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1.
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
2.
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
3.
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
4.
Science ; 369(6504): 638, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32764064
5.
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
6.
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
7.
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
8.
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
9.
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
10.
Nat Commun ; 11(1): 2716, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483118

RESUMO

How do brain mechanisms create maladaptive attractions? Here intense maladaptive attractions are created in laboratory rats by pairing optogenetic channelrhodopsin (ChR2) stimulation of central nucleus of amygdala (CeA) in rats with encountering either sucrose, cocaine, or a painful shock-delivering object. We find that pairings make the respective rats pursue either sucrose exclusively, or cocaine exclusively, or repeatedly self-inflict shocks. CeA-induced maladaptive attractions, even to the painful shock-rod, recruit mesocorticolimbic incentive-related circuitry. Shock-associated cues also gain positive incentive value and are pursued. Yet the motivational effects of paired CeA stimulation can be reversed to negative valence in a Pavlovian fear learning situation, where CeA ChR2 pairing increases defensive reactions. Finally, CeA ChR2 valence can be switched to neutral by pairing with innocuous stimuli. These results reveal valence plasticity and multiple modes for motivation via mesocorticolimbic circuitry under the control of CeA activation.


Assuntos
Encéfalo/fisiologia , Núcleo Central da Amígdala/fisiologia , Channelrhodopsins/fisiologia , Dor/fisiopatologia , Recompensa , Animais , Núcleo Central da Amígdala/metabolismo , Channelrhodopsins/metabolismo , Cocaína/administração & dosagem , Sinais (Psicologia) , Feminino , Aprendizagem/efeitos dos fármacos , Aprendizagem/fisiologia , Masculino , Motivação/fisiologia , Optogenética/métodos , Ratos Sprague-Dawley , Sacarose/administração & dosagem
12.
PLoS One ; 15(5): e0232880, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401761

RESUMO

The southern king crab (SKC) Lithodes santolla is an important commercial species in southern South America. Fishing pressure has caused the deterioration of its stocks. Currently, culture techniques are being developed for producing SKC juveniles to enhance the natural population and to recover the fishing stock. Therefore, it is necessary to know about physiology, energetic and nutritional requirements for SKC maintenance in hatchery. Thus, this study aims to evaluate the biochemical and physiological changes in the midgut gland, muscle and hemolymph of juveniles, pre-adults and adults of wild SKC. The energetic reserves, digestive enzymes activity, amino acid profile and energy were quantified in twelve juveniles, ten pre-adult, and ten adult crabs. Juveniles showed high glycogen and low lipids in the midgut gland, and low proteins and low lactate in muscle. In the hemolymph, juveniles had high lipids. Pre-adults had high glycogen and lipids in the midgut gland, and both high protein and lactate in muscle. In the hemolymph, pre-adults had high lipids. Adults had low glycogen and high lipids in midgut gland, and both high proteins and high lactate in muscle. In hemolymph, adults had high glucose and lactate. Juveniles and pre-adults had high proteinase activity, whereas adults had high lipase activity. Major essential amino acids of SKC were arginine, methionine, and tryptophan, and the non-essential amino acids were glycine, aspartic acid and glutamic acid. On another hand, SKC had similar energy in the midgut gland and muscle, regardless of the ontogenetic stage. Moreover, we demonstrated that the biochemical energy calculation underestimates the actual measured values by a calorimeter. Thus, our results help to understand the physiological changes, energetic and nutritional requirements of L. santolla, and this study is a baseline for research on diet formulation for maintaining this species under culture conditions.


Assuntos
Anomuros/fisiologia , Aquicultura/métodos , Proteínas de Artrópodes/genética , Optogenética/métodos , Aminoácidos/análise , Ração Animal , Animais , Anomuros/citologia , Anomuros/crescimento & desenvolvimento , Dieta , Metabolismo Energético , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hemolinfa , Masculino , Músculos/química
13.
Neuron ; 107(2): 351-367.e19, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32433908

