Light and Calcium Help You Label Active Neurons in the Brain

Whenever a memory is formed, neural cells or called neurons must first be activated to participate in this process. The activity of a neuron is usually defined as rapid changing of cell membrane voltage. And this kind of change of membrane voltage, or called depolarization [1], is often coupled with more drastic changing of calcium ion concentration inside the cell. Calcium ions have critical roles in the entire life span of a neuron [2], especially during the depolarization when the neuron is active. As a result, scientists have developed and utilized many tools, which are dependent on this voltage-dependent calcium rising inside a cell to label and monitor a neuron’s activity. For example, GCaMPs [3], or called genetically encoded calcium indicators, are a group of human engineered calcium sensor proteins, have been widely used in the neuroscience field as real-time fluorescent indicators of activity patterns of neurons both in vitro or in vivo (outside or inside a living animal).

However, that being said, those tools still have one major limitation – one can only observe how neurons behave with the help of calcium ions, but one cannot really do anything to those neurons they’re looking at in order to further investigate how their activities contribute to a certain physiological process or even a certain behavior in a live animal. Any ways to solve this problem? In the last issue of Nature Biotechnology (6/26/17), two groups published two back-to-back articles [4][5] showing us that actually, calcium ions can also help us control gene expression so that we can use those genetically encoded protein tools (such as opsins [6]) to manipulate neurons that interest us.

Although these two groups gave two different fancy names respectively to the systems they have developed (one is called FLARE, the other Cal-Light), the basic principles underlying them are quite similar. As is shown by the illustration drawn by me, these two systems are both combinations of Calmodulin, the TEV protease and the LOV2 proteins. These names may sound rather complicated to people who’re not experts in this field at first, but I will introduce them to you one by one.

First of all, the major switch of these systems is the TEV protease [7]. The human engineered transcription factors (TFs) [8], which could initiate the expression of our desired genetically encoded tools (fluorescent proteins, opsins, etc.), are first tethered to the inside of the cell membrane by a certain protein sequence so they cannot do their jobs. The TEV protease just recognizes that special protein sequence (not any other proteins inside the cell) and cleaves it so that our TFs could be freed to execute their tasks. Second, the TEV protease is fused to Calmodulin (CaM) [9] or its target peptide that binds to it. CaM is such a special protein so that only when there’s high concentration of calcium will it binds to its target peptide. In this way, only when the neuron is active, will the TEV protease be brought to the vicinity of the special protein sequence it can cut. Third, since neuron frequently fires thus the calcium rise inside the cell is also pretty usual, people need to bring in a temporal control so that the switch only works in our defined time period. This is when the LOV2 protein comes in. It is fused to the special sequence which would be cut by the TEV protease, and normally without blue light it will just bind to that sequence tightly and protect it from being cut. So this calcium-dependent gene expression switch is only working when the experimenter shines blue light on the neuron which has all the parts of it.

In both of these two articles, these calcium-dependent systems are successfully tested in vitro and in vivo. They’re shown to be able to trigger fluorescent protein expression only in active neurons either in culture neurons or in the cortices of an animal. In one of the articles (Lee et al.), investigators even proved that they can inhibit the lever-pressing behavior of mice taking advantage of inhibitory opsins expressed under the control of this calcium-dependent system.

Reading these articles can easily make researchers feel very excited because there would be so many research projects in which these calcium-dependent expression systems could make great contributions to. Also, we can imagine that maybe in one day there will be a voltage-dependent gene expression system that can be used to label and manipulate active neurons more precisely. I personally believe that day won’t be so far away since there’s already been a lot of studies on engineering voltage sensors like the Optopatch system [10].

More Information:
[1] Depolarization, hyperpolarization and action potential of a neuron:
[2] Neuronal calcium signaling:
[3] GCaMPs:
[4] A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.
[5] A calcium- and light-gated switch to induce gene expression in activated neurons. (A very fun video introducing this system)
[6] Brief introduction of stimulating the brain by opsins:
[7] The TEV protease:
[8] Transcription factors can control certain gene’s expression:
[9] Calmodulin:
[10] Optopatch:


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