BRCA1 and ELK-1 regulate neural progenitor cell fate in the optic tectum in response to visual experience in Xenopus laevis tadpoles.

In developing Xenopus tadpoles, the optic tectum begins to receive patterned visual input while visuomotor circuits are still undergoing neurogenesis and circuit assembly. This visual input regulates neural progenitor cell fate decisions such that maintaining tadpoles in the dark increases proliferation, expanding the progenitor pool, while visual stimulation promotes neuronal differentiation. To identify regulators of activity-dependent neural progenitor cell fate, we profiled the transcriptomes of proliferating neural progenitor cells and newly differentiated neurons using RNA-Seq. We used advanced bioinformatic analysis of 1,130 differentially expressed transcripts to identify six differentially regulated transcriptional regulators, including Breast Cancer 1 (BRCA1) and the ETS-family transcription factor, ELK-1, which are predicted to regulate the majority of the other differentially expressed transcripts. BRCA1 is known for its role in cancers, but relatively little is known about its potential role in regulating neural progenitor cell fate. ELK-1 is a multifunctional transcription factor which regulates immediate early gene expression. We investigated the potential functions of BRCA1 and ELK-1 in activity-regulated neurogenesis in the tadpole visual system using in vivo time-lapse imaging to monitor the fate of GFP-expressing SOX2+ neural progenitor cells in the optic tectum. Our longitudinal in vivo imaging analysis showed that knockdown of either BRCA1 or ELK-1 altered the fates of neural progenitor cells and furthermore that the effects of visual experience on neurogenesis depend on BRCA1 and ELK-1 expression. These studies provide insight into the potential mechanisms by which neural activity affects neural progenitor cell fate.

Postsynaptic receptors regulate presynaptic transmitter stability through transsynaptic bridges.

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABAA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABAA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

BigNeuron: a resource to benchmark and predict performance of algorithms for automated tracing of neurons in light microscopy datasets.

BigNeuron is an open community bench-testing platform with the goal of setting open standards for accurate and fast automatic neuron tracing. We gathered a diverse set of image volumes across several species that is representative of the data obtained in many neuroscience laboratories interested in neuron tracing. Here, we report generated gold standard manual annotations for a subset of the available imaging datasets and quantified tracing quality for 35 automatic tracing algorithms. The goal of generating such a hand-curated diverse dataset is to advance the development of tracing algorithms and enable generalizable benchmarking. Together with image quality features, we pooled the data in an interactive web application that enables users and developers to perform principal component analysis, t-distributed stochastic neighbor embedding, correlation and clustering, visualization of imaging and tracing data, and benchmarking of automatic tracing algorithms in user-defined data subsets. The image quality metrics explain most of the variance in the data, followed by neuromorphological features related to neuron size. We observed that diverse algorithms can provide complementary information to obtain accurate results and developed a method to iteratively combine methods and generate consensus reconstructions. The consensus trees obtained provide estimates of the neuron structure ground truth that typically outperform single algorithms in noisy datasets. However, specific algorithms may outperform the consensus tree strategy in specific imaging conditions. Finally, to aid users in predicting the most accurate automatic tracing results without manual annotations for comparison, we used support vector machine regression to predict reconstruction quality given an image volume and a set of automatic tracings.

Neuronal membrane proteasomes regulate neuronal circuit activity in vivo and are required for learning-induced behavioral plasticity.

Protein degradation is critical for brain function through processes that remain incompletely understood. Here, we investigated the in vivo function of the 20S neuronal membrane proteasome (NMP) in the brain of Xenopus laevis tadpoles. With biochemistry, immunohistochemistry, and electron microscopy, we demonstrated that NMPs are conserved in the tadpole brain and preferentially degrade neuronal activity-induced newly synthesized proteins in vivo. Using in vivo calcium imaging in the optic tectum, we showed that acute NMP inhibition rapidly increased spontaneous neuronal activity, resulting in hypersynchronization across tectal neurons. At the circuit level, inhibiting NMPs abolished learning-dependent improvement in visuomotor behavior in live animals and caused a significant deterioration in basal behavioral performance following visual training with enhanced visual experience. Our data provide in vivo characterization of NMP functions in the vertebrate nervous system and suggest that NMP-mediated degradation of activity-induced nascent proteins may serve as a homeostatic modulatory mechanism in neurons that is critical for regulating neuronal activity and experience-dependent circuit plasticity.

Activity-Induced Cortical Glutamatergic Neuron Nascent Proteins.

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function.SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms.

Quantitative BONCAT Allows Identification of Newly Synthesized Proteins after Optic Nerve Injury.

Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here, we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine, a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in more than 1000 proteins at 1 or 5 d after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.SIGNIFICANCE STATEMENT Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.

Nutrient restriction causes reversible G2 arrest in Xenopus neural progenitors.

Nutrient status affects brain development; however, the effects of nutrient availability on neural progenitor cell proliferation in vivo are poorly understood. Without food, Xenopus laevis tadpoles enter a period of stasis during which neural progenitor proliferation is drastically reduced, but resumes when food becomes available. Here, we investigate how neural progenitors halt cell division in response to nutrient restriction and subsequently re-enter the cell cycle upon feeding. We demonstrate that nutrient restriction causes neural progenitors to arrest in G2 of the cell cycle with increased DNA content, and that nutrient availability triggers progenitors to re-enter the cell cycle at M phase. Initiation of the nutrient restriction-induced G2 arrest is rapamycin insensitive, but cell cycle re-entry requires mTOR. Finally, we show that activation of insulin receptor signaling is sufficient to increase neural progenitor cell proliferation in the absence of food. A G2 arrest mechanism provides an adaptive strategy to control brain development in response to nutrient availability by triggering a synchronous burst of cell proliferation when nutrients become available. This may be a general cellular mechanism that allows developmental flexibility during times of limited resources.

Exosomes regulate neurogenesis and circuit assembly.

Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development.

Enhanced visual experience rehabilitates the injured brain in Xenopus tadpoles in an NMDAR-dependent manner.

Traumatic brain injuries introduce functional and structural circuit deficits that must be repaired for an organism to regain function. We developed an injury model in which Xenopus laevis tadpoles are given a penetrating stab wound that damages the optic tectal circuit and impairs visuomotor behavior. In tadpoles, as in other systems, injury induces neurogenesis. The newly generated neurons are thought to integrate into the existing circuit; however, whether they integrate via the same mechanisms that govern normal neuronal maturation during development is not understood. Development of the functional visuomotor circuit in Xenopus is driven by sensory activity. We hypothesized that enhanced visual experience would improve recovery from injury by facilitating integration of newly generated neurons into the tectal circuit. We labeled newly generated neurons in the injured tectum by green fluorescent protein expression and examined their circuit integration using electrophysiology and in vivo imaging. Providing animals with brief bouts of enhanced visual experience starting 24 h after injury increased synaptogenesis and circuit integration of new neurons and facilitated behavioral recovery. To investigate mechanisms of neuronal integration and behavioral recovery after injury, we interfered with N-methyl-d-aspartate (NMDA) receptor function. Ifenprodil, which blocks GluN2B-containing NMDA receptors, impaired dendritic arbor elaboration. GluN2B blockade inhibited functional integration of neurons generated in response to injury and prevented behavioral recovery. Furthermore, tectal GluN2B knockdown blocked the beneficial effects of enhanced visual experience on functional plasticity and behavioral recovery. We conclude that visual experience-mediated rehabilitation of the injured tectal circuit occurs by GluN2B-containing NMDA receptor-dependent integration of newly generated neurons. NEW & NOTEWORTHY Recovery from brain injury is difficult in most systems. The study of regenerative animal models that are capable of injury repair can provide insight into cellular and circuit mechanisms underlying repair. Using Xenopus tadpoles, we show enhanced sensory experience rehabilitates the injured visual circuit and that this experience-dependent recovery depends on N-methyl-d-aspartate receptor function. Understanding the mechanisms of rehabilitation in this system may facilitate recovery in brain regions and systems where repair is currently impossible.

Direct intertectal inputs are an integral component of the bilateral sensorimotor circuit for behavior in Xenopus tadpoles.

