Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
Cell ; 183(7): 2003-2019.e16, 2020 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-33308478

RESUMEN

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE's modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.


Asunto(s)
Conducta Animal , Calcio/metabolismo , Cuerpo Estriado/metabolismo , Animales , Femenino , Células HEK293 , Humanos , Cinética , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Optogenética , Ratas , Análisis de la Célula Individual , Transcriptoma/genética
2.
Cell ; 170(5): 1013-1027.e14, 2017 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-28823561

RESUMEN

Reward-seeking behavior is fundamental to survival, but suppression of this behavior can be essential as well, even for rewards of high value. In humans and rodents, the medial prefrontal cortex (mPFC) has been implicated in suppressing reward seeking; however, despite vital significance in health and disease, the neural circuitry through which mPFC regulates reward seeking remains incompletely understood. Here, we show that a specific subset of superficial mPFC projections to a subfield of nucleus accumbens (NAc) neurons naturally encodes the decision to initiate or suppress reward seeking when faced with risk of punishment. A highly resolved subpopulation of these top-down projecting neurons, identified by 2-photon Ca2+ imaging and activity-dependent labeling to recruit the relevant neurons, was found capable of suppressing reward seeking. This natural activity-resolved mPFC-to-NAc projection displayed unique molecular-genetic and microcircuit-level features concordant with a conserved role in the regulation of reward-seeking behavior, providing cellular and anatomical identifiers of behavioral and possible therapeutic significance.


Asunto(s)
Recompensa , Animales , Conducta Animal , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas , Neuroimagen , Corteza Prefrontal/citología , Corteza Prefrontal/metabolismo , Castigo
3.
Nat Methods ; 21(9): 1725-1735, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39103446

RESUMEN

Intracellular calcium (Ca2+) is ubiquitous to cell signaling across biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated split-TurboID (CaST) labels activated cells within 10 min with an exogenously delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST readout can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.


Asunto(s)
Calcio , Animales , Ratones , Calcio/metabolismo , Neuronas/metabolismo , Humanos , Biotina/química , Biotina/metabolismo , Señalización del Calcio , Corteza Prefrontal/metabolismo , Corteza Prefrontal/citología , Ratones Endogámicos C57BL , Masculino
4.
Nat Methods ; 20(6): 908-917, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37188954

RESUMEN

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.


Asunto(s)
Mitocondrias , Proteómica , Retículo Endoplásmico , Biotina
5.
Nature ; 565(7741): 645-649, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30651638

RESUMEN

Categorically distinct basic drives (for example, for social versus feeding behaviour1-3) can exert potent influences on each other; such interactions are likely to have important adaptive consequences (such as appropriate regulation of feeding in the context of social hierarchies) and can become maladaptive (such as in clinical settings involving anorexia). It is known that neural systems regulating natural and adaptive caloric intake, and those regulating social behaviours, involve related circuitry4-7, but the causal circuit mechanisms of these drive adjudications are not clear. Here we investigate the causal role in behaviour of cellular-resolution experience-specific neuronal populations in the orbitofrontal cortex, a major reward-processing hub that contains diverse activity-specific neuronal populations that respond differentially to various aspects of caloric intake8-13 and social stimuli14,15. We coupled genetically encoded activity imaging with the development and application of methods for optogenetic control of multiple individually defined cells, to both optically monitor and manipulate the activity of many orbitofrontal cortex neurons at the single-cell level in real time during rewarding experiences (caloric consumption and social interaction). We identified distinct populations within the orbitofrontal cortex that selectively responded to either caloric rewards or social stimuli, and found that activity of individually specified naturally feeding-responsive neurons was causally linked to increased feeding behaviour; this effect was selective as, by contrast, single-cell resolution activation of naturally social-responsive neurons inhibited feeding, and activation of neurons responsive to neither feeding nor social stimuli did not alter feeding behaviour. These results reveal the presence of potent cellular-level subnetworks within the orbitofrontal cortex that can be precisely engaged to bidirectionally control feeding behaviours subject to, for example, social influences.


