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1.
Nat Commun ; 11(1): 4491, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901033

RESUMO

The functionality of the nervous system requires transmission of information along axons with high speed and precision. Conductance velocity depends on axonal diameter whereas signaling precision requires a block of electrical crosstalk between axons, known as ephaptic coupling. Here, we use the peripheral nervous system of Drosophila larvae to determine how glia regulates axonal properties. We show that wrapping glial differentiation depends on gap junctions and FGF-signaling. Abnormal glial differentiation affects axonal diameter and conductance velocity and causes mild behavioral phenotypes that can be rescued by a sphingosine-rich diet. Ablation of wrapping glia does not further impair axonal diameter and conductance velocity but causes a prominent locomotion phenotype that cannot be rescued by sphingosine. Moreover, optogenetically evoked locomotor patterns do not depend on conductance speed but require the presence of wrapping glial processes. In conclusion, our data indicate that wrapping glia modulates both speed and precision of neuronal signaling.


Assuntos
Drosophila melanogaster/fisiologia , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Diferenciação Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Larva/citologia , Larva/fisiologia , Locomoção/fisiologia , Modelos Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neuroglia/citologia , Neuroglia/fisiologia , Optogenética , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/fisiologia , Fenótipo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais
2.
Nat Commun ; 11(1): 4358, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868768

RESUMO

Learned fear and safety are associated with distinct oscillatory states in the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC). To determine if and how these network states support the retrieval of competing memories, we mimicked endogenous oscillatory activity through optogenetic stimulation of parvalbumin-expressing interneurons in mice during retrieval of contextual fear and extinction memories. We found that exogenously induced 4 Hz and 8 Hz oscillatory activity in the BLA exerts bi-directional control over conditioned freezing behavior in an experience- and context-specific manner, and that these oscillations have an experience-dependent ability to recruit distinct functional neuronal ensembles. At the network level we demonstrate, via simultaneous manipulation of BLA and mPFC, that experience-dependent 4 Hz resonance across BLA-mPFC circuitry supports post-extinction fear memory retrieval. Our findings reveal that post-extinction fear memory retrieval is supported by local and interregional experience-dependent resonance, and suggest novel approaches for interrogation and therapeutic manipulation of acquired fear circuitry.


Assuntos
Tonsila do Cerebelo/fisiologia , Extinção Psicológica , Medo/fisiologia , Memória/fisiologia , Animais , Complexo Nuclear Basolateral da Amígdala/fisiologia , Condicionamento Psicológico , Eletrofisiologia/métodos , Aprendizagem/fisiologia , Camundongos , Optogenética/métodos , Córtex Pré-Frontal/fisiologia
3.
Nat Commun ; 11(1): 4559, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917906

RESUMO

The temporal embryonic origins of cortical GABA neurons are critical for their specialization. In the neonatal hippocampus, GABA cells born the earliest (ebGABAs) operate as 'hubs' by orchestrating population synchrony. However, their adult fate remains largely unknown. To fill this gap, we have examined CA1 ebGABAs using a combination of electrophysiology, neurochemical analysis, optogenetic connectivity mapping as well as ex vivo and in vivo calcium imaging. We show that CA1 ebGABAs not only operate as hubs during development, but also maintain distinct morpho-physiological and connectivity profiles, including a bias for long-range targets and local excitatory inputs. In vivo, ebGABAs are activated during locomotion, correlate with CA1 cell assemblies and display high functional connectivity. Hence, ebGABAs are specified from birth to ensure unique functions throughout their lifetime. In the adult brain, this may take the form of a long-range hub role through the coordination of cell assemblies across distant regions.


Assuntos
Neurônios GABAérgicos/fisiologia , Hipocampo/fisiologia , Animais , Axônios , Encéfalo , Região CA1 Hipocampal/fisiologia , Feminino , Masculino , Camundongos , Modelos Animais , Vias Neurais/fisiologia , Optogenética , Sinapses/fisiologia
4.
Nat Commun ; 11(1): 4410, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879310

RESUMO

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCNVIP) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCNVIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCNVIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCNVIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCNVIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCNVIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCNVIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCNVIP subtypes.


