RESUMO
Natural cell death pathways such as apoptosis and pyroptosis play dual roles: they eliminate harmful cells and modulate the immune system by dampening or stimulating inflammation. Synthetic protein circuits capable of triggering specific death programs in target cells could similarly remove harmful cells while appropriately modulating immune responses. However, cells actively influence their death modes in response to natural signals, making it challenging to control death modes. Here, we introduce naturally inspired "synpoptosis" circuits that proteolytically regulate engineered executioner proteins and mammalian cell death. These circuits direct cell death modes, respond to combinations of protease inputs, and selectively eliminate target cells. Furthermore, synpoptosis circuits can be transmitted intercellularly, offering a foundation for engineering synthetic killer cells that induce desired death programs in target cells without self-destruction. Together, these results lay the groundwork for programmable control of mammalian cell death.
Assuntos
Morte Celular , Humanos , Apoptose , Caspases/metabolismo , Células HEK293 , Proteólise , Piroptose/efeitos dos fármacos , Biologia Sintética/métodos , Células CultivadasRESUMO
In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.
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Public health studies indicate that artificial light is a high-risk factor for metabolic disorders. However, the neural mechanism underlying metabolic modulation by light remains elusive. Here, we found that light can acutely decrease glucose tolerance (GT) in mice by activation of intrinsically photosensitive retinal ganglion cells (ipRGCs) innervating the hypothalamic supraoptic nucleus (SON). Vasopressin neurons in the SON project to the paraventricular nucleus, then to the GABAergic neurons in the solitary tract nucleus, and eventually to brown adipose tissue (BAT). Light activation of this neural circuit directly blocks adaptive thermogenesis in BAT, thereby decreasing GT. In humans, light also modulates GT at the temperature where BAT is active. Thus, our work unveils a retina-SON-BAT axis that mediates the effect of light on glucose metabolism, which may explain the connection between artificial light and metabolic dysregulation, suggesting a potential prevention and treatment strategy for managing glucose metabolic disorders.
Assuntos
Tecido Adiposo Marrom , Hipotálamo , Camundongos , Animais , Humanos , Tecido Adiposo Marrom/metabolismo , Hipotálamo/metabolismo , Termogênese/fisiologia , Retina , Células Ganglionares da Retina , Glucose/metabolismoRESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.
Assuntos
Terapia Genética , Doença de Parkinson , Animais , Humanos , Camundongos , Corpo Estriado/metabolismo , Levodopa/uso terapêutico , Levodopa/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/terapia , Primatas , Receptores de Dopamina D1/metabolismo , Modelos Animais de DoençasRESUMO
Human reasoning depends on reusing pieces of information by putting them together in new ways. However, very little is known about how compositional computation is implemented in the brain. Here, we ask participants to solve a series of problems that each require constructing a whole from a set of elements. With fMRI, we find that representations of novel constructed objects in the frontal cortex and hippocampus are relational and compositional. With MEG, we find that replay assembles elements into compounds, with each replay sequence constituting a hypothesis about a possible configuration of elements. The content of sequences evolves as participants solve each puzzle, progressing from predictable to uncertain elements and gradually converging on the correct configuration. Together, these results suggest a computational bridge between apparently distinct functions of hippocampal-prefrontal circuitry and a role for generative replay in compositional inference and hypothesis testing.
Assuntos
Hipocampo , Córtex Pré-Frontal , Humanos , Encéfalo , Lobo Frontal , Hipocampo/fisiologia , Imageamento por Ressonância Magnética/métodos , Vias Neurais , Córtex Pré-Frontal/fisiologiaRESUMO
Animals with complex nervous systems demand sleep for memory consolidation and synaptic remodeling. Here, we show that, although the Caenorhabditis elegans nervous system has a limited number of neurons, sleep is necessary for both processes. In addition, it is unclear if, in any system, sleep collaborates with experience to alter synapses between specific neurons and whether this ultimately affects behavior. C. elegans neurons have defined connections and well-described contributions to behavior. We show that spaced odor-training and post-training sleep induce long-term memory. Memory consolidation, but not acquisition, requires a pair of interneurons, the AIYs, which play a role in odor-seeking behavior. In worms that consolidate memory, both sleep and odor conditioning are required to diminish inhibitory synaptic connections between the AWC chemosensory neurons and the AIYs. Thus, we demonstrate in a living organism that sleep is required for events immediately after training that drive memory consolidation and alter synaptic structures.
