RESUMO
Because G protein coupled receptors (GPCRs) represent the largest family of drug targets in clinical trials, GPCR signaling cascades are closely related to various physiological phenomena, attracting significant attention in pharmaceutical science. Opsins (also known as animal rhodopsins) are photoreceptive proteins containing retinal as a chromophore, which function as GPCRs and underlie the molecular basis of photoreception in animals. Recently, opsins have been progressively applied in an innovative technology called optogenetics to regulate biological activities using light. A wide variety of opsins have been identified in metazoans and characterized at the molecular and physiological levels, providing a foundation for their optogenetic applications. In this review, I briefly introduce the diversity of opsins in terms of their molecular functions, including G protein selectivity and photoreaction properties. This diversity provides a significant advantage for optically manipulating a wide variety of GPCR signaling cascades with high temporal resolution. Additionally, I discuss the rich array of opsin-based optogenetic tools used to control various physiological processes and their potential as therapeutic tools for vision restoration. Based on the introduction, I expect that the optogenetic approach will offer powerful tools to provide valuable insights into the molecular mechanisms of various physiological phenomena and next-generation treatment options for diseases beyond the capacity of traditional drugs.
Assuntos
Opsinas , Optogenética , Receptores Acoplados a Proteínas G , Optogenética/métodos , Animais , Humanos , Opsinas/metabolismo , Opsinas/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , LuzRESUMO
In the primary visual cortex area V1 activation of inhibitory interneurons, which provide negative feedback for excitatory pyramidal neurons, can improve visual response reliability and orientation selectivity. Moreover, optogenetic activation of one class of interneurons, parvalbumin (PV) positive cells, reduces the receptive field (RF) width. These data suggest that in V1 the negative feedback improves visual information processing. However, according to information theory, noise can limit information content in a signal, and to the best of our knowledge, in V1 signal-to-noise ratio (SNR) has never been estimated following either pyramidal or inhibitory neuron activation. Therefore, we optogenetically activated pyramidal or PV neurons in the deep layers of cortical area V1 and measured the SNR and RF area in nearby pyramidal neurons. Activation of pyramidal or PV neurons increased the SNR by 267% and 318%, respectively, and reduced the RF area to 60.1% and 77.5%, respectively, of that of the control. A simple integrate-and-fire neuron model demonstrated that an improved SNR and a reduced RF area can increase the amount of information encoded by neurons. We conclude that in V1 activation of pyramidal neurons improves visual information processing since the location of the visual stimulus can be pinpointed more accurately (via a reduced RF area), and more information is encoded by neurons (due to increased SNR).
Assuntos
Estimulação Luminosa , Células Piramidais , Córtex Visual , Animais , Células Piramidais/fisiologia , Ratos , Córtex Visual/fisiologia , Córtex Visual/citologia , Masculino , Percepção Visual/fisiologia , Parvalbuminas/metabolismo , Razão Sinal-Ruído , Optogenética , Interneurônios/fisiologia , Córtex Visual Primário/fisiologiaRESUMO
The recent assembly of the adult Drosophila melanogaster central brain connectome, containing more than 125,000 neurons and 50 million synaptic connections, provides a template for examining sensory processing throughout the brain1,2. Here we create a leaky integrate-and-fire computational model of the entire Drosophila brain, on the basis of neural connectivity and neurotransmitter identity3, to study circuit properties of feeding and grooming behaviours. We show that activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation4. In addition, using the model to activate neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing5-a testable hypothesis that we validate by optogenetic activation and behavioural studies. Activating different classes of gustatory neurons in the model makes accurate predictions of how several taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. Additionally, we applied this model to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit, and accurately describes the circuit response upon activation of different mechanosensory subtypes6-10. Our results demonstrate that modelling brain circuits using only synapse-level connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can describe complete sensorimotor transformations.
