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

Summary

This protocol describes brain preparation and calcium imaging procedures for the measurement of calcium dynamics in heterogeneous cortical networks with neuronal subtypes that are genetically-labeled with red fluorescent protein.

Abstract

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.

Introduction

Spontaneous physiological activity regulates brain development via the actions of calcium, which serves as a second messenger to affect processes such as neurogenesis and cellular migration¹. Physiological activity may elicit changes in cellular calcium levels in different ways. For example, during the first postnatal week of mouse development, neurons of the cerebral cortex exhibit spontaneous action potential firing, which propagates as a wave through a large subset of cortical neurons^2,3. Calcium influx occurs through voltage and ligand-gated ion channels and triggers further calcium....

Protocol

All steps described below are in compliance with the animal use and regulation policies of the University of Washington and IACUC.

1. Advanced Preparations

1. Prepare 5% agar in 100 mL of water and autoclave for 20 min at 120 °C to dissolve the agar. Immediately remove the agar from the autoclave to prevent the solution from solidifying. Portion it into 5-mL petri dishes and cover them with lids. Place the dishes in a refrigerator.
2. Make 1 L of artificial cerebrospinal f.......

Discussion

The combination of confocal imaging of fluorescent proteins to label individual cells with widefield epifluorescent calcium imaging uses important features of each technique. Epifluorescence imaging provides several benefits over other fluorescence imaging methods. Compared to confocal imaging, it allows for fast image acquisition at low light levels, as removal of out-of-focus light reduces the overall fluorescence signal. The use of high signal-to-noise fluorescent indicators (such as Fluo4 AM) allows for the resolutio.......

Materials

Name	Company	Catalog Number	Comments
Inverted Microscope	Olympus	IX81	With DSU
Camera	Hamamatsu	Orca-flash2.8 CMOS
Light Source	Sutter	Lambda XL
40x Objective	Olympus	Uapo N 340 40x water	NA 1.15, WD 0.25mm
Calcium indicator - Fluo-4 AM	ThermoFisher	F14201
Cre dependent tdTomato mouse	Jackson Labs	007914	Ai14 mouse from Allen Institute for Brain Science
Dlx5/6 Cre mice	Jackson Labs	008199	Labels inhibitory interneurons. Use a different Cre driver to label different cell types
Sterilizing Filter	Corning	09-761-112	500mL, 0.22 micron bottle-top filter
Millicell Cell Culture Inserts	EMD Millipore	PICM0RG50	30mm diameter, 0.4 micron
slice resting chamber	Warner Instruments	BSC-PC	Prechamber meant to rest slices before imaging
Neorbasal-A Medium	ThermoFisher	10888022	cell culture basal medium
NIS Elements AR	Nikon		image acquisition software