to other ototoxic exposures later in life. Here, male mice were exposed to PCBs in utero, and later as adults were exposed to 45min of high-intensity noise. We then examined the impacts of the two exposures on hearing and the organization of the auditory midbrain using two-photon imaging and analysis of the expression of mediators of oxidative stress. We observed that developmental exposure to PCBs blocked hearing recovery from acoustic trauma. In vivo two-photon imaging of the inferior colliculus (IC) revealed that this lack of recovery was associated with disruption of the tonotopic organization and reduction of inhibition in the auditory midbrain. In addition, expression analysis in the inferior colliculus revealed that reduced GABAergic inhibition was more prominent in animals with a lower capacity to mitigate oxidative stress. These data suggest that combined PCBs and noise exposure act nonlinearly to damage hearing and that this damage is associated with synaptic reorganization, and reduced capacity to limit oxidative stress. In addition, this work provides a new paradigm by which to understand nonlinear interactions between combinations of environmental toxins. [ABSTRACT FROM AUTHOR]

Monitoring the early stage of atherosclerosis (AS) without plaque formation is of great significance. Herein, we developed a metal organic framework (MOF)‐based fluorescence nanoprobe to analyze the progression of AS by assessing the levels of protein phosphorylation and glucose in blood and tissue. The probe was prepared by post‐modification of the MOF with iodine (I3−)‐rhodamine B (RhB) associate, which realizes the specific recognition of target object through the metal joint ZrIV and I3−‐RhB, respectively. We investigated different stages of target object changes in the early non‐plaque stage of AS in blood. It was found that the levels of phosphate and glucose in the blood were higher than those of the normal mice. The results of two‐photon images showed that early AS mice had higher levels of protein phosphorylation and glucose than that of the normal mice. The present study provides a suitable fluorescence tool for further revealing the pathogenesis and progression of AS. [ABSTRACT FROM AUTHOR]

Dynamic changes in sensory representations have been basic tenants of studies in neural coding and plasticity. In olfaction, relatively little is known about the dynamic range of changes in odor representations under different brain states and over time. Here, we used time-lapse in vivo two-photon calcium imaging to describe changes in odor representation by mitral cells, the output neurons of the mouse olfactory bulb. Using anesthetics as a gross manipulation to switch between different brain states (wakefulness and under anesthesia), we found that odor representations by mitral cells undergo significant re-shaping across states but not over time within state. Odor representations were well balanced across the population in the awake state yet highly diverse under anesthesia. To evaluate differences in odor representation across states, we used linear classifiers to decode odor identity in one state based on training data from the other state. Decoding across states resulted in nearly chance-level accuracy. In contrast, repeating the same procedure for data recorded within the same state but in different time points, showed that time had a rather minor impact on odor representations. Relative to the differences across states, odor representations remained stable over months. Thus, single mitral cells can change dynamically across states but maintain robust representations across months. These findings have implications for sensory coding and plasticity in the mammalian brain. [ABSTRACT FROM AUTHOR]

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small‐molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small‐molecule probe that selectively detects nuclear ATP through reversible binding, with 25‐fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1–3.3‐fold and 3.9–7.8‐fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP‐associated biology. [ABSTRACT FROM AUTHOR]

Nitrogen (N) doped CDs (N-CDs) were prepared using citric acid and hexamethylenetetramine by a one-step hydrothermal method, characterized by transmission electron microscopy (TEM), Powder X-ray Diffractometer (PXRD), Nuclear Magnetic Resonance (13C NMR), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), and ultraviolet–visible (UV–vis) absorption spectra. The doping mechanism and drug-loading characteristics of N-CDs were also studied. The results showed that N-CDs had both down- and up-conversion fluorescence properties and the average particle size was about 4.28 nm. N-CDs could be used as a drug carrier for doxorubicin (DOX) (N-CDs-DOX) via the formation of the NC bond, and the drug-loading rate of N-CDs-DOX was 35.88%. The N-CDs-DOX had pH-targeted properties and low cytotoxicity. In vitro release of N-CDs-DOX belonged to the Weibull model and Fick diffusion. The results showed that N-CDs could deliver DOX successfully, and collocate with DOX in the SH-SY5Y cells through two-photon imaging. The N-CDs-DOX system had sustained-release and pH-targeted properties. N-CDs were expected to deliver a type of drugs containing CO group and be used in cancer treatment.