Assuntos
Caenorhabditis elegans , Odorantes , Animais , Caenorhabditis elegans/fisiologia , Olfato , Sono/fisiologia , Sinapses/fisiologiaRESUMO
Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.
Assuntos
Antivirais , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Animais , Antivirais/farmacologia , Farmacorresistência Viral , Redes Reguladoras de Genes/efeitos dos fármacos , Camundongos , SARS-CoV-2/efeitos dos fármacos , Replicação ViralRESUMO
In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution. Surprisingly, statistical analysis of this much larger dataset revealed that the olfactory cortex connectivity is spatially structured. Single olfactory bulb neurons targeting a particular location along the anterior-posterior axis of piriform cortex also project to matched, functionally distinct, extra-piriform targets. Moreover, single neurons from the targeted piriform locus also project to the same matched extra-piriform targets, forming triadic circuit motifs. Thus, as in other sensory modalities, olfactory information is routed at early stages of processing to functionally diverse targets in a coordinated manner.
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Córtex Olfatório , Condutos Olfatórios , Camundongos , Animais , Bulbo Olfatório , Neurônios/fisiologia , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.
Assuntos
Condutos Olfatórios/citologia , Condutos Olfatórios/diagnóstico por imagem , Imagem com Lapso de Tempo , Animais , Axônios/fisiologia , Células Cultivadas , Dendritos/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Microtúbulos/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Fatores de TempoRESUMO
In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.
Assuntos
Movimento/fisiologia , Rede Nervosa/fisiologia , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Cerebelo/fisiologia , Sincronização Cortical , Membro Anterior/fisiologia , Interneurônios/fisiologia , Aprendizagem , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Atividade Motora/fisiologia , Núcleo Olivar/fisiologia , Optogenética , Células de Purkinje/fisiologia , Comportamento Estereotipado , Análise e Desempenho de TarefasRESUMO
Lateral intraparietal (LIP) neurons represent formation of perceptual decisions involving eye movements. In circuit models for these decisions, neural ensembles that encode actions compete to form decisions. Consequently, representation and readout of the decision variables (DVs) are implemented similarly for decisions with identical competing actions, irrespective of input and task context differences. Further, DVs are encoded as partially potentiated action plans through balance of activity of action-selective ensembles. Here, we test those core principles. We show that in a novel face-discrimination task, LIP firing rates decrease with supporting evidence, contrary to conventional motion-discrimination tasks. These opposite response patterns arise from similar mechanisms in which decisions form along curved population-response manifolds misaligned with action representations. These manifolds rotate in state space based on context, indicating distinct optimal readouts for different tasks. We show similar manifolds in lateral and medial prefrontal cortices, suggesting similar representational geometry across decision-making circuits.
Assuntos
Tomada de Decisões , Percepção de Movimento/fisiologia , Lobo Parietal/fisiologia , Animais , Comportamento Animal , Julgamento , Macaca mulatta , Masculino , Modelos Neurológicos , Neurônios/fisiologia , Estimulação Luminosa , Córtex Pré-Frontal/fisiologia , Psicofísica , Análise e Desempenho de Tarefas , Fatores de TempoRESUMO
The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.
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Potenciais de Ação/fisiologia , Bainha de Mielina/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Nós Neurofibrosos/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Masculino , Modelos Neurológicos , Fibras Nervosas Mielinizadas/metabolismo , Técnicas de Patch-Clamp/métodos , Células Piramidais/fisiologia , Ratos , Ratos WistarRESUMO
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.
Assuntos
Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Proteômica/métodos , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/metabolismo , Neurogênese/fisiologia , Nervo Olfatório/metabolismo , Condutos Olfatórios/citologia , Condutos Olfatórios/fisiologia , Receptores de Lipoproteínas/metabolismo , Olfato/fisiologiaRESUMO
Cortical layer 1 (L1) interneurons have been proposed as a hub for attentional modulation of underlying cortex, but the transformations that this circuit implements are not known. We combined genetically targeted voltage imaging with optogenetic activation and silencing to study the mechanisms underlying sensory processing in mouse barrel cortex L1. Whisker stimuli evoked precisely timed single spikes in L1 interneurons, followed by strong lateral inhibition. A mild aversive stimulus activated cholinergic inputs and evoked a bimodal distribution of spiking responses in L1. A simple conductance-based model that only contained lateral inhibition within L1 recapitulated the sensory responses and the winner-takes-all cholinergic responses, and the model correctly predicted that the network would function as a spatial and temporal high-pass filter for excitatory inputs. Our results demonstrate that all-optical electrophysiology can reveal basic principles of neural circuit function in vivo and suggest an intuitive picture for how L1 transforms sensory and modulatory inputs. VIDEO ABSTRACT.