RESUMO

To advance the measurement of distributed neuronal population representations of targeted motor actions on single trials, we developed an optical method (COSMOS) for tracking neural activity in a largely uncharacterized spatiotemporal regime. COSMOS allowed simultaneous recording of neural dynamics at ∼30 Hz from over a thousand near-cellular resolution neuronal sources spread across the entire dorsal neocortex of awake, behaving mice during a three-option lick-to-target task. We identified spatially distributed neuronal population representations spanning the dorsal cortex that precisely encoded ongoing motor actions on single trials. Neuronal correlations measured at video rate using unaveraged, whole-session data had localized spatial structure, whereas trial-averaged data exhibited widespread correlations. Separable modes of neural activity encoded history-guided motor plans, with similar population dynamics in individual areas throughout cortex. These initial experiments illustrate how COSMOS enables investigation of large-scale cortical dynamics and that information about motor actions is widely shared between areas, potentially underlying distributed computations.


Assuntos
Córtex Cerebral/fisiologia , Neuroimagem/instrumentação , Neuroimagem/métodos , Observação/métodos , Algoritmos , Animais , Comportamento Animal/fisiologia , Mapeamento Encefálico , Condicionamento Operante , Craniotomia , Camundongos , Neocórtex/citologia , Neocórtex/fisiologia , Neurônios , Optogenética/métodos , Desempenho Psicomotor , Procedimentos Cirúrgicos Robóticos/instrumentação , Procedimentos Cirúrgicos Robóticos/métodos , Razão Sinal-Ruído
14.
Neuron ; 107(1): 38-51.e8, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32353253

RESUMO

Optogenetics is among the most widely employed techniques to manipulate neuronal activity. However, a major drawback is the need for invasive implantation of optical fibers. To develop a minimally invasive optogenetic method that overcomes this challenge, we engineered a new step-function opsin with ultra-high light sensitivity (SOUL). We show that SOUL can activate neurons located in deep mouse brain regions via transcranial optical stimulation and elicit behavioral changes in SOUL knock-in mice. Moreover, SOUL can be used to modulate neuronal spiking and induce oscillations reversibly in macaque cortex via optical stimulation from outside the dura. By enabling external light delivery, our new opsin offers a minimally invasive tool for manipulating neuronal activity in rodent and primate models with fewer limitations on the depth and size of target brain regions and may further facilitate the development of minimally invasive optogenetic tools for the treatment of neurological disorders.


Assuntos
Opsinas , Optogenética/métodos , Animais , Encéfalo/fisiologia , Macaca , Camundongos , Modelos Animais , Neurônios/fisiologia
15.
PLoS One ; 15(4): e0232451, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353050

RESUMO

Numerous neuronal properties including the synaptic vesicle release process, neurotransmitter receptor complement, and postsynaptic ion channels are involved in transforming synaptic inputs into postsynaptic spiking. Temperature is a significant influencer of neuronal function and synaptic integration. Changing temperature can affect neuronal physiology in a diversity of ways depending on how it affects different members of the cell's ion channel complement. Temperature's effects on neuronal function are critical for pathological states such as fever, which can trigger seizure activity, but are also important in interpreting and comparing results of experiments conducted at room vs physiological temperature. The goal of this study was to examine the influence of temperature on synaptic properties and ion channel function in thalamocortical (TC) relay neurons in acute brain slices of the dorsal lateral geniculate nucleus, a key synaptic target of retinal ganglion cells in the thalamus. Warming the superfusate in patch clamp experiments with acutely-prepared brain slices led to an overall inhibition of synaptically-driven spiking behavior in TC neurons in response to a retinal ganglion cell spike train. Further study revealed that this was associated with an increase in presynaptic synaptic vesicle release probability and synaptic depression and altered passive and active membrane properties. Additionally, warming the superfusate triggered activation of an inwardly rectifying potassium current and altered the voltage-dependence of voltage-gated Na+ currents and T-type calcium currents. This study highlights the importance of careful temperature control in ex vivo physiological experiments and illustrates how numerous properties such as synaptic inputs, active conductances, and passive membrane properties converge to determine spike output.


Assuntos
Corpos Geniculados/fisiologia , Temperatura Alta/efeitos adversos , Optogenética/métodos , Terminações Pré-Sinápticas/fisiologia , Células Ganglionares da Retina/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo T/metabolismo , Feminino , Corpos Geniculados/citologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Transmissão Sináptica/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo
16.
Nat Commun ; 11(1): 2381, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404906

RESUMO

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.