The circuit controlling visually guided behavior in nonmammalian vertebrates, such as Xenopus tadpoles, includes retinal projections to the contralateral optic tectum, where visual information is processed, and tectal motor outputs projecting ipsilaterally to hindbrain and spinal cord. Tadpoles have an intertectal commissure whose function is unknown, but it might transfer information between the tectal lobes. Differences in visual experience between the two eyes have profound effects on the development and function of visual circuits in animals with binocular vision, but the effects on animals with fully crossed retinal projections are not clear. We tested the effect of monocular visual experience on the visuomotor circuit in Xenopus tadpoles. We show that cutting the intertectal commissure or providing visual experience to one eye (monocular visual experience) is sufficient to disrupt tectally mediated visual avoidance behavior. Monocular visual experience induces asymmetry in tectal circuit activity across the midline. Repeated exposure to monocular visual experience drives maturation of the stimulated retinotectal synapses, seen as increased AMPA-to-NMDA ratios, induces synaptic plasticity in intertectal synaptic connections, and induces bilaterally asymmetric changes in the tectal excitation-to-inhibition ratio (E/I). We show that unilateral expression of peptides that interfere with AMPA or GABAA receptor trafficking alters E/I in the transfected tectum and is sufficient to degrade visuomotor behavior. Our study demonstrates that monocular visual experience in animals with fully crossed visual systems produces asymmetric circuit function across the midline and degrades visuomotor behavior. The data further suggest that intertectal inputs are an integral component of a bilateral visuomotor circuit critical for behavior. NEW & NOTEWORTHY The developing optic tectum of Xenopus tadpoles represents a unique circuit in which laterally positioned eyes provide sensory input to a circuit that is transiently monocular, but which will be binocular in the animal's adulthood. We challenge the idea that the two lobes of tadpole optic tectum function independently by testing the requirement of interhemispheric communication and demonstrate that unbalanced sensory input can induce structural and functional plasticity in the tectum sufficient to disrupt function.

Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis.

UNLABELLED: Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. SIGNIFICANCE STATEMENT: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning-induced behavioral plasticity in vivo.

Thyroid Hormone Acts Locally to Increase Neurogenesis, Neuronal Differentiation, and Dendritic Arbor Elaboration in the Tadpole Visual System.

Thyroid hormone (TH) regulates many cellular events underlying perinatal brain development in vertebrates. Whether and how TH regulates brain development when neural circuits are first forming is less clear. Furthermore, although the molecular mechanisms that impose spatiotemporal constraints on TH action in the brain have been described, the effects of local TH signaling are poorly understood. We determined the effects of manipulating TH signaling on development of the optic tectum in stage 46-49 Xenopus laevis tadpoles. Global TH treatment caused large-scale morphological effects in tadpoles, including changes in brain morphology and increased tectal cell proliferation. Either increasing or decreasing endogenous TH signaling in tectum, by combining targeted DIO3 knockdown and methimazole, led to corresponding changes in tectal cell proliferation. Local increases in TH, accomplished by injecting suspensions of tri-iodothyronine (T3) in coconut oil into the midbrain ventricle or into the eye, selectively increased tectal or retinal cell proliferation, respectively. In vivo time-lapse imaging demonstrated that local TH first increased tectal progenitor cell proliferation, expanding the progenitor pool, and subsequently increased neuronal differentiation. Local T3 also dramatically increased dendritic arbor growth in neurons that had already reached a growth plateau. The time-lapse data indicate that the same cells are differentially sensitive to T3 at different time points. Finally, TH increased expression of genes pertaining to proliferation and neuronal differentiation. These experiments indicate that endogenous TH locally regulates neurogenesis at developmental stages relevant to circuit assembly by affecting cell proliferation and differentiation and by acting on neurons to increase dendritic arbor elaboration. SIGNIFICANCE STATEMENT: Thyroid hormone (TH) is a critical regulator of perinatal brain development in vertebrates. Abnormal TH signaling in early pregnancy is associated with significant cognitive deficits in humans; however, it is difficult to probe the function of TH in early brain development in mammals because of the inaccessibility of the fetal brain in the uterine environment and the challenge of disambiguating maternal versus fetal contributions of TH. The external development of tadpoles allows manipulation and direct observation of the molecular and cellular mechanisms underlying TH's effects on brain development in ways not possible in mammals. We find that endogenous TH locally regulates neurogenesis at developmental stages relevant to circuit assembly by affecting neural progenitor cell proliferation and differentiation and by acting on neurons to enhance dendritic arbor elaboration.

Optic flow instructs retinotopic map formation through a spatial to temporal to spatial transformation of visual information.

Retinotopic maps are plastic in response to changes in sensory input; however, the experience-dependent instructive cues that organize retinotopy are unclear. In animals with forward-directed locomotion, the predominant anterior to posterior optic flow activates retinal ganglion cells in a stereotyped temporal to nasal sequence. Here we imaged retinotectal axon arbor location and structural plasticity to assess map refinement in vivo while exposing Xenopus tadpoles to visual stimuli. We show that the temporal sequence of retinal activity driven by natural optic flow organizes retinotopy by regulating axon arbor branch dynamics, whereas the opposite sequence of retinal activity prevents map refinement. Our study demonstrates that a spatial to temporal to spatial transformation of visual information controls experience-dependent topographic map plasticity. This organizational principle is likely to apply to other sensory modalities and projections in the brain.

An in vivo screen to identify candidate neurogenic genes in the developing Xenopus visual system.

Neurogenesis in the brain of Xenopus laevis continues throughout larval stages of development. We developed a 2-tier screen to identify candidate genes controlling neurogenesis in Xenopus optic tectum in vivo. First, microarray and NanoString analyses were used to identify candidate genes that were differentially expressed in Sox2-expressing neural progenitor cells or their neuronal progeny. Then an in vivo, time-lapse imaging-based screen was used to test whether morpholinos against 34 candidate genes altered neural progenitor cell proliferation or neuronal differentiation over 3 days in the optic tectum of intact Xenopus tadpoles. We co-electroporated antisense morpholino oligonucleotides against each of the candidate genes with a plasmid that drives GFP expression in Sox2-expressing neural progenitor cells and quantified the effects of morpholinos on neurogenesis. Of the 34 morpholinos tested, 24 altered neural progenitor cell proliferation or neuronal differentiation. The candidates which were tagged as differentially expressed and validated by the in vivo imaging screen include: actn1, arl9, eif3a, elk4, ephb1, fmr1-a, fxr1-1, fbxw7, fgf2, gstp1, hat1, hspa5, lsm6, mecp2, mmp9, and prkaca. Several of these candidates, including fgf2 and elk4, have known or proposed neurogenic functions, thereby validating our strategy to identify candidates. Genes with no previously demonstrated neurogenic functions, gstp1, hspa5 and lsm6, were identified from the morpholino experiments, suggesting that our screen successfully revealed unknown candidates. Genes that are associated with human disease, such as such as mecp2 and fmr1-a, were identified by our screen, providing the groundwork for using Xenopus as an experimental system to probe conserved disease mechanisms. Together the data identify candidate neurogenic regulatory genes and demonstrate that Xenopus is an effective experimental animal to identify and characterize genes that regulate neural progenitor cell proliferation and differentiation in vivo.

Experience-dependent plasticity of excitatory and inhibitory intertectal inputs in Xenopus tadpoles.

Communication between optic tecta/superior colliculi is thought to be required for sensorimotor behaviors by comparing inputs across the midline, however the development of and the role of visual experience in the function and plasticity of intertectal connections are unclear. We combined neuronal tracing, in vivo time-lapse imaging, and electrophysiology to characterize the structural and functional development of intertectal axons and synapses in Xenopus tadpole optic tectum. We find that intertectal connections are established early during optic tectal circuit development. We determined the neurotransmitter identity of intertectal neurons using both rabies virus-mediated tracing combined with post-hoc immunohistochemistry, and electrophysiology. Excitatory and inhibitory intertectal neuronal somata are similarly distributed throughout the tectum. Excitatory and inhibitory intertectal axons are structurally similar and elaborate broadly in the contralateral tectum. We demonstrate that intertectal and retinotectal axons converge onto tectal neurons by recording postsynaptic currents after stimulating intertectal and retinotectal inputs. Cutting the intertectal commissure removes synaptic responses to contralateral tectal stimulation. In vivo time-lapse imaging demonstrated that visual experience drives plasticity in intertectal bouton size and dynamics. Finally, visual experience coordinately drives the maturation of excitatory and inhibitory intertectal inputs by increasing AMPA- and GABA-receptor mediated currents, comparable to experience-dependent maturation of retinotectal inputs. These data indicate that visual experience regulates plasticity of excitatory and inhibitory intertectal inputs, maintaining the excitatory: inhibitory ratio of intertectal input. These studies place intertectal inputs as key players in tectal circuit development and suggest that they may play a role in sensory information processing critical to sensorimotor behaviors.

Direct detection of biotinylated proteins by mass spectrometry.

Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called "Direct Detection of Biotin-containing Tags" (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h.

Visual neurons recognize complex image transformations.

Natural visual scenes are dominated by sequences of transforming images. Spatial visual information is thought to be processed by detection of elemental stimulus features which are recomposed into scenes. How image information is integrated over time is unclear. We explored visual information encoding in the optic tectum. Unbiased stimulus presentation shows that the majority of tectal neurons recognize image sequences. This is achieved by temporally dynamic response properties, which encode complex image transitions over several hundred milliseconds. Calcium imaging reveals that neurons that encode spatiotemporal image sequences fire in spike sequences that predict a logical diagram of spatiotemporal information processing. Furthermore, the temporal scale of visual information is tuned by experience. This study indicates how neurons recognize dynamic visual scenes that transform over time.

Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

A small molecule accelerates neuronal differentiation in the adult rat.

Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.