RESUMO
Optogenetics has revolutionised neuroscience research, but at the same time has brought a plethora of new variables to consider when designing an experiment with AAV-based targeted gene delivery. Some concerns have been raised regarding the impact of AAV injection volume and expression time in relation to longitudinal experimental designs. In this study, we investigated the efficiency of optically evoked post-synaptic responses in connection to two variables: the volume of the injected virus and the expression time of the virus. For this purpose, we expressed the blue-shifted ChR2, oChIEF, employing a widely used AAV vector delivery strategy. We found that the volume of the injected virus has a minimal impact on the efficiency of optically-evoked postsynaptic population responses. The expression time, on the other hand, has a pronounced effect, with a gradual reduction in the population responses beyond 4 weeks of expression. We strongly advise to monitor time-dependent expression profiles when planning or conducting long-term experiments that depend on successful and stable channelrhodopsin expression.
Assuntos
Terapia Genética , Vetores Genéticos , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Optogenética , Dependovirus/metabolismoRESUMO
A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vectors (AAV) driven by the CaMKIIα promoter in both male and female mice. We tested the AAV5 and AAV9 serotype of viruses with either Channelrhodopsin 2 (ChR2)-mCherry or Archaerhodopsin-T-green fluorescent protein (GFP) constructs, with different dilutions. We show that in all cases, the reporter proteins can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and cholecystokinin (CCK)-containing GABAergic cells, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the immunodetectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors.
Assuntos
Interneurônios , Células Piramidais , Camundongos , Masculino , Feminino , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Células Piramidais/fisiologia , Interneurônios/fisiologia , Neurônios/metabolismo , Colecistocinina/metabolismo , Parvalbuminas/metabolismoRESUMO
The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.
Assuntos
Degeneração Retiniana , Camundongos , Humanos , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Degeneração Retiniana/metabolismo , Optogenética/métodos , Opsinas de Bastonetes/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
OBJECTIVE: Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice. METHODS: To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used. RESULTS: Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and metabolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis. CONCLUSIONS: Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis.
Assuntos
Insulina , Receptor de Insulina , Animais , Camundongos , Ratos , Channelrhodopsins/metabolismo , Comportamento Alimentar , Hiperfagia/metabolismo , Insulina/metabolismo , Camundongos Transgênicos , Obesidade/metabolismo , Receptor de Insulina/metabolismo , Rombencéfalo/metabolismoAssuntos
Técnicas de Transferência de Genes , Terapia Genética , Frequência Cardíaca , Optogenética , Taquicardia Ventricular/terapia , Ultrassonografia de Intervenção , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos , Vírus da Hepatite B da Marmota/genética , Preparação de Coração Isolado , Camundongos Transgênicos , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Sinais de Poliadenilação na Ponta 3' do RNA , Recuperação de Função Fisiológica , Elementos Reguladores de Transcrição , Vírus 40 dos Símios/genética , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologiaRESUMO
The incidence of chronic pain is especially high in women, but the underlying mechanisms remain poorly understood. Interleukin-23 (IL-23) is a pro-inflammatory cytokine and contributes to inflammatory diseases (e.g., arthritis and psoriasis) through dendritic/T cell signaling. Here we examined the IL-23 involvement in sexual dimorphism of pain, using an optogenetic approach in transgenic mice expressing channelrhodopsin-2 (ChR2) in TRPV1-positive nociceptive neurons. In situ hybridization revealed that compared to males, females had a significantly larger portion of small-sized (100-200 µm2) Trpv1+ neurons in dorsal root ganglion (DRG). Blue light stimulation of a hindpaw of transgenic mice induced intensity-dependent spontaneous pain. At the highest intensity, females showed more intense spontaneous pain than males. Intraplantar injection of IL-23 (100 ng) induced mechanical allodynia in females only but had no effects on paw edema. Furthermore, intraplantar IL-23 only potentiated blue light-induced pain in females, and intrathecal injection of IL-23 also potentiated low-dose capsaicin (500 ng) induced spontaneous pain in females but not males. IL-23 expresses in DRG macrophages of both sexes. Intrathecal injection of IL-23 induced significantly greater p38 phosphorylation (p-p38), a marker of nociceptor activation, in DRGs of female mice than male mice. In THP-1 human macrophages estrogen and chemotherapy co-application increased IL-23 secretion, and furthermore, estrogen and IL-23 co-application, but not estrogen and IL-23 alone, significantly increased IL-17A release. These findings suggest a novel role of IL-23 in macrophage signaling and female-dominant pain, including C-fiber-mediated spontaneous pain. Our study has also provided new insight into cytokine-mediated macrophage-nociceptor interactions, in a sex-dependent manner.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Interleucina-23/toxicidade , Fibras Nervosas Amielínicas/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Dor/induzido quimicamente , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Humanos , Interleucina-17/metabolismo , Luz , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Nervosas Amielínicas/metabolismo , Nociceptores/metabolismo , Optogenética , Dor/genética , Dor/metabolismo , Dor/fisiopatologia , Caracteres Sexuais , Células THP-1 , Canais de Cátion TRPV/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.