Asunto(s)
Conducta Alimentaria/fisiología , Vías Nerviosas/fisiología , Neuronas/citología , Neuronas/fisiología , Corteza Prefrontal/citología , Corteza Prefrontal/fisiología , Conducta Social , Animales , Condicionamiento Operante/fisiología , Ingestión de Energía , Masculino , Ratones , Ratones Endogámicos C57BL , Optogenética , Recompensa , Análisis de la Célula Individual
6.
Nature ; 549(7672): 345-350, 2017 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-28902833

RESUMEN

Identification of neural circuit changes that contribute to behavioural plasticity has routinely been conducted on candidate circuits that were preselected on the basis of previous results. Here we present an unbiased method for identifying experience-triggered circuit-level changes in neuronal ensembles in mice. Using rabies virus monosynaptic tracing, we mapped cocaine-induced global changes in inputs onto neurons in the ventral tegmental area. Cocaine increased rabies-labelled inputs from the globus pallidus externus (GPe), a basal ganglia nucleus not previously known to participate in behavioural plasticity triggered by drugs of abuse. We demonstrated that cocaine increased GPe neuron activity, which accounted for the increase in GPe labelling. Inhibition of GPe activity revealed that it contributes to two forms of cocaine-triggered behavioural plasticity, at least in part by disinhibiting dopamine neurons in the ventral tegmental area. These results suggest that rabies-based unbiased screening of changes in input populations can identify previously unappreciated circuit elements that critically support behavioural adaptations.


Asunto(s)
Cocaína/farmacología , Globo Pálido/efectos de los fármacos , Globo Pálido/fisiología , Plasticidad Neuronal/efectos de los fármacos , Virus de la Rabia/genética , Coloración y Etiquetado , Animales , Ganglios Basales/efectos de los fármacos , Ganglios Basales/fisiología , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Área Tegmental Ventral/citología , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/fisiología
7.
Nat Rev Neurosci ; 18(4): 222-235, 2017 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-28303019

RESUMEN

Modern optogenetics can be tuned to evoke activity that corresponds to naturally occurring local or global activity in timing, magnitude or individual-cell patterning. This outcome has been facilitated not only by the development of core features of optogenetics over the past 10 years (microbial-opsin variants, opsin-targeting strategies and light-targeting devices) but also by the recent integration of optogenetics with complementary technologies, spanning electrophysiology, activity imaging and anatomical methods for structural and molecular analysis. This integrated approach now supports optogenetic identification of the native, necessary and sufficient causal underpinnings of physiology and behaviour on acute or chronic timescales and across cellular, circuit-level or brain-wide spatial scales.


Asunto(s)
Neurociencias/métodos , Optogenética/métodos , Animales , Electrofisiología/métodos , Neuroanatomía/métodos , Neuroimagen/métodos , Neurociencias/tendencias , Optogenética/tendencias
8.
Nat Chem Biol ; 15(2): 101-110, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30659298

RESUMEN

To understand how the brain relates to behavior, it is essential to record neural activity in awake, behaving animals. To achieve this goal, a large variety of genetically encoded sensors have been developed to monitor and record the series of events following neuronal firing, including action potentials, intracellular calcium rise, neurotransmitter release and immediate early gene expression. In this Review, we discuss the existing genetically encoded tools for detecting and integrating neuronal activity in animals and highlight the remaining challenges and future opportunities for molecular biologists.


Asunto(s)
Mapeo Encefálico/métodos , Imagen Molecular/métodos , Neuronas/fisiología , Animales , Encéfalo/diagnóstico por imagen , Humanos , Neuronas/metabolismo
9.
Nature ; 526(7575): 653-9, 2015 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-26436451

RESUMEN

Top-down prefrontal cortex inputs to the hippocampus have been hypothesized to be important in memory consolidation, retrieval, and the pathophysiology of major psychiatric diseases; however, no such direct projections have been identified and functionally described. Here we report the discovery of a monosynaptic prefrontal cortex (predominantly anterior cingulate) to hippocampus (CA3 to CA1 region) projection in mice, and find that optogenetic manipulation of this projection (here termed AC-CA) is capable of eliciting contextual memory retrieval. To explore the network mechanisms of this process, we developed and applied tools to observe cellular-resolution neural activity in the hippocampus while stimulating AC-CA projections during memory retrieval in mice behaving in virtual-reality environments. Using this approach, we found that learning drives the emergence of a sparse class of neurons in CA2/CA3 that are highly correlated with the local network and that lead synchronous population activity events; these neurons are then preferentially recruited by the AC-CA projection during memory retrieval. These findings reveal a sparsely implemented memory retrieval mechanism in the hippocampus that operates via direct top-down prefrontal input, with implications for the patterning and storage of salient memory representations.


