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.

A prevailing hypothesis is that neurofibrillary tangles play a causal role in driving cognitive decline in Alzheimer\'s disease (AD) because tangles correlate anatomically with areas that undergo neuronal loss. We used two-photon longitudinal imaging to directly test this hypothesis and observed the fate of individual neurons in two mouse models. At any time point, neurons without tangles died at >3 times the rate as neurons with tangles. Additionally, prior to dying, they became >20% more distant from neighboring neurons across imaging sessions. Similar microstructural changes were evident in a population of non-tangle-bearing neurons in Alzheimer\'s donor tissues. Together, these data suggest that nonfibrillar tau puts neurons at high risk of death, and surprisingly, the presence of a tangle reduces this risk. Moreover, cortical microstructure changes appear to be a better predictor of imminent cell death than tangle status is and a promising tool for identifying dying neurons in Alzheimer\'s.

Object recognition often involves the brain segregating objects from their surroundings. Neurophysiological studies of figure-ground texture segregation have yielded inconsistent results, particularly on whether V1 neurons can perform figure-ground texture segregation or just detect texture borders. To address this issue from a population perspective, we utilized two-photon calcium imaging to simultaneously record the responses of large samples of V1 and V4 neurons to figure-ground texture stimuli in awake, fixating macaques. The average response changes indicate that V1 neurons mainly detect texture borders, while V4 neurons are involved in figure-ground segregation. However, population analysis (SVM decoding of PCA-transformed neuronal responses) reveal that V1 neurons not only detect figure-ground borders, but also contribute to figure-ground texture segregation, although requiring substantially more principal components than V4 neurons to reach a 75 % decoding accuracy. Individually, V1/V4 neurons showing larger (negative/positive) figure-ground response differences contribute more to figure-ground segregation. But for V1 neurons, the contribution becomes significant only when many principal components are considered. We conclude that V1 neurons participate in figure-ground segregation primarily by defining the figure borders, and the poorly structured figure-ground information V1 neurons carry could be further utilized by V4 neurons to accomplish figure-ground segregation.

Cells need to migrate along gradients of chemicals (chemotaxis) in the course of development, wound healing, or immune responses. Neutrophils are prototypical migratory cells that are rapidly recruited to injured or infected tissues from the bloodstream. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein. Neutrophil chemotaxis has been largely studied in vitro; few assays have been developed to monitor gradient responses in complex living tissues. Here, we describe a laser-wound assay to generate focal injury in zebrafish larvae and monitor changes in behaviour and cytoskeletal dynamics. The first step is to cross adult fish and collect and rear embryos expressing a relevant fluorescent reporter (for example, Lifeact-mRuby, which labels dynamic actin) to an early larval stage. Subsequently, larvae are mounted and prepared for live imaging and wounding under a two-photon microscope. Finally, the resulting data are processed and used for cell segmentation and quantification of actin dynamics. Altogether, this assay allows the visualisation of cellular dynamics in response to acute injury at high resolution and can be combined with other manipulations, such as genetic or chemical perturbations. Key features • This protocol is designed to trigger laser wound in zebrafish larvae using two-photon intravital microscopy. • The ability to wound while imaging makes it possible to monitor the behaviour and actin changes of the cells immediately after gradient exposure. • The protocol requires a two-photon microscope for best results. Compared with one-photon laser wounding, the injury is more precise and has better tissue penetration. • The focal nature of the wounds is suitable for studies of neutrophil swarming/aggregation and can be further adapted to infectious settings.

In classical cerebellar learning, Purkinje cells (PkCs) associate climbing fiber (CF) error signals with predictive granule cells (GrCs) that were active just prior (∼150 ms). The cerebellum also contributes to behaviors characterized by longer timescales. To investigate how GrC-CF-PkC circuits might learn seconds-long predictions, we imaged simultaneous GrC-CF activity over days of forelimb operant conditioning for delayed water reward. As mice learned reward timing, numerous GrCs developed anticipatory activity ramping at different rates until reward delivery, followed by widespread time-locked CF spiking. Relearning longer delays further lengthened GrC activations. We computed CF-dependent GrC→PkC plasticity rules, demonstrating that reward-evoked CF spikes sufficed to grade many GrC synapses by anticipatory timing. We predicted and confirmed that PkCs could thereby continuously ramp across seconds-long intervals from movement to reward. Learning thus leads to new GrC temporal bases linking predictors to remote CF reward signals-a strategy well suited for learning to track the long intervals common in cognitive domains.