Assuntos
Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Bactérias Gram-Negativas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/microbiologia , Células Eucarióticas/microbiologia , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/fisiologia , Humanos , Luz , Microscopia de Fluorescência , Optogenética/métodos , Transporte Proteico/efeitos da radiação , Análise Espacial , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/fisiologia
17.
Nat Commun ; 11(1): 2388, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404907

RESUMO

Deep brain stimulation (DBS) of the subthalamic nucleus is a symptomatic treatment of Parkinson's disease but benefits only to a minority of patients due to stringent eligibility criteria. To investigate new targets for less invasive therapies, we aimed at elucidating key mechanisms supporting deep brain stimulation efficiency. Here, using in vivo electrophysiology, optogenetics, behavioral tasks and mathematical modeling, we found that subthalamic stimulation normalizes pathological hyperactivity of motor cortex pyramidal cells, while concurrently activating somatostatin and inhibiting parvalbumin interneurons. In vivo opto-activation of cortical somatostatin interneurons alleviates motor symptoms in a parkinsonian mouse model. A computational model highlights that a decrease in pyramidal neuron activity induced by DBS or by a stimulation of cortical somatostatin interneurons can restore information processing capabilities. Overall, these results demonstrate that activation of cortical somatostatin interneurons may constitute a less invasive alternative than subthalamic stimulation.


Assuntos
Estimulação Encefálica Profunda/métodos , Levodopa/uso terapêutico , Transtornos Parkinsonianos/terapia , Somatostatina/metabolismo , Algoritmos , Animais , Antiparkinsonianos/uso terapêutico , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Córtex Motor/fisiopatologia , Optogenética/métodos , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/fisiopatologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Núcleo Subtalâmico/efeitos dos fármacos , Núcleo Subtalâmico/metabolismo , Núcleo Subtalâmico/fisiopatologia
18.
J Neurosci ; 40(22): 4323-4334, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32312888

RESUMO

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective therapy for the motor symptoms of Parkinson's disease (PD). However, the neural elements mediating symptom relief are unclear. A previous study concluded that direct optogenetic activation of STN neurons was neither necessary nor sufficient for relief of parkinsonian symptoms. However, the kinetics of the channelrhodopsin-2 (ChR2) used for cell-specific activation are too slow to follow the high rates required for effective DBS, and thus the contribution of activation of STN neurons to the therapeutic effects of DBS remains unclear. We quantified the behavioral and neuronal effects of optogenetic STN DBS in female rats following unilateral 6-hydroxydopamine (6-OHDA) lesion using an ultrafast opsin (Chronos). Optogenetic STN DBS at 130 pulses per second (pps) reduced pathologic circling and ameliorated deficits in forelimb stepping similarly to electrical DBS, while optogenetic STN DBS with ChR2 did not produce behavioral effects. As with electrical DBS, optogenetic STN DBS exhibited a strong dependence on stimulation rate; high rates produced symptom relief while low rates were ineffective. High-rate optogenetic DBS generated both increases and decreases in firing rates of single neurons in STN, globus pallidus externa (GPe), and substantia nigra pars reticular (SNr), and disrupted ß band oscillatory activity in STN and SNr. High-rate optogenetic STN DBS can indeed ameliorate parkinsonian motor symptoms through reduction of abnormal oscillatory activity in the STN-associated neural circuit, and these results highlight that the kinetic properties of opsins have a strong influence on the effects of optogenetic stimulation.SIGNIFICANCE STATEMENT Whether STN local cells contribute to the therapeutic effects of subthalamic nucleus (STN) deep brain stimulation (DBS) in Parkinson's disease (PD) remains unclear. We re-examined the role of STN local cells in mediating the symptom-relieving effects of STN DBS using cell type-specific optogenetic stimulation with a much faster opsin, Chronos. Direct optogenetic stimulation of STN neurons was effective in treating the symptoms of parkinsonism in the 6-hydroxydopamine (6-OHDA) lesion rat. These results highlight that the kinetic properties of opsins can have a strong influence on the effects of optogenetic activation/inhibition and must be considered when employing optogenetic to study high-rate neural stimulation.


