Measuring the Calcium Dynamics of Individual, Genetically-labeled Neurons of the Developing Mouse Neocortex

Spontaneous activity in the developing mammalian cortex is necessary for proper network formation. Such activity may be intrinsic to individual cells or driven by network interactions, and different types of activity may affect distinct components of development. A striking feature of cortical development is the propagating waves of activity that cause simultaneous action potential firing in neurons across broad cortical regions. Waves have been proposed to play roles in patterning connections, such as those between the cortex and thalamus, as well as in placing inhibitory interneurons into the correct cortical layers. Calcium signaling induced by waves is likely to mediate these effects on development. Calcium imaging techniques in brain slice preparations may be used to visualize wave activity propagating between brain structures and to examine the contribution of individual cells to population activity. Slices have an advantage over dissociated cultures because of the ability to examine cellular activity in a setting with preserved network features, such as cortical layering. However, slice preparation for the physiological examination of developing cells can be difficult. The slicing process reduces network connectivity and injures cells. High potassium ringer solutions are often necessary to produce the synchronous activity that is normally present in vivo. This work describes a set of methods for brain slice preparation that allow for the measurement of the physiological patterns of synchronous activity without increasing potassium by using short-term organotypic slice cultures to increase cell health. Methods to identify genetically-labeled neuronal subpopulations in the cortical plate of these slices while conducting calcium imaging of heterogeneous neurons in the cortical network are presented. An overview of the slice preparation and imaging techniques of the developing cortex, which are useful for assaying both single-cell and population-level activity patterns, are presented. These methods may be adapted to many different neuronal subtypes and anatomical regions.