Assuntos
Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Núcleo Supraquiasmático , Animais , Mapeamento Encefálico , Relógios Circadianos/fisiologia , Locomoção/fisiologia , Camundongos , Optogenética/métodos , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/metabolismo
5.
Nat Commun ; 11(1): 4395, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879322

RESUMO

The formation and maintenance of spatial representations within hippocampal cell assemblies is strongly dictated by patterns of inhibition from diverse interneuron populations. Although it is known that inhibitory synaptic strength is malleable, induction of long-term plasticity at distinct inhibitory synapses and its regulation of hippocampal network activity is not well understood. Here, we show that inhibitory synapses from parvalbumin and somatostatin expressing interneurons undergo long-term depression and potentiation respectively (PV-iLTD and SST-iLTP) during physiological activity patterns. Both forms of plasticity rely on T-type calcium channel activation to confer synapse specificity but otherwise employ distinct mechanisms. Since parvalbumin and somatostatin interneurons preferentially target perisomatic and distal dendritic regions respectively of CA1 pyramidal cells, PV-iLTD and SST-iLTP coordinate a reprioritisation of excitatory inputs from entorhinal cortex and CA3. Furthermore, circuit-level modelling reveals that PV-iLTD and SST-iLTP cooperate to stabilise place cells while facilitating representation of multiple unique environments within the hippocampal network.


Assuntos
Hipocampo/fisiologia , Interneurônios/metabolismo , Células Piramidais/fisiologia , Potenciais de Ação , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Canais de Cálcio Tipo T/metabolismo , Channelrhodopsins/metabolismo , Hipocampo/citologia , Camundongos , Optogenética/métodos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Transdução de Sinais , Somatostatina/metabolismo , Sinapses/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(32): 19590-19598, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32732431

RESUMO

During rapid eye movement (REM) sleep, behavioral unresponsiveness contrasts strongly with intense brain-wide neural network dynamics. Yet, the physiological functions of this cellular activation remain unclear. Using in vivo calcium imaging in freely behaving mice, we found that inhibitory neurons in the lateral hypothalamus (LHvgat) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LHvgat cells induced a reorganization of these activity patterns during subsequent feeding behaviors accompanied by decreased food intake. Our findings provide evidence for a role for REM sleep in the maintenance of cellular representations of feeding behavior.


Assuntos
Comportamento Alimentar/fisiologia , Região Hipotalâmica Lateral/fisiologia , Sono REM/fisiologia , Animais , Mapeamento Encefálico , Masculino , Camundongos , Rede Nervosa , Inibição Neural , Neurônios/metabolismo , Neurônios/fisiologia , Optogenética , Sono/fisiologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
7.
Nat Commun ; 11(1): 4045, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792484

RESUMO

Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.


Assuntos
Luz , Optogenética/métodos , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ligação Proteica/efeitos da radiação , Proteínas/química , Proteínas/metabolismo
8.
Nat Commun ; 11(1): 3834, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737309

RESUMO

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.


Assuntos
Escherichia coli/efeitos dos fármacos , Optogenética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Tetraciclina/farmacologia , Tetraciclinas/farmacologia , Transcrição Genética/efeitos dos fármacos , Clonagem Molecular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fotólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Raios Ultravioleta
9.
Yakugaku Zasshi ; 140(8): 993-1000, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741873

RESUMO

The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Engenharia Genética , Luz , Optogenética , Ativação Transcricional , Animais , Diferenciação Celular , DNA/genética , Edição de Genes , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Recombinação Genética
10.
Nat Commun ; 11(1): 3935, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769979