Assuntos
Encéfalo , Drosophila melanogaster , Modelos Neurológicos , Paladar , Animais , Drosophila melanogaster/fisiologia , Encéfalo/fisiologia , Encéfalo/citologia , Paladar/fisiologia , Comportamento Alimentar/fisiologia , Asseio Animal/fisiologia , Sinapses/fisiologia , Simulação por Computador , Optogenética , Neurônios Motores/fisiologia , Feminino , Masculino , ConectomaRESUMO
A goal of neuroscience is to obtain a causal model of the nervous system. The recently reported whole-brain fly connectome1-3 specifies the synaptic paths by which neurons can affect each other, but not how strongly they do affect each other in vivo. To overcome this limitation, we introduce a combined experimental and statistical strategy for efficiently learning a causal model of the fly brain, which we refer to as the 'effectome'. Specifically, we propose an estimator for a linear dynamical model of the fly brain that uses stochastic optogenetic perturbation data to estimate causal effects and the connectome as a prior to greatly improve estimation efficiency. We validate our estimator in connectome-based linear simulations and show that it recovers a linear approximation to the nonlinear dynamics of more biophysically realistic simulations. We then analyse the connectome to propose circuits that dominate the dynamics of the fly nervous system. We discover that the dominant circuits involve only relatively small populations of neurons-thus, neuron-level imaging, stimulation and identification are feasible. This approach also re-discovers known circuits and generates testable hypotheses about their dynamics. Overall, we provide evidence that fly whole-brain dynamics are generated by a large collection of small circuits that operate largely independently of each other. This implies that a causal model of a brain can be feasibly obtained in the fly.
Assuntos
Encéfalo , Conectoma , Drosophila melanogaster , Modelos Neurológicos , Neurônios , Animais , Encéfalo/fisiologia , Drosophila melanogaster/fisiologia , Neurônios/fisiologia , Optogenética , Modelos Lineares , Masculino , Feminino , Processos Estocásticos , Reprodutibilidade dos TestesRESUMO
Novel stimulation methods are needed to overcome the limitations of contemporary cochlear implants. Optogenetics is a technique that confers light sensitivity to neurons via the genetic introduction of light-sensitive ion channels. By controlling neural activity with light, auditory neurons can be activated with higher spatial precision. Understanding the behaviour of opsins at high stimulation rates is an important step towards their translation. To elucidate this, we compared the temporal characteristics of auditory nerve and inferior colliculus responses to optogenetic, electrical, and combined optogenetic-electrical stimulation in virally transduced mice expressing one of two channelrhodopsins, ChR2-H134R or ChIEF, at stimulation rates up to 400 pulses per second (pps). At 100 pps, optogenetic responses in ChIEF mice demonstrated higher fidelity, less change in latency, and greater response stability compared to responses in ChR2-H134R mice, but not at higher rates. Combined stimulation improved the response characteristics in both cohorts at 400 pps, although there was no consistent facilitation of electrical responses. Despite these results, day-long stimulation (up to 13 h) led to severe and non-recoverable deterioration of the optogenetic responses. The results of this study have significant implications for the translation of optogenetic-only and combined stimulation techniques for hearing loss.
Assuntos
Vias Auditivas , Channelrhodopsins , Estimulação Elétrica , Optogenética , Animais , Optogenética/métodos , Camundongos , Vias Auditivas/fisiologia , Vias Auditivas/metabolismo , Channelrhodopsins/metabolismo , Channelrhodopsins/genética , Estimulação Elétrica/métodos , Colículos Inferiores/fisiologia , Colículos Inferiores/metabolismo , Nervo Coclear/fisiologia , Nervo Coclear/metabolismo , Cinética , Implantes CoclearesRESUMO
Response gain is a crucial means by which modulatory systems control the impact of sensory input. In the visual cortex, the serotonergic 5-HT2A receptor is key in such modulation. However, due to its expression across different cell types and lack of methods that allow for specific activation, the underlying network mechanisms remain unsolved. Here we optogenetically activate endogenous G protein-coupled receptor (GPCR) signaling of a single receptor subtype in distinct mouse neocortical subpopulations in vivo. We show that photoactivation of the 5-HT2A receptor pathway in pyramidal neurons enhances firing of both excitatory neurons and interneurons, whereas 5-HT2A photoactivation in parvalbumin interneurons produces bidirectional effects. Combined photoactivation in both cell types and cortical network modelling demonstrates a conductance-driven polysynaptic mechanism that controls the gain of visual input without affecting ongoing baseline levels. Our study opens avenues to explore GPCRs neuromodulation and its impact on sensory-driven activity and ongoing neuronal dynamics.