Synchronous neuronal activity is organized into neuronal oscillations with various frequency and time domains across different brain areas and brain states. For example, hippocampal theta, gamma and sharp wave oscillations are critical for memory formation and communication between hippocampal subareas and the cortex. In this study, we investigated the neuronal activity of the dentate gyrus (DG) with electrophysiological and optical imaging tools during sleep-wake cycles. We found that the activity of major glutamatergic cell populations in the DG is organized into in-fraslow oscillations (0.01 - 0.03 Hz) during NREM sleep. Although the DG is considered a sparsely active network during wakefulness, we found that 50% of granule cells and about 25% of mossy cells exhibit increased activity during NREM sleep. Further experiments revealed that the infraslow oscillation in the DG is modulated by rhythmic serotonin release during sleep, which oscillates at the same frequency but in an opposite phase. Genetic manipulation of 5-HT receptors revealed that this neuromodulatory regulation is mediated by 5-HT1a receptors and the knockdown of these receptors leads to memory impairment. Together, our results provide novel mechanistic insights into how the 5-HT system can influence hippocampal activity patterns during sleep.

Fatty acid amide hydrolase (FAAH) plays a crucial role in the metabolism of the endocannabinoid system by hydrolyzing a series of bioactive amides, whose abnormal levels are associated with neuronal disorders including Alzheimer\'s disease (AD). However, due to the lack of suitable quantitative sensing tools, real-time and accurate monitoring of the activity of FAAH in living systems remains unresolved. Herein, a novel enzyme-activated near-infrared two-photon ratiometric fluorescent probe (CANP) based on a naphthylvinylpyridine monofluorophore is successfully developed, in which the electron-withdrawing amide moiety is prone to be hydrolyzed to an electron-donating amine group under the catalysis of FAAH, leading to the activation of the intramolecular charge transfer process and the emergence of a new 80 nm red-shifted emission, thereby achieving a ratiometric luminescence response. Benefiting from the high selectivity, high sensitivity, and ratiometric response to FAAH, the probe CANP is successfully used to quantitatively monitor and image the FAAH levels in living neurons, by which an amyloid β (Aβ)-induced upregulation of endogenous FAAH activity is observed. Similar increases in FAAH activity are found in various brain regions of AD model mice, indicating a potential fatty acid amide metabolite-involved pathway for the pathological deterioration of AD. Moreover, our quantitative FAAH inhibition experiments further demonstrate the great value of CANP as an efficient visual probe for in situ and precise assessment of FAAH inhibitors in complex living systems, assisting the discovery of FAAH-related therapeutic agents.

How newly formed memories are preserved while brain plasticity is ongoing has been a source of debate. One idea is that synapses which experienced recent plasticity become resistant to further plasticity, a type of metaplasticity often referred to as saturation. Here, we probe the local dendritic mechanisms that limit plasticity at recently potentiated synapses. We show that recently potentiated individual synapses exhibit a synapse-specific refractory period for further potentiation. We further found that the refractory period is associated with reduced postsynaptic CaMKII signaling; however, stronger synaptic activation only partially restored the ability for further plasticity. Importantly, the refractory period is released after one hour, a timing that coincides with the enrichment of several postsynaptic proteins to pre-plasticity levels. Notably, increasing the level of the postsynaptic scaffolding protein, PSD95, but not of PSD93, overcomes the refractory period. Our results support a model in which potentiation at a single synapse is sufficient to initiate a synapse-specific refractory period that persists until key postsynaptic proteins regain their steady-state synaptic levels.

Neurons in the primary cortex carry sensory- and behavior-related information, but it remains an open question how this information emerges and intersects together during learning. Current evidence points to two possible learning-related changes: sensory information increases in the primary cortex or sensory information remains stable, but its readout efficiency in association cortices increases. We investigated this question by imaging neuronal activity in mouse primary somatosensory cortex before, during, and after learning of an object localization task. We quantified sensory- and behavior-related information and estimated how much sensory information was used to instruct perceptual choices as learning progressed. We find that sensory information increases from the start of training, while choice information is mostly present in the later stages of learning. Additionally, the readout of sensory information becomes more efficient with learning as early as in the primary sensory cortex. Together, our results highlight the importance of primary cortical neurons in perceptual learning.

BACKGROUND: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging.
RESULTS: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL).
CONCLUSIONS: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.