two-photon calcium imaging of mouse superior colliculus neurons

上丘的双光子和宽场钙成像

The superior colliculus (SC) is an important visual center in all vertebrates. In mammals, it receives direct inputs from the retina and the visual cortex1. While optical recording has been widely applied to the cortex2,3,4,5, its application in the SC is hindered by poor optical accesss6,7,8,9,10,11,12,13,14,15,16,17,18,19. The goal of this protocol is to provide details about two complementary methods for optical recording of the neural activity in the SC.
The SC is located beneath the cortex and transverse sinus, which limits optical access to the collicular neurons. One approach to overcome this limitation is to aspirate the overlaying cortex and expose the anterior-lateral SC7,9,10,13,14,19. However, because the SC receives cortical inputs, such an operation might affect how the SC neurons respond to visual stimuli. To overcome this limitation, we detail here an alternative protocol to image the superficial layer of the posterior-medial SC with a silicon plug, while leaving the cortex intact8,11. Specifically, to achieve single-cell resolution, we applied two-photon microscopy to image calcium responses in the posterior-medial SC of wild-type mice. In addition, to achieve broad coverage, we applied wide-field microscopy to image the entire SC of a mutant mouse whose posterior cortex has not developed20.
The two methods described in this protocol are complementary to each other. The two-photon calcium imaging without ablating the cortex is appropriate for recording neural activity at&#160;single-cell resolution with intact cortical inputs. The wide-field calcium imaging is appropriate for recording neural activity in the entire SC while sacrificing spatial resolution.

作者聚焦：揭示上丘的神经编码和视觉处理机制 

小鼠上丘的钙成像

Watch this Scientific Journal Video about Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons at JoVE.com

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons  

小鼠上丘神经元的双光子钙成像

植入头板和塞子后，将动物固定在带有头板的旋转跑步机上。将跑步机放在双光子显微镜下方，并将高度调整到合适的位置。在物镜和头板之间放置一个黑色铝制锥体，以防止显示器的光污染，从而进行视觉刺激。要进行双光子成像，请在 20 到 80 毫瓦之间调整样品平面的激光功率。以 4.8 赫兹扫描 600 x 600 微米的视场，空间分辨率为每像素 2.4 微米，图像神经活动深度可达 350 微米。使用旋转编码器，记录鼠标在跑步机上的运动。使用带有 50 毫米镜头的相机记录其瞳孔大小和位置。最后，通过记录刺激计算机发送的触发器，将这些记录和图像采集与视觉刺激同步。给出了野生型小鼠SC神经元的钙反应和荧光在图像中的标准偏差投影。

two-photon-calcium-imaging-of-mouse-superior-colliculus-neurons

Research

JoVE Journal

Neuroscience

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons

280 次观看. 植入头板和塞子后，将动物固定在带有头板的旋转跑步机上。将跑步机放在双光子显微镜下方，并将高度调整到合适的位置。在物镜和头板之间放置一个黑色铝制锥体，以防止显示器的光污染，从而进行视觉刺激。要进行双光子成像，请在 20 到 80 毫瓦之间调整样品平面的激光功率。以 4.8 赫兹扫描 600 x 600 微米的视场，空间分辨率为每像素 2.4 微米，图像神经活动深度?...

视频: Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons  

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...