Assuntos
Channelrhodopsins/genética , Variação Genética , Células Bipolares da Retina/metabolismo , Opsinas de Bastonetes/genética , Animais , Channelrhodopsins/metabolismo , Dependovirus/genética , Humanos , Injeções Intravítreas , Camundongos , Optogenética , Opsinas de Bastonetes/metabolismo , Transdução GenéticaRESUMO
Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467 nm) were applied for up to 24 h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p < 0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.
Assuntos
Sobrevivência Celular/fisiologia , Cálcio/metabolismo , Ciclo Celular/fisiologia , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Células HeLa , Humanos , Optogenética , TransfecçãoRESUMO
Advances in neuroscience have relied on the development of techniques that examine neuronal cell activities. One major challenge involves the limitations in labeling and controlling neuronal activities relating to the cell's activation state. In this study, the modified human codon-optimized channelrhodopsin-2 photoreceptor hChR2(C128S) was integrated into function with inducible gene expression methods and materials: the Tet system and the highly efficient minimum promoter of Arc/Arg3.1. The system successfully expressed the target fusion gene exclusively in activated SH-SY5Y human neuroblastoma cells while maintaining the essential characteristics of ChR2. The expression of the channelrhodopsin construct was observed, while the expression duration was refined by treatment with doxycycline. The optogenetic construct here tested the application of the minimum Arc/Arg3.1 promoter, an advanced immediate-early gene promoter, for the expression of the channelrhodopsin gene. Along with its noninvasive nature, this expression system promises to serve dual functions as a cell activity indicator and cell actuator, creating the possibility for researchers to precisely label cells according to their activation state and control the activities of specific neuronal cell populations.
Assuntos
Neuroblastoma , Neurônios , Channelrhodopsins/metabolismo , Humanos , Neuroblastoma/genética , Plasmídeos , Regiões Promotoras GenéticasRESUMO
An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.
Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Movimento , Optogenética , Animais , Arteríolas/metabolismo , Comportamento Animal , Capilares/metabolismo , Channelrhodopsins/metabolismo , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Neurônios/metabolismo , Fatores de Tempo , Vênulas/metabolismoRESUMO
Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1ß production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.
Assuntos
Dor Crônica/etiologia , Microglia/fisiologia , Nervos Espinhais/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Channelrhodopsins/metabolismo , Dor Crônica/fisiopatologia , Feminino , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Optogenética/métodos , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Nervos Espinhais/fisiologiaRESUMO
Although optogenetics has revolutionized rodent neuroscience, it is still rarely used in other model organisms as the efficiencies of viral gene transfer differ between species and comprehensive viral transduction studies are rare. However, for comparative research, birds offer valuable model organisms as they have excellent visual and cognitive capabilities. Therefore, the following study establishes optogenetics in pigeons on histological, physiological, and behavioral levels. We show that AAV1 is the most efficient viral vector in various brain regions and leads to extensive anterograde and retrograde ChR2 expression when combined with the CAG promoter. Furthermore, transient optical stimulation of ChR2 expressing cells in the entopallium decreases pigeons' contrast sensitivity during a grayscale discrimination task. This finding demonstrates causal evidence for the involvement of the entopallium in contrast perception as well as a proof of principle for optogenetics in pigeons and provides the groundwork for various other methods that rely on viral gene transfer in birds.