Assuntos
Interneurônios , Optogenética , Células Piramidais , Receptor 5-HT2A de Serotonina , Córtex Visual , Animais , Córtex Visual/metabolismo , Córtex Visual/fisiologia , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/genética , Camundongos , Interneurônios/metabolismo , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Parvalbuminas/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia , FemininoRESUMO
Spiral waves in cardiac tissue have been identified as a significant factor leading to life-threatening arrhythmias and ventricular fibrillation. Consequently, understanding the mechanisms underlying the dynamics of such waves and exploring strategies for their elimination have garnered substantial interest and emerged as crucial research objectives. Spiral waves often become pinned (trapped) at anatomical obstacles in cardiac tissue, resulting in increased stability and posing challenges for their elimination. The unpinning of spiral waves can be achieved through the application of an external electric field and has been the subject of previous research. Recently, optogenetics has emerged as an alternative method to modulate electrical activity by illumination of cardiac tissue. In this paper, we employ mathematical modeling to investigate the potential of utilizing local illumination to unpin and eliminate spiral waves in cardiac tissue. We also extend this methodology to explore the effects of more complex turbulent excitation patterns. We conduct simulations using low-dimensional (Barkley) and ionic (Fenton-Karma) models of cardiac tissue, incorporating optogenetical channels. Our findings demonstrate that local suprathreshold illumination can successfully unpin spiral waves in 100% of cases. Notably, unlike unpinning by electrical field stimulation, this approach does not necessitate precise timing of stimulus application during a specific phase of rotation. Additionally, we demonstrate that periodic optogenetical stimulation can effectively eliminate both unpinned spiral waves and turbulence by moving them toward the boundary via an antitachycardia pacing mechanism.
Assuntos
Optogenética , Modelos Cardiovasculares , Coração/fisiologiaRESUMO
The reinforcing nature of social interactions is necessary for the maintenance of appropriate social behavior. However, the neural substrates underlying social reward processing and how they might differ based on the sex and internal state of the animal remains unknown. It is also unclear whether these neural substrates are shared with those involved in nonsocial rewarding processing. We developed a fully automated, two choice (social-sucrose) operant assay in which mice choose between social and nonsocial rewards to directly compare the reward-related behaviors associated with two competing stimuli. We performed cellular resolution calcium imaging of medial prefrontal cortex (mPFC) neurons in male and female mice across varying states of water restriction and social isolation. We found that mPFC neurons maintain largely non-overlapping, flexible representations of social and nonsocial reward that vary with internal state in a sex-dependent manner. Additionally, optogenetic manipulation of mPFC activity during the reward period of the assay disrupted reward-seeking behavior across male and female mice. Thus, using a two choice operant assay, we have identified sex-dependent, non-overlapping neural representations of social and nonsocial reward in the mPFC that vary with internal state and that are essential for appropriate reward-seeking behavior.
Assuntos
Neurônios , Optogenética , Córtex Pré-Frontal , Recompensa , Comportamento Social , Animais , Córtex Pré-Frontal/fisiologia , Feminino , Masculino , Neurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Caracteres Sexuais , Comportamento Animal/fisiologia , Condicionamento Operante/fisiologia , Comportamento de Escolha/fisiologia , Isolamento Social/psicologiaRESUMO
BACKGROUND: The lateral hypothalamus (LHA) is an evolutionarily conserved structure that regulates basic functions of an organism, particularly wakefulness. To clarify the function of LHAGABA neurons and their projections on regulating general anesthesia is crucial for understanding the excitatory and inhibitory effects of anesthetics on the brain. The aim of the present study is to investigate whether LHAGABA neurons play either an inhibitory or a facilitatory role in sevoflurane-induced anesthetic arousal regulation. METHODS: We used fiber photometry and immunofluorescence staining to monitor changes in neuronal activity during sevoflurane anesthesia. Opto-/chemogenetic modulations were employed to study the effect of neurocircuit modulations during the anesthesia. Anterograde tracing was used to identify a GABAergic projection from the LHA to a periaqueductal gray (PAG) subregion. RESULTS: c-Fos staining showed that LHAGABA activity was inhibited by induction of sevoflurane anesthesia. Anterograde tracing revealed that LHAGABA neurons project to multiple arousal-associated brain areas, with the lateral periaqueductal gray (LPAG) being one of the dense projection areas. Optogenetic experiments showed that activation of LHAGABA neurons and their downstream target LPAG reduced the burst suppression ratio (BSR) during continuous sevoflurane anesthesia. Chemogenetic experiments showed that activation of LHAGABA and its projection to LPAG neurons prolonged the anesthetic induction time and promoted wakefulness. CONCLUSIONS: In summary, we show that an inhibitory projection from LHAGABA to LPAGGABA neurons promotes arousal from sevoflurane-induced loss of consciousness, suggesting a complex control of wakefulness through intimate interactions between long-range connections.