RESUMO

GABAA/glycine-mediated neuronal inhibition critically depends on intracellular chloride (Cl-) concentration which is mainly regulated by the K+-Cl- co-transporter 2 (KCC2) in the adult central nervous system (CNS). KCC2 heterogeneity thus affects information processing across CNS areas. Here, we uncover a gradient in Cl- extrusion capacity across the superficial dorsal horn (SDH) of the spinal cord (laminae I-II: LI-LII), which remains concealed under low Cl- load. Under high Cl- load or heightened synaptic drive, lower Cl- extrusion is unveiled in LI, as expected from the gradient in KCC2 expression found across the SDH. Blocking TrkB receptors increases KCC2 in LI, pointing to differential constitutive TrkB activation across laminae. Higher Cl- lability in LI results in rapidly collapsing inhibition, and a form of activity-dependent synaptic plasticity expressed as a continuous facilitation of excitatory responses. The higher metaplasticity in LI as compared to LII differentially affects sensitization to thermal and mechanical input. Thus, inconspicuous heterogeneity of Cl- extrusion across laminae critically shapes plasticity for selective nociceptive modalities.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Cloretos/metabolismo , Plasticidade Neuronal/fisiologia , Nociceptividade/fisiologia , Células do Corno Posterior/fisiologia , Animais , Células Cultivadas , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Neurológicos , Optogenética , Cultura Primária de Células , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptor trkB/antagonistas & inibidores , Receptor trkB/metabolismo , Simportadores/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(33): 20265-20273, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747555

RESUMO

Huntington disease (HD) is an ideal model for investigating selective neurodegeneration, as expanded polyQ repeats in the ubiquitously expressed huntingtin (HTT) cause the preferential neurodegeneration in the striatum of the HD patient brains. Here we report that adeno-associated virus (AAV) transduction-mediated depletion of Hap1, the first identified huntingtin-associated protein, in adult HD knock-in (KI) mouse brains leads to selective neuronal loss in the striatum. Further, Hap1 depletion-mediated neuronal loss via AAV transduction requires the presence of mutant HTT. Rhes, a GTPase that is enriched in the striatum and sumoylates mutant HTT to mediate neurotoxicity, binds more N-terminal HTT when Hap1 is deficient. Consistently, more soluble and sumoylated N-terminal HTT is presented in HD KI mouse striatum when HAP1 is absent. Our findings suggest that both Rhes and Hap1 as well as cellular stress contribute to the preferential neurodegeneration in HD, highlighting the involvement of multiple factors in selective neurodegeneration.


Assuntos
Corpo Estriado/patologia , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Optogenética , Animais , Corpo Estriado/metabolismo , Dependovirus , Regulação da Expressão Gênica , Doença de Huntington/genética , Lasers , Luz , Camundongos , Rede Nervosa , Proteínas do Tecido Nervoso/genética
12.
Nat Commun ; 11(1): 3996, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778725

RESUMO

Psychomotor stimulants increase dopamine levels in the striatum and promote locomotion; however, their effects on striatal pathway function in vivo remain unclear. One model that has been proposed to account for these motor effects suggests that stimulants drive hyperactivity via activation and inhibition of direct and indirect pathway striatal neurons, respectively. Although this hypothesis is consistent with the cellular actions of dopamine receptors and received support from optogenetic and chemogenetic studies, it has been rarely tested with in vivo recordings. Here, we test this model and observe that cocaine increases the activity of both pathways in the striatum of awake mice. These changes are linked to a dopamine-dependent cocaine-induced strengthening of upstream orbitofrontal cortex (OFC) inputs to the dorsomedial striatum (DMS) in vivo. Finally, depressing OFC-DMS pathway with a high frequency stimulation protocol in awake mice over-powers the cocaine-induced potentiation of OFC-DMS pathway and attenuates the expression of locomotor sensitization, directly linking OFC-DMS potentiation to cocaine-induced hyperactivity.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Hipercinese/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Animais , Comportamento Animal , Modelos Animais de Doenças , Dopamina , Feminino , Hipercinese/induzido quimicamente , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Optogenética
13.
PLoS Biol ; 18(8): e3000762, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760088

RESUMO

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.


Assuntos
Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interfase , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Optogenética/métodos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
14.
Nat Commun ; 11(1): 4044, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792536

RESUMO

A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.