Assuntos
Channelrhodopsins/metabolismo , Columbidae/genética , Dependovirus , Optogenética , Telencéfalo/metabolismo , AnimaisRESUMO
The ability of a single Ca2+ ion to play an important role in cell biology is highlighted by the need for cells to form Ca2+ signals in the dimensions of space, time, and amplitude. Thus, spatial and temporal changes in intracellular Ca2+ concentration are important for determining cell fate. Optogenetic technology has been developed to provide more precise and targeted stimulation of cells. Here, U2OS cells overexpressing Ca2+ translocating channelrhodopsin (CatCh) were used to mediate Ca2+ influx through blue light illumination with various parameters, such as intensity, frequency, duty cycle, and duration. We identified that several Ca2+ -dependent transcription factors and certain kinases can be activated by specific Ca2+ waves. Using a wound-healing assay, we found that low-frequency Ca2+ oscillations increased cell migration through the activation of NF-κB. This study explores the regulation of cell migration by Ca2+ signals. Thus, we can choose optical parameters to modulate Ca2+ waves and achieve activation of specific signaling pathways. This novel methodology can be applied to clarify related cell-signaling mechanisms in the future.
Assuntos
Neoplasias Ósseas/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Movimento Celular , Optogenética , Osteossarcoma/metabolismo , Análise de Célula Única , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Humanos , Microscopia de Fluorescência , NF-kappa B/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , Fatores de Tempo , Imagem com Lapso de TempoRESUMO
Channelrhodopsins (ChRs) are light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. The currently characterized ChR families include green algal and cryptophyte cation-conducting ChRs (CCRs) and cryptophyte, haptophyte, and stramenopile anion-conducting ChRs (ACRs). Here, we report the discovery of a new family of phylogenetically distinct ChRs encoded by marine giant viruses and acquired from their unicellular green algal hosts. These previously unknown viral and green algal ChRs act as ACRs when expressed in cultured neuroblastoma-derived cells and are likely involved in behavioral responses to light.
Assuntos
Channelrhodopsins/genética , Clorófitas/genética , Transferência Genética Horizontal , Genes Virais , Vírus Gigantes/genética , Animais , Ânions/metabolismo , Linhagem Celular , Channelrhodopsins/metabolismo , Clorófitas/metabolismo , Clorófitas/efeitos da radiação , Clorófitas/virologia , Vírus Gigantes/metabolismo , Células Híbridas , Luz , Metagenômica , Camundongos , Optogenética , Filogenia , RatosAssuntos
Barreira Hematoencefálica , Técnicas de Transferência de Genes , Vetores Genéticos , Neurociências/métodos , Opsinas/genética , Optogenética , Engenharia de Proteínas/métodos , Animais , Channelrhodopsins/metabolismo , Dependovirus , Dosagem de Genes , Camundongos , Sinucleinopatias/terapia , TransgenesRESUMO
Bioluminescence has long been used to image biological processes in vivo. This technology features luciferase enzymes and luciferin small molecules that produce visible light. Bioluminescent photons can be detected in tissues and live organisms, enabling sensitive and noninvasive readouts on physiological function. Traditional applications have focused on tracking cells and gene expression patterns, but new probes are pushing the frontiers of what can be visualized. The past few years have also seen the merger of bioluminescence with optogenetic platforms. Luciferase-luciferin reactions can drive light-activatable proteins, ultimately triggering signal transduction and other downstream events. This review highlights these and other recent advances in bioluminescence technology, with an emphasis on tool development. We showcase how new luciferins and engineered luciferases are expanding the scope of optical imaging. We also highlight how bioluminescent systems are being leveraged not just for sensing-but also controlling-biological processes.
Assuntos
Medições Luminescentes/métodos , Animais , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Imidazóis/química , Luz , Luciferases/genética , Luciferases/metabolismo , Microscopia de Fluorescência , Neoplasias/diagnóstico por imagem , Imagem Óptica , Pirazinas/químicaRESUMO
In plants, environmental stressors trigger plasma membrane depolarizations. Being electrically interconnected via plasmodesmata, proper functional dissection of electrical signaling by electrophysiology is basically impossible. The green alga Chlamydomonas reinhardtii evolved blue light-excited channelrhodopsins (ChR1, 2) to navigate. When expressed in excitable nerve and muscle cells, ChRs can be used to control the membrane potential via illumination. In Arabidopsis plants, we used the algal ChR2-light switches as tools to stimulate plasmodesmata-interconnected photosynthetic cell networks by blue light and monitor the subsequent plasma membrane electrical responses. Blue-dependent stimulations of ChR2 expressing mesophyll cells, resting around -160 to -180 mV, reproducibly depolarized the membrane potential by 95 mV on average. Following excitation, mesophyll cells recovered their prestimulus potential not without transiently passing a hyperpolarization state. By combining optogenetics with voltage-sensing microelectrodes, we demonstrate that plant plasma membrane AHA-type H+-ATPase governs the gross repolarization process. AHA2 protein biochemistry and functional expression analysis in Xenopus oocytes indicates that the capacity of this H+ pump to recharge the membrane potential is rooted in its voltage- and pH-dependent functional anatomy. Thus, ChR2 optogenetics appears well suited to noninvasively expose plant cells to signal specific depolarization signatures. From the responses we learn about the molecular processes, plants employ to channel stress-associated membrane excitations into physiological responses.