Assuntos
Anestésicos Inalatórios , Nível de Alerta , Neurônios GABAérgicos , Vias Neurais , Substância Cinzenta Periaquedutal , Sevoflurano , Animais , Sevoflurano/farmacologia , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/fisiologia , Camundongos , Anestésicos Inalatórios/farmacologia , Masculino , Nível de Alerta/efeitos dos fármacos , Nível de Alerta/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Camundongos Endogâmicos C57BL , Região Hipotalâmica Lateral/efeitos dos fármacos , Região Hipotalâmica Lateral/fisiologia , Camundongos Transgênicos , Optogenética , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismoRESUMO
Circumventing the limitations of current bioassays, we introduce a light-controlled assay, OptoAssay, toward wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bidirectional movement of assay components, only by changing the wavelength of light. Demonstrating exceptional versatility, the OptoAssay showcases its efficacy on various substrates, delivering a dynamic bioassay format. The applicability of the OptoAssay is successfully demonstrated by the calibration of a competitive model assay, resulting in a superior limit of detection of 8 pg ml-1, which is beyond those of conventional ELISA tests. In the future, combined with smartphones, OptoAssays could obviate the need for external flow control systems such as pumps or valves and signal readout devices, enabling on-site analysis in resource-limited settings.
Assuntos
Bioensaio , Luz , Optogenética , Optogenética/métodos , Bioensaio/métodos , HumanosRESUMO
To determine how neuronal circuits encode and drive behavior, it is often necessary to measure and manipulate different aspects of neurochemical signaling in awake animals. Optogenetics and calcium sensors have paved the way for these types of studies, allowing for the perturbation and readout of spiking activity within genetically defined cell types. However, these methods lack the ability to further disentangle the roles of individual neuromodulator and neuropeptides on circuits and behavior. We review recent advances in chemical biology tools that enable precise spatiotemporal monitoring and control over individual neuroeffectors and their receptors in vivo. We also highlight discoveries enabled by such tools, revealing how these molecules signal across different timescales to drive learning, orchestrate behavioral changes, and modulate circuit activity.
Assuntos
Técnicas Biossensoriais , Vias Neurais , Neuropeptídeos , Neurotransmissores , Animais , Cálcio/metabolismo , Cálcio/análise , Aprendizagem , Neurônios/metabolismo , Neurônios/fisiologia , Neuropeptídeos/análise , Neurotransmissores/análise , Optogenética , Vias Neurais/fisiologiaRESUMO
Anatomical connectivity and lesion-deficit studies have shown that the dorsal and ventral hippocampi contribute to cognitive and emotional processes, respectively. However, the role of the dorsal hippocampus (dHP) in emotional or stress-related behaviors remains unclear. Here, we showed that neuronal activity in the dHP affects stress-coping behaviors in mice via excitatory projections to the medial prefrontal cortex (mPFC). The antidepressant ketamine rapidly induced c-Fos expression in both the dorsal and ventral hippocampi. The suppression of GABAergic transmission in the dHP-induced molecular changes similar to those induced by ketamine administration, including eukaryotic elongation factor 2 (eEF2) dephosphorylation, brain-derived neurotrophic factor (BDNF) elevation, and extracellular signal-regulated kinase (ERK) phosphorylation. These synaptic and molecular changes in the dHP induced a reduction in the immobility time of the mice in the tail-suspension and forced swim tests without affecting anxiety-related behavior. Conversely, pharmacological and chemogenetic potentiation of inhibitory neurotransmission in the dHP CA1 region induced passive coping behaviors during the tests. Transneuronal tracing and electrophysiology revealed monosynaptic excitatory connections between dHP CA1 neurons and mPFC neurons. Optogenetic stimulation of dHP CA1 neurons in freely behaving mice produced c-Fos induction and spike firing in the mPFC neurons. Chemogenetic activation of the dHP-recipient mPFC neurons reversed the passive coping behaviors induced by suppression of dHP CA1 neuronal activity. Collectively, these results indicate that neuronal activity in the dHP modulates stress-coping strategies to inescapable stress and contributes to the antidepressant effects of ketamine via the dHP-mPFC circuit.