The auditory cortex (AC) modulates the activity of upstream pathways in the auditory brainstem via descending (corticofugal) projections. This feedback system plays an important role in the plasticity of the auditory system by shaping response properties of neurons in many subcortical nuclei. The majority of layer (L) 5 corticofugal neurons project to the inferior colliculus (IC). This corticocollicular (CC) pathway is involved in processing of complex sounds, auditory-related learning, and defense behavior. Partly due to their location in deep cortical layers, CC neuron population activity patterns within neuronal AC ensembles remain poorly understood. We employed two-photon imaging to record the activity of hundreds of L5 neurons in anesthetized as well as awake animals. CC neurons are broader tuned than other L5 pyramidal neurons and display weaker topographic order in core AC subfields. Network activity analyses revealed stronger clusters of CC neurons compared to non-CC neurons, which respond more reliable and integrate information over larger distances. However, results obtained from secondary auditory cortex (A2) differed considerably. Here CC neurons displayed similar or higher topography, depending on the subset of neurons analyzed. Furthermore, specifically in A2, CC activity clusters formed in response to complex sounds were spatially more restricted compared to other L5 neurons. Our findings indicate distinct network mechanism of CC neurons in analyzing sound properties with pronounced subfield differences, demonstrating that the topography of sound-evoked responses within AC is neuron-type dependent.

Neurons in cortical circuits are often coactivated as ensembles, yet it is unclear whether ensembles play a functional role in behavior. Some ensemble neurons have pattern completion properties, triggering the entire ensemble when activated. Using two-photon holographic optogenetics in mouse primary visual cortex, we tested whether recalling ensembles by activating pattern completion neurons alters behavioral performance in a visual task. Disruption of behaviorally relevant ensembles by activation of non-selective neurons decreased performance, whereas activation of only two pattern completion neurons from behaviorally relevant ensembles improved performance, by reliably recalling the whole ensemble. Also, inappropriate behavioral choices were evoked by the mistaken activation of behaviorally relevant ensembles. Finally, in absence of visual stimuli, optogenetic activation of two pattern completion neurons could trigger behaviorally relevant ensembles and correct behavioral responses. Our results demonstrate a causal role of neuronal ensembles in a visually guided behavior and suggest that ensembles implement internal representations of perceptual states.

Heteroatom-doping carbon dots (CDs) have excellent fluorescence properties. In the paper, nitrogen (N) and phosphorus (P) co-doped CDs (N, P-CDs) were prepared through the hydrothermal method and characterized by TEM, PXRD, FT-IR, 13C NMR, XPS, UV–vis, and fluorescence spectra. The optical properties and drug (doxorubicin) - delivery performance of N, P-CDs were also studied. The results showed that the average size of N, P-CDs was 3.64 nm and the quantum yield was 30 %. The N, P-CDs had down- and up-conversion fluorescence properties, and the luminescence mechanism of the N, P-CDs was explained. The drug loading capacity of N, P-CDs was 39.11 %, and the drug delivery system (N, P-CDs-DOX) had low cytotoxicity, sustained release, and pH-targeted properties. The in vitro release of N, P-CDs-DOX belonged to the Weibull model and Fick diffusion, exhibiting the same release model and mechanism with free DOX. The N, P-CDs could deliver DOX successfully, which was demonstrated by the co-collocation of N, P-CDs and DOX in SH-SY5Y cells through single/two-photon imaging. N, P-CDs could potentially be used in drug-targeted delivery and cell imaging.

Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways.

Dynamic changes in sensory representations have been basic tenants of studies in neural coding and plasticity. In olfaction, relatively little is known about the dynamic range of changes in odor representations under different brain states and over time. Here, we used time-lapse in vivo two-photon calcium imaging to describe changes in odor representation by mitral cells, the output neurons of the mouse olfactory bulb. Using anesthetics as a gross manipulation to switch between different brain states (wakefulness and under anesthesia), we found that odor representations by mitral cells undergo significant re-shaping across states but not over time within state. Odor representations were well balanced across the population in the awake state yet highly diverse under anesthesia. To evaluate differences in odor representation across states, we used linear classifiers to decode odor identity in one state based on training data from the other state. Decoding across states resulted in nearly chance-level accuracy. In contrast, repeating the same procedure for data recorded within the same state but in different time points, showed that time had a rather minor impact on odor representations. Relative to the differences across states, odor representations remained stable over months. Thus, single mitral cells can change dynamically across states but maintain robust representations across months. These findings have implications for sensory coding and plasticity in the mammalian brain.

Herein, a reversible pH fluorescent sensor was developed using caffeic acid as the precursor by one-step solvothermal synthesis method. The carbon dots-based sensor (CA-CDs) exhibited pH-dependent increase in fluorescence intensity and showed linear relationship in the range of pH 6.60 and 8.00. Notably, the fluorescence sensor has a reversible response to pH change. Finally, the CA-CDs has been successfully applied for two-photon imaging of the pH in liver and kidney of diabetic mice. Imaging results showed that the pH value in kidney of diabetic mice was lower than that of the normal mice, while the pH value in liver of diabetic mice was almost the same as that of the normal mice. The present study provides a simple analytical method for pH detection suitable for in vivo. [ABSTRACT FROM AUTHOR]

The inflammatory microenvironment involves changes in pH and protein phosphorylation state and is closely related to the occurrence and development of atherosclerosis (AS). Herein, we constructed a dual‐detection fluorescence nanosensor PCN‐NP‐HPZ based on post modification of MOFs, which realized the simultaneous detection and imaging of pH and phosphorylation through the pH‐sensitive group piperazine and the ZrIV node of the MOFs. The sensors were used to monitor changes in blood pH and phosphate levels at different time stages during atherosclerotic plaque formation. Two‐photon fluorescence imaging was also performed in the vascular endothelium. Blood tests combined with two‐photon fluorescence images indicated that in the early stage of AS, blood and tissue pH levels were lower than that of the normal mice, while phosphate and tissue phosphorylation levels were higher than that of the normal mice. The present study provides a new analysis method for the assessment of early atherosclerotic disease. [ABSTRACT FROM AUTHOR]

The cerebrovasculature plays an essential role in neurovascular and homeostatic functions in health and disease conditions. Many efforts have been made for developing vascular thrombosis methods to study vascular dysfunction in vivo, while technical challenges remain, such as accuracy and depth‐selectivity to target a single vessel in the cerebral cortex. Herein, this paper first demonstrates the evaluation and quantification of the feasibility and effects of Rose Bengal (RB)‐induced photothrombosis with 720–1070 nm ultrafast lasers in a raster scan. A flexible and reproducible approach is then proposed to employ a 1070 nm ultrafast laser with a spiral scan for producing RB‐induced occlusion, which is described as precision ultrafast laser‐induced photothrombosis (PLP). Combine with two‐photon microscopy imaging, this PLP displays highly precise and fast occlusion induction of various vessel types, sizes, and depths, which enhances the precision and power of the photothrombosis protocol. Overall, the PLP method provides a real‐time, practical, precise, and depth‐selected single‐vessel photothrombosis technology in the cerebral cortex with commercially available optical equipment, which is crucial for exploring brain vascular function with high spatial‐temporal resolution in the brain. [ABSTRACT FROM AUTHOR]