Asunto(s)
Memoria/fisiología , Neocórtex/citología , Neocórtex/fisiología , Vías Nerviosas/fisiología , Animales , Condicionamiento Psicológico , Miedo , Giro del Cíngulo/fisiología , Hipocampo/citología , Hipocampo/fisiología , Aprendizaje/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Neuronas/fisiología , Optogenética , Corteza Prefrontal/fisiología , Interfaz Usuario-Computador
10.
Nat Methods ; 13(4): 325-8, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26878381

RESUMEN

Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP), which we used to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We explored the versatility of the FIP microscope by quantifying real-time activity relationships among many brain regions during social behavior, simultaneously recording activity along multiple axonal pathways during sensory experience, performing simultaneous two-color activity recording, and applying optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior.


Asunto(s)
Mapeo Encefálico/métodos , Encéfalo/fisiología , Señalización del Calcio , Vías Nerviosas , Fotometría/métodos , Conducta Social , Animales , Encéfalo/citología , Ratones
11.
Nature ; 496(7444): 219-23, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23515158

RESUMEN

Behavioural states in mammals, such as the anxious state, are characterized by several features that are coordinately regulated by diverse nervous system outputs, ranging from behavioural choice patterns to changes in physiology (in anxiety, exemplified respectively by risk-avoidance and respiratory rate alterations). Here we investigate if and how defined neural projections arising from a single coordinating brain region in mice could mediate diverse features of anxiety. Integrating behavioural assays, in vivo and in vitro electrophysiology, respiratory physiology and optogenetics, we identify a surprising new role for the bed nucleus of the stria terminalis (BNST) in the coordinated modulation of diverse anxiety features. First, two BNST subregions were unexpectedly found to exert opposite effects on the anxious state: oval BNST activity promoted several independent anxious state features, whereas anterodorsal BNST-associated activity exerted anxiolytic influence for the same features. Notably, we found that three distinct anterodorsal BNST efferent projections-to the lateral hypothalamus, parabrachial nucleus and ventral tegmental area-each implemented an independent feature of anxiolysis: reduced risk-avoidance, reduced respiratory rate, and increased positive valence, respectively. Furthermore, selective inhibition of corresponding circuit elements in freely moving mice showed opposing behavioural effects compared with excitation, and in vivo recordings during free behaviour showed native spiking patterns in anterodorsal BNST neurons that differentiated safe and anxiogenic environments. These results demonstrate that distinct BNST subregions exert opposite effects in modulating anxiety, establish separable anxiolytic roles for different anterodorsal BNST projections, and illustrate circuit mechanisms underlying selection of features for the assembly of the anxious state.


Asunto(s)
Ansiedad/fisiopatología , Vías Nerviosas/fisiología , Núcleos Septales/fisiopatología , Potenciales de Acción , Animales , Ansiedad/patología , Electrofisiología , Ratones , Optogenética , Núcleos Septales/anatomía & histología , Núcleos Septales/citología
12.
Opt Express ; 23(25): 32573-81, 2015 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-26699047

RESUMEN

Phase spatial light modulators (SLMs) are widely used for generating multifocal three-dimensional (3D) illumination patterns, but these are limited to a field of view constrained by the pixel count or size of the SLM. Further, with two-photon SLM-based excitation, increasing the number of focal spots penalizes the total signal linearly--requiring more laser power than is available or can be tolerated by the sample. Here we analyze and demonstrate a method of using galvanometer mirrors to time-sequentially reposition multiple 3D holograms, both extending the field of view and increasing the total time-averaged two-photon signal. We apply our approach to 3D two-photon in vivo neuronal calcium imaging.


Asunto(s)
Holografía/métodos , Imagenología Tridimensional , Iluminación/métodos , Optometría/métodos , Humanos , Estimulación Luminosa/métodos
13.
bioRxiv ; 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38798353

RESUMEN

Intracellular calcium (Ca2+) is ubiquitous to cell signaling across all biology. While existing fluorescent sensors and reporters can detect activated cells with elevated Ca2+ levels, these approaches require implants to deliver light to deep tissue, precluding their noninvasive use in freely-behaving animals. Here we engineered an enzyme-catalyzed approach that rapidly and biochemically tags cells with elevated Ca2+ in vivo. Ca2+-activated Split-TurboID (CaST) labels activated cells within 10 minutes with an exogenously-delivered biotin molecule. The enzymatic signal increases with Ca2+ concentration and biotin labeling time, demonstrating that CaST is a time-gated integrator of total Ca2+ activity. Furthermore, the CaST read-out can be performed immediately after activity labeling, in contrast to transcriptional reporters that require hours to produce signal. These capabilities allowed us to apply CaST to tag prefrontal cortex neurons activated by psilocybin, and to correlate the CaST signal with psilocybin-induced head-twitch responses in untethered mice.