Assuntos
Hipocampo , Córtex Pré-Frontal , Estresse Psicológico , Animais , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiologia , Camundongos , Hipocampo/metabolismo , Estresse Psicológico/metabolismo , Masculino , Neurônios/metabolismo , Adaptação Psicológica/fisiologia , Ketamina/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transmissão Sináptica , Camundongos Endogâmicos C57BL , Comportamento Animal , Optogenética/métodos , Capacidades de EnfrentamentoRESUMO
The central amygdala (CeA) is a crucial hub in the processing of affective itch, containing a diverse array of neuronal populations. Among these components, Neuropeptide Y (NPY) and its receptors, such as NPY2R, affect various physiological and psychological processes. Despite this broad impact, the precise role of NPY2R+ CeA neurons in itch modulation remains unknown, particularly concerning any potential lateralization effects. To address this, we employed optogenetics to selectively stimulate NPY2R+ CeA neurons in mice, investigating their impact on itch modulation. Optogenetic activation of NPY2R+ CeA neurons reduced scratching behavior elicited by pruritogens without exhibiting any lateralization effects. Electrophysiological recordings confirmed increased neuronal activity upon stimulation. However, this modulation did not affect thermal sensitivity, mechanical sensitivity, or formalin-induced hyperalgesia. Additionally, no alterations in anxiety-like behaviors or locomotion were observed upon stimulation. Projection tracing revealed connections of NPY2R+ CeA neurons to brain regions implicated in itch processing. Overall, this comprehensive study highlights the role of NPY2R+ CeA neurons in itch regulation without any lateralization effects.
Assuntos
Tonsila do Cerebelo , Neurônios , Prurido , Receptores de Neuropeptídeo Y , Animais , Receptores de Neuropeptídeo Y/metabolismo , Prurido/metabolismo , Prurido/induzido quimicamente , Camundongos , Neurônios/metabolismo , Tonsila do Cerebelo/metabolismo , Comportamento Animal , Masculino , Optogenética , Neuropeptídeo Y/metabolismo , Camundongos Endogâmicos C57BL , Núcleo Central da Amígdala/metabolismoRESUMO
New memories are integrated into prior knowledge of the world. But what if consecutive memories exert opposing demands on the host brain network? We report that acquiring a robust (food-context) memory constrains the mouse hippocampus within a population activity space of highly correlated spike trains that prevents subsequent computation of a flexible (object-location) memory. This densely correlated firing structure developed over repeated mnemonic experience, gradually coupling neurons in the superficial sublayer of the CA1 stratum pyramidale to whole-population activity. Applying hippocampal theta-driven closed-loop optogenetic suppression to mitigate this neuronal recruitment during (food-context) memory formation relaxed the topological constraint on hippocampal coactivity and restored subsequent flexible (object-location) memory. These findings uncover an organizational principle for the peer-to-peer coactivity structure of the hippocampal cell population to meet memory demands.
Assuntos
Região CA1 Hipocampal , Memória , Optogenética , Ritmo Teta , Animais , Masculino , Potenciais de Ação , Região CA1 Hipocampal/fisiologia , Região CA1 Hipocampal/citologia , Memória/fisiologia , Neurônios/fisiologia , Células Piramidais/fisiologiaRESUMO
Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.
