This post is the first in a series that aims to educate readers about the tools that are used in neuroscience research.
Decades of neurobiology research have shown that calcium ions (Ca2+) are crucial second mesengers in neurons. For instance, they regulate gene expression, bring about neurotransmitter release and facilitate synaptic plasticity. Ca2+ ions can enter the cytoplasm of a neuron from two main sources: the extracellular environment and intracellular stores.
The first route is mediated primarily by voltage-gated calcium channels (“C” in diagram). When a neuron becomes active, its membrane is depolarized and this allows Ca2+ ions to enter the cytoplasm.
The second route is mediated by Ca2+ channels on the endoplasmic reticulum. When Ca2+ ions enter the cytoplasm via voltage-gated calcium channels, SERCA proteins pump them into the endoplasmic reticulum at high concentrations. This intracellular store of Ca2+ can be released when G-protein coupled receptors on the cell surface produce inositol triphosphate (IP3) via the action of Phospholipase C (PLC). IP3 stimulates calcium channels called IP3 receptors (“IP3R” in diagram) on the endoplasmic reticulum, which raise the concentration of Ca2+ ions in the cytoplasm dramatically.
Altered intracellular Ca2+ signaling has been implicated in a variety of disease states such as schizophrenia, Alzheimer’s and Huntington’s. In order to understand these diseases, it is important to be able to visualize the flux of Ca2+ ions within a neuron. Calcium imaging makes this goal a feasible one by allowing Ca2+ concentration to be detected as changes in fluorescence.
A molecule is called fluorescent if it absorbs light at a different wavelength than it emits light. If you shine one color light at a fluorophore, it will emit a different color, usually with a longer wavelength. The specific wavelengths at which a fluorophore absorbs and emits light are highly sensitive to the molecule’s structure. Because molecules often undergo conformational changes when they bind another chemical, this binding event also has the potential to change the properties of the fluorophore.
In 1985, Roger Tsien’s group at Berkeley was trying to chemically link a molecule that could bind Ca2+ ions to a molecule with fluorescent properties. They hoped that the resulting molecule would have different fluorescent properties depending on whether Ca2+ was bound or not. One of the compounds that they synthesized, called fura-2, is still very popular today.
The figure to the right is copied from Roger Tsien’s 1985 paper and it depicts the how fura-2’s excitation spectrum changes in the presence or absence of Ca2+ ions. Regardless of whether fura-2 binds Ca2+, it emits light at ~510 nm. However, the wavelength at which it absorbs light is dependent on whether Ca2+ is bound. In the absence of Ca2+, fura-2 is excited by 360 nm light; when saturated with Ca2+ ions, fura-2 is excited by 330 nm light. Therefore, if you compare the intensity of 510 nm light that is emitted when you shine 360 nm light on your biological sample to the intensity of 510 nm light that is emitted when you shine 330 nm light on your sample, you can calculate the concentration of Ca2+ ions. Using high-resolution microscopes, it is even possible to localize the changes in fluorescence within a single neuron.
Here is a beautiful video that was made using using Ca2+-sensitive dyes. It shows how Ca2+ release from the endoplasmic reticulum propogates down the dendrites of a neuron toward the cell body in a wave-like fashion. It also shows how these waves interact at a network level:
Interested in learning more about Ca2+ imaging? Here are some recent papers that use the technique:
- Hagenston et al. examine how Ca2+ waves alter the membrane excitability of cortical neurons. Full disclosure: I used to work in this lab.
- Tang et al. show how calcium signaling is involved in Huntington’s disease.
- Jin et al. demonstrate a novel form of long-term depression (LTD) that involves calcium signaling.