Two new papers came out last month in Nature Methods, with each describing a new Ca2+ sensor that works in vivo, and with one able to detect single action potentials. This vastly improves our ability to conduct studies employing Ca2+ imaging.

Why is Ca2+ imaging cool, at least for neuroscientists? Well, after a few years of published work by several different labs, many are now comfortable with equating a fast rise in intracellular Ca2+ with spiking activity (ideally/critically, one should know the relationship between the amplitude of the Ca2+ indicator fluorescence change and the number of action potentials). This strategy thus provides an exciting option to simultaneously visualize the activity of many cells. Electrical techniques utilizing extracellular recording methods collect simultaneous information from only a few cells (at best), and importantly, provide no spatial information regarding the location of the cells (like in relation to one another).

With these new indicators, we may soon see studies combining the strength of ensemble visualization with complex behavior.

The state of the art for imaging cell ensemble activity in vivo has been bulk-loading of Ca2+ dyes, as described here. Although a powerful technique, this bulk-loading method does not inherently allow for the identification of the cells being imaged, only provides a short window of time for data collection before the dyes are extruded from the cells (a few hours at best), and precludes chronic imaging over days. The new genetically-encoded sensors (mostly) fix these problems. Being genetically-encoded, these indicators now provide the luxury of persistently labeling a known targeted set of cells that can be imaged for days non-invasively (well, after surgery is completed to install a window…). Other genetically-coded indicators have been around (Camgaroo, GCaMp, and Cameleon as examples), but none of these worked in vivo and even in reduced preparations, could not discern single action potentials. So these new tools are definitely an improvement.

The first paper is from Hasan and colleagues, which utilizes a FRET-based sensor and ratiometric imaging of YFP and CFP fluorescence. The nice thing about using an energy transfer probe in vivo is that it helps to eliminate motion artifacts that would arise from simply imaging raw single-color fluorescence. The sensor is an updated version of the Cameleon sensor (called D3cpv). High expression levels are key to providing great signals, which the authors achieved by using viral-based vectors. The paper goes on to describe their ability to detect single action potentials in vivo in response to sensory stimulation (they were imaging in somatosensory cortex, so they looked at the response to whisker deflections), although the change in signal amplitude was not as robust as observed in vitro.

Griesbeck and colleagues used a sensor based on the troponin C molecule that also involved ratiometric imaging. Although this group did not detect single action potentials, they had the added wrinkle of being able to monitor the same population of V1 neurons responding to visual stimuli over the course of days. They were able to observe stable orientation tuning of cells over days using Ca2+ imaging, which is a remarkable feat.

Despite these significant advances, there are still plenty of problems. In a N&V feature describing the new papers, Rochefort and Konnerth summed it up best with their “wish list”:

The neuroscientist’s wish list, when it comes to dreaming of new GECIs, includes the design and construction of sensors that (i) have an improved signal-to-noise ratio, (ii) have different affinities for Ca2+ and faster kinetics, (iii) have different emission spectra, allowing the simultaneous imaging of different types of neurons, and (iv) are appropriate and efficient for imaging Ca2+ in organelles, such as the endoplasmatic reticulum, and in subcellular structures, such as spines or axon terminals.

A tall order, but given the rapid progress, wishful thinking may soon give way to reality.

Wallace, D., zum Alten Borgloh, S., Astori, S., Yang, Y., Bausen, M., Kügler, S., Palmer, A., Tsien, R., Sprengel, R., Kerr, J., Denk, W., & Hasan, M. (2008). Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor Nature Methods, 5 (9), 797-804 DOI: 10.1038/nmeth.1242

Mank, M., Santos, A., Direnberger, S., Mrsic-Flogel, T., Hofer, S., Stein, V., Hendel, T., Reiff, D., Levelt, C., Borst, A., Bonhoeffer, T., Hübener, M., & Griesbeck, O. (2008). A genetically encoded calcium indicator for chronic in vivo two-photon imaging Nature Methods, 5 (9), 805-811 DOI: 10.1038/nmeth.1243