Assuntos
Bacteriorodopsinas , Neurônios , Optogenética , Animais , Humanos , Potenciais de Ação , Bacteriorodopsinas/metabolismo , Bacteriorodopsinas/genética , Bacteriorodopsinas/química , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Channelrhodopsins/química , Criptófitas/genética , Criptófitas/metabolismo , Células HEK293 , Ativação do Canal Iônico/efeitos da radiação , Luz , Mutação , Neurônios/metabolismo , Neurônios/efeitos da radiação , Optogenética/métodos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Functional activation of the focal ischemic brain has been reported to improve outcomes by augmenting collateral blood flow. However, functional activation also increases metabolic demand and might thereby worsen outcomes. Indeed, preclinical and clinical reports have been conflicting. Here, we tested the effect of functional activation during acute ischemic stroke using distal middle cerebral artery occlusion in anesthetized mice. METHODS: Using transgenic mice expressing channelrhodopsin-2 in neurons, we delivered functional activation using physiological levels of transcranial optogenetic stimulation of the moderately ischemic cortex (ie, penumbra), identified using real-time full-field laser speckle perfusion imaging during a 1-hour distal microvascular clip of the middle cerebral artery. Neuronal activation was confirmed using evoked field potentials, and infarct volumes were measured in tissue slices 48 hours later. RESULTS: Optogenetic stimulation of the penumbra was associated with more than 2-fold larger infarcts than stimulation of the contralateral homotopic region and the sham stimulation group (n=10, 7, and 9; 11.0±5.6 versus 5.1±4.3 versus 4.1±3.7 mm3; P=0.008, 1-way ANOVA). Identical stimulation in wild-type mice that do not express channelrhodopsin-2 did not have an effect. Optogenetic stimulation was associated with a small increase in penumbral perfusion that did not explain enlarged infarcts. CONCLUSIONS: Our data suggest that increased neuronal activity during acute focal arterial occlusions can be detrimental, presumably due to increased metabolic demand, and may have implications for the clinical management of hyperacute stroke patients.
Assuntos
AVC Isquêmico , Camundongos Transgênicos , Optogenética , Animais , Camundongos , AVC Isquêmico/fisiopatologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Isquemia Encefálica/fisiopatologia , Neurônios/metabolismo , Circulação Cerebrovascular/fisiologia , Camundongos Endogâmicos C57BLRESUMO
Rationale: Optogenetically engineered facultative anaerobic bacteria exhibit a favorable tendency to colonize at solid tumor sites and spatiotemporally-programmable therapeutics release abilities, attracting extensive attention in precision tumor therapy. However, their therapeutic efficacy is moderate. Conventional photothermal agents with high tumor ablation capabilities exhibit low tumor targeting efficiency, resulting in significant off-target side effects. The combination of optogenetics and photothermal therapy may offer both tumor-targeting and excellent tumor-elimination capabilities, which unfortunately has rarely been investigated. Herein, we construct a bacteria-based cascade near-infrared optogentical-photothermal system (EcNαHL-UCNPs) for enhanced tumor therapy. Methods: EcNαHL-UCNPs consists of an optogenetically engineered Escherichia coli Nissle 1917 (EcN) conjugated with lanthanide-doped upconversion nanoparticles (UCNPs), which are capable of locally secreting α-hemolysin (αHL), a pore-forming protein, in responsive to NIR irradiation. Anti-tumor effects of EcNαHL-UCNPs were determined in both H22 and 4T1 tumors. Results: The αHL not only eliminates tumor cells, but more importantly disrupts endothelium to form thrombosis as an in situ photothermal agent in tumors. The in situ formed thrombosis significantly potentiates the photothermic ablation of H22 tumors upon subsequent NIR light irradiation. Besides, αHL secreted by EcNαHL-UCNPs under NIR light irradiation not only inhibits 4T1 tumor growth, but also suppresses metastasis of 4T1 tumor via inducing the immune response. Conclusion: Our studies highlight bacteria-based cascade optogenetical-photothermal system for precise and effective tumor therapy.