Assuntos
Optogenética/métodos , Biologia Sintética/métodos , Animais , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
15.
PLoS Comput Biol ; 16(7): e1007857, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667921

RESUMO

In many cases of inherited retinal degenerations, ganglion cells are spared despite photoreceptor cell death, making it possible to stimulate them to restore visual function. Several studies have shown that it is possible to express an optogenetic protein in ganglion cells and make them light sensitive, a promising strategy to restore vision. However the spatial resolution of optogenetically-reactivated retinas has rarely been measured, especially in the primate. Since the optogenetic protein is also expressed in axons, it is unclear if these neurons will only be sensitive to the stimulation of a small region covering their somas and dendrites, or if they will also respond to any stimulation overlapping with their axon, dramatically impairing spatial resolution. Here we recorded responses of mouse and macaque retinas to random checkerboard patterns following an in vivo optogenetic therapy. We show that optogenetically activated ganglion cells are each sensitive to a small region of visual space. A simple model based on this small receptive field predicted accurately their responses to complex stimuli. From this model, we simulated how the entire population of light sensitive ganglion cells would respond to letters of different sizes. We then estimated the maximal acuity expected by a patient, assuming it could make an optimal use of the information delivered by this reactivated retina. The obtained acuity is above the limit of legal blindness. Our model also makes interesting predictions on how acuity might vary upon changing the therapeutic strategy, assuming an optimal use of the information present in the retinal activity. Optogenetic therapy could thus potentially lead to high resolution vision, under conditions that our model helps to determinine.


Assuntos
Cegueira , Optogenética/métodos , Células Ganglionares da Retina/fisiologia , Animais , Cegueira/fisiopatologia , Cegueira/terapia , Terapia Genética , Macaca , Camundongos , Modelos Biológicos , Retina/fisiologia , Acuidade Visual/fisiologia
16.
Elife ; 92020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32609086

RESUMO

A new genetic marker enables precise control over a group of inhibitory neurons in monkeys.


Assuntos
Macaca , Optogenética , Animais , Córtex Cerebral , Neurônios GABAérgicos , Primatas
17.
PLoS Biol ; 18(7): e3000712, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32663220

RESUMO

Tools enabling closed-loop experiments are crucial to delineate causal relationships between the activity of genetically labeled neurons and specific behaviors. We developed the Raspberry Pi Virtual Reality (PiVR) system to conduct closed-loop optogenetic stimulation of neural functions in unrestrained animals. PiVR is an experimental platform that operates at high temporal resolution (70 Hz) with low latencies (<30 milliseconds), while being affordable (

Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/fisiologia , Optogenética , Córtex Sensório-Motor/fisiologia , Realidade Virtual , Animais , Quimiotaxia , Larva/fisiologia , Luz , Locomoção , Masculino , Neurônios/fisiologia , Odorantes , Sensação/fisiologia , Software , Paladar/fisiologia , Peixe-Zebra
18.
Nature ; 583(7816): 421-424, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641825

RESUMO

The suprachiasmatic nucleus (SCN) serves as the body's master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day-night cycle1-4. For example, the SCN opposes overnight adipsia by driving water intake before sleep5,6, and by driving the secretion of anti-diuretic hormone7,8 and lowering body temperature9,10 to reduce water loss during sleep11. These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase12-16. However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19-a time at which SCNVP (vasopressin) neurons are inactive-excited SCNVP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCNVP neurons and mimicked by optogenetic stimulation of SCNVP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLTGAD) relay this information to SCNVP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLTGAD neuron axon terminals excited SCNVP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLTGAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLTGAD→ SCNVP pathway and was prevented by chemogenetic inhibition of OVLTGAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks.


Assuntos
Relógios Circadianos/fisiologia , Vias Neurais , Neurônios/metabolismo , Sódio/metabolismo , Núcleo Supraquiasmático/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Ingestão de Líquidos/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Vias Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Optogenética , Organum Vasculosum/citologia , Organum Vasculosum/efeitos dos fármacos , Organum Vasculosum/enzimologia , Organum Vasculosum/fisiologia , Concentração Osmolar , Solução Salina Hipertônica/administração & dosagem , Solução Salina Hipertônica/metabolismo , Solução Salina Hipertônica/farmacologia , Sódio/administração & dosagem , Sódio/farmacologia , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Vasopressinas/metabolismo
20.
PLoS Biol ; 18(7): e3000794, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730242

RESUMO

The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.


Assuntos
Reatores Biológicos , Técnicas de Cultura/instrumentação , Optogenética/instrumentação , Automação , Biofilmes , Proliferação de Células , Simulação por Computador , Software
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