The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Long-range corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing somatotopic specificity as injections move from sensory to motor and frontal areas. Somatotopic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher somatotopic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. Thus, as predicted by a long-standing basal ganglia model, corticostriatal projections of interconnected cortical areas form parallel circuits in basal ganglia-thalamus-cortex loops.

Cellular ultrastructures for signal integration are unknown in any nervous system. The ellipsoid body (EB) of the Drosophila brain is thought to control locomotion upon integration of various modalities of sensory signals with the animal internal status. However, the expected excitatory and inhibitory input convergence that virtually all brain centres exhibit is not yet described in the EB. Based on the EB expression domains of genetic constructs from the choline acetyl transferase (Cha), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) genes, we identified a new set of neurons with the characteristic ring-shaped morphology (R neurons) which are presumably cholinergic, in addition to the existing GABA-expressing neurons. The R1 morphological subtype is represented in the Cha- and TH-expressing classes. In addition, using transmission electron microscopy, we identified a novel type of synapse in the EB, which exhibits the precise array of two independent active zones over the same postsynaptic dendritic domain, that we named 'agora'. This array is compatible with a coincidence detector role, and represents ~8% of all EB synapses in Drosophila. Presumably excitatory R neurons contribute to coincident synapses. Functional silencing of EB neurons by driving genetically tetanus toxin expression either reduces walking speed or alters movement orientation depending on the targeted R neuron subset, thus revealing functional specialisations in the EB for locomotion control.

Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. Here, we identify a population of sexually dimorphic aIPg neurons in the adult central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. Our work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Dendritic cells (DCs) are specialized sentinel and APCs coordinating innate and adaptive immunity. Through proteins on their cell surface, DCs sense changes in the environment, internalize pathogens, present processed Ags, and communicate with other immune cells. By combining chemical labeling and quantitative mass spectrometry, we systematically profiled and compared the cell-surface proteomes of human primary conventional DCs (cDCs) in their resting and activated states. TLR activation by a lipopeptide globally reshaped the cell-surface proteome of cDCs, with >100 proteins upregulated or downregulated. By simultaneously elevating positive regulators and reducing inhibitory signals across multiple protein families, the remodeling creates a cell-surface milieu promoting immune responses. Still, cDCs maintain the stimulatory-to-inhibitory balance by leveraging a distinct set of inhibitory molecules. This analysis thus uncovers the molecular complexity and plasticity of the cDC cell surface and provides a roadmap for understanding cDC activation and signaling.

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

Repeated exposure to stressors is known to produce large-scale remodeling of neurons within the prefrontal cortex (PFC). Recent work suggests stress-related forms of structural plasticity can interact with aging to drive distinct patterns of pyramidal cell morphological changes. However, little is known about how other cellular components within PFC might be affected by these challenges. Here, we examined the effects of stress exposure and aging on medial prefrontal cortical glial subpopulations. Interestingly, we found no changes in glial morphology with stress exposure but a profound morphological change with aging. Furthermore, we found an upregulation of non-nuclear glucocorticoid receptors (GR) with aging, while nuclear levels remained largely unaffected. Both changes are selective for microglia, with no stress or aging effect found in astrocytes. Lastly, we show that the changes found within microglia inversely correlated with the density of dendritic spines on layer III pyramidal cells. These findings suggest microglia play a selective role in synaptic health within the aging brain.

Adaptive movements are critical to animal survival. To guide future actions, the brain monitors different outcomes, including achievement of movement and appetitive goals. The nature of outcome signals and their neuronal and network realization in motor cortex (M1), which commands the performance of skilled movements, is largely unknown. Using a dexterity task, calcium imaging, optogenetic perturbations, and behavioral manipulations, we studied outcome signals in murine M1. We find two populations of layer 2-3 neurons, “success”- and “failure” related neurons that develop with training and report end-result of trials. In these neurons, prolonged responses were recorded after success or failure trials, independent of reward and kinematics. In contrast, the initial state of layer-5 pyramidal tract neurons contains a memory trace of the previous trial’s outcome. Inter-trial cortical activity was needed to learn new task requirements. These M1 reflective layer-specific performance outcome signals, can support reinforcement motor learning of skilled behavior.