The brain contains over a billion neurons, each with complex networks of connections throughout the brain. Patterns of neural activity are believed to generate specific aspects of behaviour and cognition, but how?
In order to decode patterns of neural activity, neuroscientists need to visualize hundreds of neurons simultaneously—not just an individual neuron—in freely-behaving animals. From a technology standpoint, this is no easy feat.
Current techniques, such as in vivo electrophysiology, can record neural activity with spike-timed precision, but lacks the ability to localize the activity of large populations of individual cells and identify cell types.
Is there a method to visualize neural activity in vivo?
What is In Vivo Calcium Imaging?
Calcium imaging enables neuroscientists to visualize activity in hundreds of individual neurons simultaneously using fluorescent activity sensors. Changes in fluorescence indicate fluctuations in intracellular calcium, which is an indirect indicator of neural activity (1).
The development of genetically encoded calcium indicators (GECIs) has enabled neuroscientists to study specific cell types (e.g. excitatory or inhibitory neurons).
GCaMP, a green fluorescent GECI, is commonly used in calcium imaging experiments since it has been optimized over many generations (currently GCaMP7) for speed, signal-to-noise ratio, expression, and changes in fluorescence (2). The calcium imaging toolbox is constantly growing with the development of new GECIs, such as RCaMP and XCaMP (3,4).
Why Use In Vivo Calcium Imaging?
Recently, there has been a surge in the use of calcium imaging to study neural circuits in vivo. This popularization has led to optimized biological methods and better technology, allowing more neuroscientists to adopt the technique.
Aside from popularity, are their other reasons neuroscientists are drawn to calcium imaging?
Calcium imaging provides neuroscientists with the means to study specific populations of cells within or across brain regions in freely-behaving animals. In doing so, neuroscientists can investigate how neural activity may be linked to aspects of behaviour and cognition, effectively linking genetically-identified cells with function.
Behaviour and cognition cannot be characterized by an isolated firing pattern of neurons because they are learned or adapted over time. Calcium imaging can be used to track the activity of neurons over time and investigate how networks grow or change during learning. This is especially important for the longitudinal study of animal models of brain disease. Neuroscientists can begin to understand the development of activity-related changes in these models and assess the long-term effects of pharmacological interventions.
With any new technique, there are associated pitfalls. As we’ve known for years, neural activity is associated with action potentials — changes in voltage across the cell membrane. Calcium imaging measures changes in intra-cellular calcium concentration, providing an indirect indicator of neural activity. Compared to changes in voltage, fluctuations in calcium levels are much slower and may reflect a summation of signals rather than individual spikes (5). That is to say the temporal resolution of calcium imaging may be limited for fast-spiking neurons, such as interneurons.
The gradual development of genetically encoded voltage indicators (GEVIs), designed to detect changes in voltage, are expected to overcome these limitations (6). As with any new technology, widespread application of GEVIs will take time, requiring validation and optimization for use in vivo.
Despite this limitation of calcium imaging, this technique has still greatly advanced our understanding of how neural activity relates to behaviour and cognition.
- Grienberger, C & Konnerth A. (2012). Imaging calcium in neurons. Neuron, 73(5), 862-885.
- Dana, H et al. (2019). High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nature Methods, 16, 649-657.
- Akerboom, J et al. (2013). Genetically encoded calcium indicators for multi-colour neural activity imaging and combination optogenetics. Frontiers in Molecular Neuroscience.
- Inoue, M et al. (2019). Rational engineering of XCaMPS, a multicolour GECI suite for in vivo imaging of complex brain circuit dynamics. Cell, 177(5), 1346-1360.
- Wei, Z et al. (2019). A comparison of neuronal population dynamics measured with calcium imaging and electrophysiology. Biorxiv.
- Knopfel, T & Song C. (2019). Optical voltage imaging in neurons: moving from technology development to practical tool. Nature Neuroscience, 20, 719-727.