Assuntos
Membrana Celular/metabolismo , Channelrhodopsins/metabolismo , Bombas de Próton/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Algas/metabolismo , Channelrhodopsins/fisiologia , Chlamydomonas reinhardtii/metabolismo , Cor , Concentração de Íons de Hidrogênio , Luz , Potenciais da Membrana/fisiologia , Optogenética/métodos , Bombas de Próton/fisiologia , Rodopsina/metabolismo , Transdução de SinaisRESUMO
Optogenetics has become an indispensable tool for investigating brain functions. Although non-human primates are particularly useful models for understanding the functions and dysfunctions of the human brain, application of optogenetics to non-human primates is still limited. In the present study, we generate an effective adeno-associated viral vector serotype DJ to express channelrhodopsin-2 (ChR2) under the control of a strong ubiquitous CAG promoter and inject into the somatotopically identified forelimb region of the primary motor cortex in macaque monkeys. ChR2 is strongly expressed around the injection sites, and optogenetic intracortical microstimulation (oICMS) through a homemade optrode induces prominent cortical activity: Even single-pulse, short-duration oICMS evokes long-lasting repetitive firings of cortical neurons. In addition, oICMS elicits distinct forelimb movements and muscle activity, which are comparable to those elicited by conventional electrical ICMS. The present study removes obstacles to optogenetic manipulation of neuronal activity and behaviors in non-human primates.
Assuntos
Membro Anterior/fisiologia , Córtex Motor/fisiologia , Movimento/fisiologia , Optogenética , Animais , Channelrhodopsins/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Macaca , Neurônios/fisiologia , Estimulação FísicaRESUMO
Hypothalamic tanycytes are chemosensitive glial cells that contact the cerebrospinal fluid in the third ventricle and send processes into the hypothalamic parenchyma. To test whether they can activate neurons of the arcuate nucleus, we targeted expression of a Ca2+-permeable channelrhodopsin (CatCh) specifically to tanycytes. Activation of tanycytes ex vivo depolarized orexigenic (neuropeptide Y/agouti-related protein; NPY/AgRP) and anorexigenic (proopiomelanocortin; POMC) neurons via an ATP-dependent mechanism. In vivo, activation of tanycytes triggered acute hyperphagia only in the fed state during the inactive phase of the light-dark cycle.
Assuntos
Núcleo Arqueado do Hipotálamo/fisiopatologia , Células Ependimogliais/fisiologia , Hiperfagia/fisiopatologia , Neurônios/fisiologia , Proteína Relacionada com Agouti/metabolismo , Animais , Apetite/fisiologia , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/diagnóstico por imagem , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Metabolismo Energético/fisiologia , Comportamento Alimentar/fisiologia , Feminino , Genes Reporter , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Rede Nervosa/fisiologia , Neuropeptídeo Y/metabolismo , Imagem Óptica , Optogenética , Técnicas de Patch-Clamp , Pró-Opiomelanocortina/metabolismo , Técnicas EstereotáxicasRESUMO
Itch and pain are distinct unpleasant sensations that can be triggered from the same receptive fields in the skin, raising the question of how pruriception and nociception are coded and discriminated. Here, we tested the multimodal capacity of peripheral first-order neurons, focusing on the genetically defined subpopulation of mouse C-fibers that express the chloroquine receptor MrgprA3. Using optogenetics, chemogenetics, and pharmacology, we assessed the behavioral effects of their selective stimulation in a wide variety of conditions. We show that metabotropic Gq-linked stimulation of these C-afferents, through activation of native MrgprA3 receptors or DREADDs, evokes stereotypical pruriceptive rather than nocifensive behaviors. In contrast, fast ionotropic stimulation of these same neurons through light-gated cation channels or native ATP-gated P2X3 channels predominantly evokes nocifensive rather than pruriceptive responses. We conclude that C-afferents display intrinsic multimodality, and we provide evidence that optogenetic and chemogenetic interventions on the same neuronal populations can drive distinct behavioral outputs.