Assuntos
Estimulação Encefálica Profunda/métodos , Movimento , Optogenética/métodos , Transtornos Parkinsonianos/fisiopatologia , Núcleo Subtalâmico/fisiopatologia , Animais , Ritmo beta , Potenciais Evocados , Feminino , Globo Pálido/fisiopatologia , Opsinas/genética , Opsinas/metabolismo , Transtornos Parkinsonianos/terapia , Ratos , Ratos Sprague-Dawley , Substância Negra/fisiopatologia , Núcleo Subtalâmico/metabolismo
19.
J Clin Neurosci ; 77: 199-202, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32336639

RESUMO

The thoracic sympathetic chain is implicated in several disease processes including palmar hyperhidrosis and complex regional pain syndrome. These diseases are often medically refractory and require surgical treatments including sympathectomies and ganglion blocks. The use of chemogenetic or optogenetic technologies to modulate sympathetic chain activity may be a potential treatment for these diseases. However, there is no established thoracoscopic surgical approach to deliver viral vectors into the thoracic sympathetic chain and ganglia. Our objective was to evaluate the feasibility of thoracoscopic injection of the swine sympathetic chain. Two Landrace farm pigs underwent a novel procedure for thoracoscopic sympathetic chain injections. One was non-survival and one was a five-day survival surgery. Both procedures successfully delivered methylene blue in the thoracic sympathetic chain. Over the five-day postoperative period, the animal displayed stable vital signs. Thoracoscopic targeted injections of the sympathetic chain is a feasible approach to deliver therapeutics in swine. Future studies should investigate the use of transgene expression as a potential means to control sympathetic output for the development of novel therapies for palmar or axillary hyperhidrosis, thoracic neuropathic pain syndromes and select peripheral vascular diseases.


Assuntos
Gânglios Simpáticos/cirurgia , Toracoscopia/métodos , Animais , Vias Autônomas , Feminino , Optogenética/métodos , Suínos
20.
J Neurosci ; 40(18): 3591-3603, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32265261

RESUMO

The septo-hippocampal cholinergic system is critical for hippocampal learning and memory. However, a quantitative description of the in vivo firing patterns and physiological function of medial septal (MS) cholinergic neurons is still missing. In this study, we combined optogenetics with multichannel in vivo recording and recorded MS cholinergic neuron firings in freely behaving male mice for 5.5-72 h. We found that their firing activities were highly correlated with hippocampal theta states. MS cholinergic neurons were highly active during theta-dominant epochs, such as active exploration and rapid eye movement sleep, but almost silent during non-theta epochs, such as slow-wave sleep (SWS). Interestingly, optogenetic activation of these MS cholinergic neurons during SWS suppressed CA1 ripple oscillations. This suppression could be rescued by muscarinic M2 or M4 receptor antagonists. These results suggest the following important physiological function of MS cholinergic neurons: maintaining high hippocampal acetylcholine level by persistent firing during theta epochs, consequently suppressing ripples and allowing theta oscillations to dominate.SIGNIFICANCE STATEMENT The major source of acetylcholine in the hippocampus comes from the medial septum. Early experiments found that lesions to the MS result in the disappearance of hippocampal theta oscillation, which leads to speculation that the septo-hippocampal cholinergic projection contributing to theta oscillation. In this article, by long-term recording of MS cholinergic neurons, we found that they show a theta state-related firing pattern. However, optogenetically activating these neurons shows little effect on theta rhythm in the hippocampus. Instead, we found that activating MS cholinergic neurons during slow-wave sleep could suppress hippocampal ripple oscillations. This suppression is mediated by muscarinic M2 and M4 receptors.


Assuntos
Potenciais de Ação/fisiologia , Neurônios Colinérgicos/fisiologia , Hipocampo/fisiologia , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M4/fisiologia , Ritmo Teta/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Agonistas Colinérgicos/farmacologia , Neurônios Colinérgicos/química , Neurônios Colinérgicos/efeitos dos fármacos , Hipocampo/química , Hipocampo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Antagonistas Muscarínicos/farmacologia , Optogenética/métodos , Técnicas de Cultura de Órgãos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inibidores , Receptores Muscarínicos/fisiologia , Ritmo Teta/efeitos dos fármacos
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