RESUMEN
cAMP is a universal second messenger regulated by various upstream pathways including Ca2+ and G-protein-coupled receptors (GPCRs). To decipher in vivo cAMP dynamics, we rationally designed cAMPinG1, a sensitive genetically encoded green cAMP indicator that outperformed its predecessors in both dynamic range and cAMP affinity. Two-photon cAMPinG1 imaging detected cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds. In addition, multicolor imaging with a sensitive red Ca2+ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca2+ and cAMP in hundreds of neurons. We found Ca2+-related cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-related cAMP responses. Overall, our multicolor suite will facilitate analysis of the interaction between the Ca2+, GPCR and cAMP signaling at single-cell resolution both in vitro and in vivo.
Asunto(s)
Calcio , AMP Cíclico , Neuronas , Corteza Visual , Animales , AMP Cíclico/metabolismo , Calcio/metabolismo , Ratones , Corteza Visual/metabolismo , Corteza Visual/fisiología , Corteza Visual/citología , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Ratones Endogámicos C57BL , Señalización del Calcio , Células HEK293RESUMEN
Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method "Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing" (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.
Asunto(s)
Interneuronas , Neuronas , Animales , Ratones , Axones , Cognición , Dependovirus/genética , Proteínas Fluorescentes Verdes/genéticaRESUMEN
Information in the brain is represented by the collective and coordinated activity of single neurons. Activity is determined by a large amount of dynamic synaptic inputs from neurons in the same and/or distant brain regions. Therefore, the simultaneous recording of single neurons across several brain regions is critical for revealing the interactions among neurons that reflect the computational principles of the brain. Recently, several wide-field two-photon (2P) microscopes equipped with sizeable objective lenses have been reported. These microscopes enable large-scale in vivo calcium imaging and have the potential to make a significant contribution to the elucidation of information-processing mechanisms in the cerebral cortex. This review discusses recent reports on wide-field 2P microscopes and describes the trade-offs encountered in developing wide-field 2P microscopes. Large-scale imaging of neural activity allows us to test hypotheses proposed in theoretical neuroscience, and to identify rare but influential neurons that have potentially significant impacts on the whole-brain system.
Asunto(s)
Encéfalo , Neuronas , Encéfalo/fisiología , Calcio , Corteza Cerebral/fisiología , Microscopía , Neuronas/fisiologíaRESUMEN
We recently established a simple and versatile adeno-associated virus (AAV) induction approach that enables dense (>90% labeled neurons) and cortical-wide Ca2+ sensor expression. Here, we describe the stepwise protocol for neonatal AAV injection of a Ca2+ sensor. We also detail the steps for subsequent craniotomy to generate a chronic cranial window, followed by wide-field two-photon Ca2+ imaging in an awake mouse. This protocol serves as an alternative to the use of transgenic animals and offers translatable options for cortical-wide experiments. For complete details on the use and execution of this protocol, please refer to Ota et al. (2021).
Asunto(s)
Corteza Cerebral/diagnóstico por imagen , Dependovirus/genética , Imagen Óptica/métodos , Animales , Calcio/metabolismo , Craneotomía , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Inyecciones , Masculino , Ratones , Cráneo/cirugíaRESUMEN
Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of â¼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements.
Asunto(s)
Corteza Cerebral/fisiología , Conectoma , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Señalización del Calcio , Corteza Cerebral/citología , Femenino , Límite de Detección , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/normas , Neuronas/fisiología , Relación Señal-RuidoRESUMEN
Every organ demonstrates specific vascular characteristics and functions maintained by interactions of endothelial cells (ECs) and parenchymal cells. Particularly, brain ECs play a central role in the formation of a functional blood brain barrier (BBB). Organ-specific ECs have their own morphological features, and organ specificity must be considered when investigating interactions between ECs and other cell types constituting a target organ. Here we constructed angiogenesis-based microvascular networks with perivascular cells in a microfluidic device setting by coculturing ECs and mesenchymal stem cells (MSCs). Furthermore, we analyzed endothelial barrier functions as well as fundamental morphology, an essential step to build an in vitro BBB model. In particular, we used both brain microvascular ECs (BMECs) and human umbilical vein ECs (HUVECs) to test if organ specificity of ECs affects the formation processes and endothelial barrier functions of an engineered microvascular network. We found that microvascular formation processes differed by the source of ECs. HUVECs formed more extensive microvascular networks compared to BMECs while no differences were observed between BMECs and HUVECs in terms of both the microvascular diameter and the number of pericytes peripherally associated with the microvasculatures. To compare the endothelial barrier functions of each type of EC, we performed fluorescence dextran perfusion on constructed microvasculatures. The permeability coefficient of BMEC microvasculatures was significantly lower than that of HUVEC microvasculatures. In addition, there were significant differences in terms of tight junction protein expression. These results suggest that the organ source of ECs influences the properties of engineered microvasculature and thus is a factor to be considered in the design of organ-specific cell culture models.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Permeabilidad Capilar , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Microvasos/metabolismo , Neovascularización Fisiológica , Pericitos/metabolismo , Barrera Hematoencefálica/citología , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Dispositivos Laboratorio en un Chip , Microvasos/citología , Fenotipo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismoRESUMEN
IMPACT STATEMENT: Construction of capillary networks is a fundamental challenge for the development of three-dimensional (3D) tissue engineering. However, it is not well understood how to construct stable capillary networks that maintain a luminal size similar to that of capillary structures in vivo (i.e., <10 µm diameter). In this study, we demonstrated the construction of stable capillary networks covered by pericyte-like perivascular cells using an in vitro 3D angiogenesis model by optimizing interactions between endothelial cells and perivascular cells. Our 3D angiogenesis model can be combined with 3D culture of epithelial cells in the context of vascularization of 3D tissue-engineered constructs.
Asunto(s)
Capilares/citología , Pericitos/citología , Ingeniería de Tejidos/métodos , Membrana Basal/metabolismo , Proliferación Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Imagenología Tridimensional , Células Madre Mesenquimatosas/citología , Microfluídica , Neovascularización FisiológicaRESUMEN
Neurovascular unit (NVU) is a basic unit in the brain, including neurons, glial cells, blood vessels and extracellular matrix. This concept implies the importance of a three-dimensional (3D) culture model including these cell types for investigating brain functions. However, little is known about the construction of an in vitro 3D NVU model. In the present study, we aimed at constructing 3D neurovascular tissues by combining in vitro neurogenesis and angiogenesis models using a microfluidic platform, which is a critical step toward the NVU construction in vitro. Three gel conditions, which were fibrin gel, fibrin-Matrigel mixed gel and fibrin-hyaluronan mixed gel, were investigated to optimize the gel components in terms of neurogenesis and angiogenesis. First, fibrin-Matrigel mixed gel was found to promote neural stem cell (NSC) differentiation into neurons and neurite extension. In particular, 3D neural networks were constructed in 2-8 mg/ml fibrin-Matrigel mixed gel. Second, we found that capillary-like structures were also formed in the fibrin-Matrigel mixed gel by coculturing brain microvascular endothelial cells (BMECs) and human mesenchymal stem cells (MSCs). Finally, we combined both neural and vascular culture models and succeeded in constructing 3D neurovascular tissues with an optimized seeding condition of NSCs, BMECs and MSCs.