Intracranial Injection of Adeno-associated Viral Vectors

Lowery, Rebecca L.; Majewska, Ania K.

doi:10.3791/2140

Cited by 40 publications

(29 citation statements)

References 4 publications

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“…For example, pyramidal neurons in different cortical layers can be targeted by performing in utero electroporation at defined embryonic stages 28,29 . Similarly, injection of viral vectors engineered to express a fluorescent protein can also be used to label cells in different brain regions 30 . In addition, many lines of transgenic mice have been generated to drive the expression of Cre recombinase under cell type specific promoters 31,32 .…”

Section: Discussionmentioning

confidence: 99%

Two-Photon <em>in vivo</em> Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Zuo

2014

JoVE

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In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Two-Photon <em>in vivo</em> Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Zuo

2014

JoVE

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“…The BBB can be overcome via an invasive procedure in which a small hole is drilled into the skull. By slowly inserting a small diameter injection needle or a pulled glass capillary (with tip-diameters of around 10–20 μm), contrast agents can be directly injected into the brain region of interest [219]. The main limitation of intracranial injections is the small volume that can be delivered (usually less than 1 μl in mice).…”

Section: Dynamic Contrast Enhancement Approachesmentioning

confidence: 99%

Advanced optoacoustic methods for multiscale imaging of in vivo dynamics

et al. 2017

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Visualization of dynamic functional and molecular events in an unperturbed in vivo environment is essential for understanding the complex biology of living organisms and of disease state and progression. To this end, optoacoustic (photoacoustic) sensing and imaging have demonstrated the exclusive capacity to maintain excellent optical contrast and high resolution in deep-tissue observations, far beyond the penetration limits of modern microscopy. Yet, the time domain is paramount for the observation and study of complex biological interactions that may be invisible in single snapshots of living systems. This review focuses on the recent advances in optoacoustic imaging assisted by smart molecular labeling and dynamic contrast enhancement approaches that enable new types of multiscale dynamic observations not attainable with other bio-imaging modalities. A wealth of investigated new research topics and clinical applications is further discussed, including imaging of large-scale brain activity patterns, volumetric visualization of moving organs and contrast agent kinetics, molecular imaging using targeted and genetically expressed labels, as well as three-dimensional handheld diagnostics of human subjects.

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“…Classic cranial window surgery protocols include the removal of a piece of the scalp, leaving the newly implanted window to recover unprotected by skin (Cao et al, 2013; Goldey et al, 2014; Holtmaat et al, 2009). In addition, viruses encoding calcium indicators are typically injected intracortically in the same surgical procedure as the implantation of the cranial window (Chen et al, 2013; Lowery and Majewska, 2010; Resendez et al, 2016). Variations in intracortical injection-induced damage and viral spread increase unpredictability in the experimental yield.…”

Section: Introductionmentioning

confidence: 99%

Skin suturing and cortical surface viral infusion improves imaging of neuronal ensemble activity with head-mounted miniature microscopes

Cao

Zhang

et al. 2017

Journal of Neuroscience Methods

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Background In vivo optical imaging of neural activity provides important insights into brain functions at the single-cell level. Cranial windows and virally delivered calcium indicators are commonly used for imaging cortical activity through two-photon microscopes in head-fixed animals. Recently, head-mounted one-photon microscopes have been developed for freely behaving animals. However, minimizing tissue damage from the virus injection procedure and maintaining window clarity for imaging can be technically challenging. New method We used a wide-diameter glass pipette at the cortical surface for infusing the viral calcium reporter AAV-GCaMP6 into the cortex. After infusion, the scalp skin over the implanted optical window was sutured to facilitate postoperative recovery. The sutured scalp was removed approximately two weeks later and a miniature microscope was attached above the window to image neuronal activity in freely moving mice. Results We found that cortical surface virus infusion efficiently labeled neurons in superficial layers, and scalp skin suturing helped to maintain the long-term clarity of optical windows. As a result, several hundred neurons could be recorded in freely moving animals. Comparison with existing methods Compared to intracortical virus injection and open-scalp postoperative recovery, our methods minimized tissue damage and dura overgrowth underneath the optical window, and significantly increased the experimental success rate and the yield of identified neurons. Conclusion Our improved cranial surgery technique allows for high-yield calcium imaging of cortical neurons with head-mounted microscopes in freely behaving animals. This technique may be beneficial for other optical applications such as two-photon microscopy, multi-site imaging, and optogenetic modulation.

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Intracranial Injection of Adeno-associated Viral Vectors

Cited by 40 publications

References 4 publications

Two-Photon <em>in vivo</em> Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon <em>in vivo</em> Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Advanced optoacoustic methods for multiscale imaging of in vivo dynamics

Skin suturing and cortical surface viral infusion improves imaging of neuronal ensemble activity with head-mounted miniature microscopes

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