"Calcium vs. Activity"

Three studies have been published recently using genetically encoded calcium indicators (GECI) probes in living mice to measure intracellular calcium concentrations. The question is: do these probes work? Well the answer depends what you mean by work. There is no doubt the impact that green fluorescent protein has had on biology. A very nice history of biofluorescence was published in 2005 ("A glow in the dark", by Pieribone & Gruber) for those who want the almost full history (Antony Persechini is not mentioned in the book index). However, the impact of the vast number of GEI is mixed, many only giving rise to one or two studies. Given the committment of the NIH to transgenic mice, there is the possibility that if "really good" GECI were developed, then many labs might actually use them.

First, Bacskai and co-workers used YC3.6 driven by adenovirus injected into Layer5 mouse cortex. In a lovely study, the Boston Alzheimer group measured resting calcium levels in normal mice and mice with various transgenically encoded human AD mutations. They found that near plaques a significant percentage of dendrites have elevated calcium. Normal resting calcium was 77 nM. This average increased to 499 nM in 20% of dendrites. Further, the compartmentalisation of calcium by spine heads was lost in AD mice. Some poor graduate student manually quantified 2977 spines! (By the way, I did not know that under resting conditions, calcium showed a gradient across the spine neck?)

Using a GECI called D3spv, again driven by injected adenovirus, Hassan and co-workers could reliably detect whisker deflections in the mouse cortex. And with a bit of signal averaging, they could even see single action potentials (though the S/N is somewhat frightening). For an excellent review of GECI see Mank & Greisbeck article in Chemical Reviews 2008.

Lastly, Griesbeck and co-workers have developed a new GECI called TN-XXL. They use in utero electroporation to deliver this into neurons. Remarkably, they can repeatedly image visual stimuli presented the same mouse over many days! They compare these calcium signals with those they have obtained from acute experiments with a calcium dye. Not surprisingly the calcium performs better. However, such dyes can only be injected once into the brain to give clear signals. Griesbeck and co-workers report that they can re-image the same neurons on day 1, 3, 5 and 19 through an implanted cranial window using TN-XXL.

All three of these studies are Herculean in nature, and the authors should be congratulated for working on such a difficult and important problem. Many of us would love to be able to buy a mouse and image calcium in vivo in our cell of choice. Let's hope these three papers are really getting us closer to that day. It must be noted that in other studies it has been reported that GECI do not seem to track intracellular neuronal calcium concentrations faithfully (Svoboda and co-workers). Though in heart muscle they might (Kotlikoff and co-workers). So do GECI monitor [calcium]? Not with kinetic fidelity. However, what about activity? Yes, it seems so. The future seems exciting.

Kuchibhotla, Goldman, Lattarulo, Wu, Hyman, Bacskai Neuron (2008) 59:214-225.

Wallace, Zum Alten Borgloh, Astori, Yang, Bausen, Kügler, Palmer, Tsien, Sprengel, Kerr, Denk, Hasan Nature Methods (2008) 5:797-804.

Mank, Santos, Direnberger, Mrsic-Flogel, Hofer, Stein, Hendel, Reiff, Levelt, Borst, Bonhoeffer, Hübener, Griesbeck Nature Methods (2008) 9:805-811.

Mao, O'Connor, Scheuss, Nakai, Svoboda Plos ONE (2008) 3:e1796.

Pologrut, Yasuda, Svoboda (2004) J Neurosci 24: 9572–9579.

Mank, Griesbeck Chem. Rev. (2008) 108:1550-1564.

Tallini, Ohkura, Choi Ji, Imoto, Doran, Lee, Plan, Wilson, Xin, Sanbe, Gulick, Mathai, Robbins, Salama, Nakai, Kotlikoff (2006) Proc Natl Acad Sci U S A 103: 4753–4758.

Romoser, Hinkle, Persechini J. Biol. Chem. 272, 13270–13274 (1997).

Baylor, Chandler, Marshall J. Physiol. (1982) 331:130-177. The picture is taken from this article. Still the most helpful discussion on dye kinetics.