Assuntos
Escherichia coli , Nanopartículas , Optogenética , Terapia Fototérmica , Animais , Camundongos , Terapia Fototérmica/métodos , Escherichia coli/genética , Linhagem Celular Tumoral , Nanopartículas/química , Optogenética/métodos , Camundongos Endogâmicos BALB C , Raios Infravermelhos , Feminino , Neoplasias/terapia , Humanos , Fototerapia/métodosRESUMO
Alteration of synaptic function in the dorsal horn (DH) has been implicated as a cellular substrate for the development of neuropathic pain, but certain details remain unclear. In particular, the lack of information on the types of synapses that undergo functional changes hinders the understanding of disease pathogenesis from a synaptic plasticity perspective. Here, we addressed this issue by using optogenetic and retrograde tracing ex vivo to selectively stimulate first-order nociceptors expressing Nav1.8 (NRsNav1.8) and record the responses of spinothalamic tract neurons in spinal lamina I (L1-STTNs). We found that spared nerve injury (SNI) increased excitatory postsynaptic currents (EPSCs) in L1-STTNs evoked by photostimulation of NRsNav1.8 (referred to as Nav1.8-STTN EPSCs). This effect was accompanied by a significant change in the failure rate and paired-pulse ratio of synaptic transmission from NRsNav1.8 to L1-STTN and in the frequency (not amplitude) of spontaneous EPSCs recorded in L1-STTNs. However, no change was observed in the ratio of AMPA to NMDA receptor-mediated components of Nav1.8-STTN EPSCs or in the amplitude of unitary EPSCs constituting Nav1.8-STTN EPSCs recorded with extracellular Ca2+ replaced by Sr2+ In addition, there was a small increase (approximately 10%) in the number of L1-STTNs showing immunoreactivity for phosphorylated extracellular signal-regulated kinases in mice after SNI compared with sham. Similarly, only a small percentage of L1-STTNs showed a lower action potential threshold after SNI. In conclusion, our results show that SNI induces presynaptic modulation at NRNav1.8 (consisting of both peptidergic and nonpeptidergic nociceptors) synapses on L1-STTNs forming the lateral spinothalamic tract.
Assuntos
Potenciais Pós-Sinápticos Excitadores , Canal de Sódio Disparado por Voltagem NAV1.8 , Nociceptores , Tratos Espinotalâmicos , Transmissão Sináptica , Animais , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Nociceptores/metabolismo , Nociceptores/fisiologia , Tratos Espinotalâmicos/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Transmissão Sináptica/fisiologia , Camundongos , Optogenética , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The individuals often show consolation to distressed companions or show aggression to the intruders. The circuit mechanisms underlying switching between consolation and aggression remain unclear. In the present study, using male mandarin voles, we identified that two distinct subtypes of oxytocin receptor (OXTR) neurons in the medial amygdala (MeA) projecting to the anterior insula (AI) and ventrolateral aspect of ventromedial hypothalamus (VMHvl) response differently to stressed siblings or unfamiliar intruders using c-Fos or calcium recording. Oxytocin release and activities of PVN neurons projecting to MeA increased upon consoling and attacking. OXTR antagonist injection to the MeA reduced consoling and attacking. Apoptosis, optogenetic or pharmacogenetic manipulation of these two populations of neurons altered behavioral responses to these two social stimuli respectively. Here, we show that two subtypes of OXTR neurons in the MeA projecting to the AI or VMHvl causally control consolation or aggression that may underlie switch between consolation and aggression.
Assuntos
Agressão , Arvicolinae , Complexo Nuclear Corticomedial , Neurônios , Ocitocina , Receptores de Ocitocina , Animais , Receptores de Ocitocina/metabolismo , Receptores de Ocitocina/genética , Masculino , Agressão/fisiologia , Arvicolinae/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Ocitocina/metabolismo , Complexo Nuclear Corticomedial/metabolismo , Complexo Nuclear Corticomedial/fisiologia , Comportamento Animal/fisiologia , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiologia , Comportamento Social , Proteínas Proto-Oncogênicas c-fos/metabolismo , Vias Neurais/fisiologia , OptogenéticaRESUMO
Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5'-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.