14.
Nat Comput Sci ; 4(3): 224-236, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38532137

RESUMEN

Here we used machine learning to engineer genetically encoded fluorescent indicators, protein-based sensors critical for real-time monitoring of biological activity. We used machine learning to predict the outcomes of sensor mutagenesis by analyzing established libraries that link sensor sequences to functions. Using the GCaMP calcium indicator as a scaffold, we developed an ensemble of three regression models trained on experimentally derived GCaMP mutation libraries. The trained ensemble performed an in silico functional screen on 1,423 novel, uncharacterized GCaMP variants. As a result, we identified the ensemble-derived GCaMP (eGCaMP) variants, eGCaMP and eGCaMP+, which achieve both faster kinetics and larger ∆F/F0 responses upon stimulation than previously published fast variants. Furthermore, we identified a combinatorial mutation with extraordinary dynamic range, eGCaMP2+, which outperforms the tested sixth-, seventh- and eighth-generation GCaMPs. These findings demonstrate the value of machine learning as a tool to facilitate the efficient engineering of proteins for desired biophysical characteristics.


Asunto(s)
Señalización del Calcio , Calcio , Calcio/metabolismo , Colorantes , Indicadores y Reactivos , Aprendizaje Automático
15.
Science ; 381(6657): 495, 2023 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-37535725
16.
bioRxiv ; 2023 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-36945504

RESUMEN

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

17.
Cell Rep Methods ; 2(7): 100257, 2022 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-35880019

RESUMEN

Fiber photometry is a widely used fluorescence approach for measuring neuronal activity in freely behaving animals; however, these signals can be contaminated by hemoglobin absorption artifacts. Zhang et al. propose a computational method to quantify and correct hemoglobin photon absorption effects using spectral fiber photometry, resulting in more accurate neuronal activity measurements.


Asunto(s)
Artefactos , Fotometría , Animales , Fotometría/métodos , Neuronas/fisiología
18.
Fac Rev ; 11: 25, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36262561

RESUMEN

Synapses are specialized cellular junctions essential for communication between neurons. Synapse loss occurs in many neurodegenerative diseases. Harnessing our molecular knowledge of the development and maintenance of synapses, Suzuki et al. present the first comprehensive attempt to use a synthetic protein to bridge the pre- and postsynaptic membranes1. They show that this powerful approach can stimulate the formation of pre- and postsynaptic specializations in vitro, rescue synaptic deficits of mutant mice in vivo, and ameliorate synapse loss and behavioral abnormalities in both Alzheimer's disease and spinal cord injury mouse models.

19.
Elife ; 82019 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-30942168

RESUMEN

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested 'AND' gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.


Asunto(s)
Técnicas Citológicas/métodos , Genes Reporteros , Luciferasas/análisis , Biología Molecular/métodos , Mapeo de Interacción de Proteínas/métodos , Células HEK293 , Humanos , Luz , Luciferasas/genética , Sensibilidad y Especificidad
20.
Neuron ; 101(1): 133-151.e7, 2019 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-30503173

RESUMEN

Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed. Excitation to reward-predictive cues dominated in the NAc lateral shell and was largely absent in the vNAcMed. Moreover, we demonstrate that glutamatergic (VGLUT2-expressing) neurons in the lateral hypothalamus represent a key afferent input for providing information about aversive outcomes to vNAcMed-projecting DA neurons. Collectively, we reveal the distinct functional contributions of separate mesolimbic DA subsystems and their afferent pathways underlying motivated behaviors. VIDEO ABSTRACT.


Asunto(s)
Reacción de Prevención/fisiología , Neuronas Dopaminérgicas/metabolismo , Sistema Límbico/metabolismo , Red Nerviosa/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Sistema Límbico/citología , Masculino , Mesencéfalo/citología , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Red Nerviosa/citología , Técnicas de Cultivo de Órganos , Fotometría/métodos , Área Tegmental Ventral/citología , Proteína 2 de Transporte Vesicular de Glutamato/biosíntesis
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA