The hippocampus is essential for episodic memory, which requires single-trial learning. Although long-term potentiation (LTP) of synaptic strength is a candidate mechanism for learning, it is typically induced by using repeated synaptic activation to produce precisely timed, high-frequency, or rhythmic firing. Here we show that hippocampal synapses potentiate robustly in response to strong activation by a single burst. The induction mechanism of this single-burst LTP requires activation of NMDA receptors, L-type voltage-gated calcium channels, and dendritic spikes. Thus, dendritic spikes are a critical trigger for a form of LTP that is consistent with the function of the hippocampus in episodic memory.

Dendrites on neurons integrate synaptic inputs to determine spike timing. Dendrites also convey back-propagating action potentials (bAPs) which interact with synaptic inputs to produce plateau potentials and to mediate synaptic plasticity. The biophysical rules which govern the timing, spatial structures, and ionic character of dendritic excitations are not well understood. We developed molecular, optical, and computational tools to map sub-millisecond voltage dynamics throughout the dendritic trees of CA1 pyramidal neurons under diverse optogenetic and synaptic stimulus patterns, in acute brain slices. We observed history-dependent bAP propagation in distal dendrites, driven by locally generated Na+ spikes (dSpikes). Dendritic depolarization creates a transient window for dSpike propagation, opened by A-type KV channel inactivation, and closed by slow NaV inactivation. Collisions of dSpikes with synaptic inputs triggered calcium channel and N-methyl-D-aspartate receptor (NMDAR)-dependent plateau potentials, with accompanying complex spikes at the soma. This hierarchical ion channel network acts as a spike-rate accelerometer, providing an intuitive picture of how dendritic excitations shape associative plasticity rules.Competing Interest StatementThe authors have declared no competing interest.

Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light microscopy (LM) and electron microscopy (EM). Here we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100-nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery.

Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.Competing Interest StatementThe authors have declared no competing interest.

Dense-core vesicles (DCVs) are found in various types of cells, such as neurons, pancreatic β-cells, and chromaffin cells. These vesicles release transmitters, peptides, and hormones to regulate diverse functions, such as the stress response, immune response, behavior, and blood glucose levels. In traditional electron microscopy after chemical fixation, it is often reported that the dense cores occupy a portion of the vesicle towards the center and are surrounded by a clear halo. With electron microscopy following cryo-fixation in adrenal chromaffin cells, we report here that we did not observe halos, but dense cores filling up the entire vesicles suggesting that halos are likely the product of chemical fixation. More importantly, we observed that a fraction of DCVs contained 36-168 nm clear-core vesicles. A similar fraction of DCVs labeled with fluorescent false neurotransmitter FFN 511 or the dense-core matrix protein chromogranin A (CGA) were colocalized with fluorescently labeled or endogenous CD63 or ALIX, the membrane or lumen marker of ∼40-160 nm exosomes. These results suggest that DCVs contain exosomes. Since exosomes are generally thought to reside within multivesicular bodies in the cytosol and are released to the extracellular space to mediate diverse cell-to-cell communications, our findings suggest that dense-core vesicle fusion from many cell types is a new source for releasing exosomes to mediate intercellular communications. Given that dense-core vesicle fusion mediates many physiological functions, such as stress responses, immune responses, behavior regulation, and blood glucose regulation, exosome release from dense-core vesicle fusion might contribute to mediating these important functions.

Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged µ-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

During development, cells undergo a sequence of specification events to form functional tissues and organs. To investigate complex tissue development, it is crucial to visualize how cell lineages emerge and to be able to manipulate regulatory factors with temporal control. We recently developed TEMPO (Temporal Encoding and Manipulation in a Predefined Order), a genetic tool to label with different colors and genetically manipulate consecutive cell generations in vertebrates. TEMPO relies on CRISPR to activate a cascade of fluorescent proteins which can be imaged in vivo. Here, we explain the steps to design, generate, and express TEMPO constructs in zebrafish and mice.

Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent 'standard candles' for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.