Assuntos
Eletrofisiologia/métodos , Potenciais Somatossensoriais Evocados/fisiologia , Interneurônios/fisiologia , Inibição Neural/fisiologia , Imagem Óptica/métodos , Córtex Somatossensorial/citologia , Potenciais de Ação/fisiologia , Animais , Neurônios Colinérgicos/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , Potenciais Sinápticos/fisiologia , Vibrissas/fisiologiaRESUMO
Calcium imaging using two-photon scanning microscopy has become an essential tool in neuroscience. However, in its typical implementation, the tradeoffs between fields of view, acquisition speeds, and depth restrictions in scattering brain tissue pose severe limitations. Here, using an integrated systems-wide optimization approach combined with multiple technical innovations, we introduce a new design paradigm for optical microscopy based on maximizing biological information while maintaining the fidelity of obtained neuron signals. Our modular design utilizes hybrid multi-photon acquisition and allows volumetric recording of neuroactivity at single-cell resolution within up to 1 × 1 × 1.22 mm volumes at up to 17 Hz in awake behaving mice. We establish the capabilities and potential of the different configurations of our imaging system at depth and across brain regions by applying it to in vivo recording of up to 12,000 neurons in mouse auditory cortex, posterior parietal cortex, and hippocampus.
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Microscopia/métodos , Imagem Molecular/métodos , Neuroimagem/métodos , Animais , Encéfalo/fisiologia , Cálcio/metabolismo , Feminino , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Análise de Célula Única/métodosRESUMO
Social interactions involve complex decision-making tasks that are shaped by dynamic, mutual feedback between participants. An open question is whether and how emergent properties may arise across brains of socially interacting individuals to influence social decisions. By simultaneously performing microendoscopic calcium imaging in pairs of socially interacting mice, we find that animals exhibit interbrain correlations of neural activity in the prefrontal cortex that are dependent on ongoing social interaction. Activity synchrony arises from two neuronal populations that separately encode one's own behaviors and those of the social partner. Strikingly, interbrain correlations predict future social interactions as well as dominance relationships in a competitive context. Together, our study provides conclusive evidence for interbrain synchrony in rodents, uncovers how synchronization arises from activity at the single-cell level, and presents a role for interbrain neural activity coupling as a property of multi-animal systems in coordinating and sustaining social interactions between individuals.
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Encéfalo/metabolismo , Neurônios/metabolismo , Animais , Sinalização do Cálcio , Comportamento Competitivo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Análise de Componente Principal , Predomínio SocialRESUMO
Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis. VIDEO ABSTRACT.
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Tissues are exposed to diverse inflammatory challenges that shape future inflammatory responses. While cellular metabolism regulates immune function, how metabolism programs and stabilizes immune states within tissues and tunes susceptibility to inflammation is poorly understood. Here, we describe an innate immune metabolic switch that programs long-term intestinal tolerance. Intestinal interleukin-18 (IL-18) stimulation elicited tolerogenic macrophages by preventing their proinflammatory glycolytic polarization via metabolic reprogramming to fatty acid oxidation (FAO). FAO reprogramming was triggered by IL-18 activation of SLC12A3 (NCC), leading to sodium influx, release of mitochondrial DNA, and activation of stimulator of interferon genes (STING). FAO was maintained in macrophages by a bistable switch that encoded memory of IL-18 stimulation and by intercellular positive feedback that sustained the production of macrophage-derived 2'3'-cyclic GMP-AMP (cGAMP) and epithelial-derived IL-18. Thus, a tissue-reinforced metabolic switch encodes durable immune tolerance in the gut and may enable reconstructing compromised immune tolerance in chronic inflammation.
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The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.
Assuntos
Hipocampo/metabolismo , Animais , Axônios/fisiologia , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Hipocampo/citologia , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto , Memória de Curto Prazo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Análise de Componente Principal , Células Piramidais/citologia , Células Piramidais/metabolismo , Análise de Sequência de RNA , TranscriptomaRESUMO
Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed â¼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.