Researchers at MIT have developed a calcium indicator, or sensor, that can overcome one of the main problems associated with a technique that is used to visualize, or map the activity of individual neurons in the brain. Unlike existing calcium sensors used for one-photon fluorescent imaging, the new molecule accumulates only in the body of a neuron, and so reduces background noise resulting from crosstalk from the axons and dendrites of neighboring neurons.

“People are using calcium indicators for monitoring neural activity in many parts of the brain,” said Edward Boyden, PhD, the Y. Eva Tan professor in neurotechnology and a professor of biological engineering and of brain and cognitive sciences at MIT. “Now they can get better results, obtaining more accurate neural recordings that are less contaminated by crosstalk.”

Boyden is senior author of the team’s published paper in Neuron, which is titled, “Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator.” The paper’s lead authors are research scientists Or Shemesh, PhD, Changyang Linghu, PhD, and Kiryl Piatkevich, PhD.

When neurons fire an electrical impulse, they also experience a surge of calcium ions. By measuring those surges, researchers can indirectly monitor neuron activity, helping them to study the role of individual neurons in many different brain functions. “Methods for one-photon fluorescent imaging of calcium dynamics are popular for neural activity mapping in the living brain,” the team explained. “These techniques capture, at high speeds (e.g., >20 Hz), the dynamics of hundreds of neurons across large fields of view at a low equipment complexity and cost.”

However, one drawback of this technique is that crosstalk generated by the axons and dendrites that extend from neighboring neurons generates background noise that makes it harder to get a distinctive signal from the neuron being studied. “Neuroscientists often focus on analyzing data from the cell bodies of neurons being imaged, but these signals are contaminated by those from closely passing axons and dendrites,” the researchers continued.

MIT engineers have now developed a way to overcome that issue, by creating calcium indicators, or sensors, that accumulate only in the body of a neuron. To achieve this, the researchers fused a commonly used calcium indicator called GCaMP to a short peptide that targets it to the cell body. The new molecule, which the researchers call SomaGCaMP, can be easily incorporated into existing workflows for calcium imaging, they suggested.

The GCaMP calcium indicator consists of a fluorescent protein attached to a calcium-binding protein called calmodulin, and a calmodulin-binding protein called M13 peptide. GCaMP fluoresces when it binds to calcium ions in the brain, allowing researchers to indirectly measure neuron activity. “Calcium is easy to image because it goes from a very low concentration inside the cell to a very high concentration when a neuron is active,” noted Boyden, who is also a member of MIT’s McGovern Institute for Brain Research, Media Lab, and Koch Institute for Integrative Cancer Research.

The simplest way to detect these fluorescent signals is using one-photon microscopy, a relatively inexpensive imaging technique that can image large brain samples at high speed. However, the GCaMP goes into all parts of a neuron, which is why signals from the axons of one neuron can appear as if they are coming from the cell body of a neighbor, making the signal less accurate. A more expensive technique called two-photon microscopy can partly overcome this by focusing light very narrowly onto individual neurons, but this approach requires specialized equipment and is also slower.

Boyden’s lab decided to take a different approach to overcoming the problems of one-photon microscopy, by modifying the indicator itself, rather than the imaging equipment. “We thought, rather than optically focusing light, what if we molecularly focused the indicator?” he said. “A lot of people use hardware, such as two-photon microscopes, to clean up the imaging. We’re trying to build a molecular version of what other people do with hardware.”

Boyden and his colleagues had previously reported on the use of a similar approach to reduce crosstalk between fluorescent probes that directly image neurons’ membrane voltage. In parallel, they decided to try a similar approach with calcium imaging, which is a more widely used technique. To target GCaMP exclusively to cell bodies of neurons, the researchers tried fusing GCaMP to many different proteins. Working with MIT biology professor Amy Keating, PhD, the investigators explored two types of candidates—naturally occurring proteins that are known to accumulate in the cell body, and human-designed peptides. These synthetic proteins are coiled-coil proteins, which have a distinctive structure in which multiple helices of the proteins coil together.

The researchers screened about 30 candidates in neurons grown in lab dishes, and then chose two, one artificial coiled-coil, and one naturally occurring peptide, to test in animals. Working with Misha Ahrens, PhD, who studies zebrafish at the Janelia Research Campus, they found that both proteins offered significant improvements over the original version of GCaMP. The signal-to-noise ratio increased, and activity between adjacent neurons showed reduced correlation.

Through subsequent studies in mice, performed in the lab of Xue Han, PhD, at Boston University, the researchers also found that the new indicators reduced the correlations between activity of neighboring neurons. Additional studies using a microendoscope, which they performed in the lab of Kay Tye, PhD, at the Salk Institute for Biological Studies, revealed a significant increase in signal-to-noise ratio with the new indicators. “Our new indicator makes the signals more accurate,” Boyden commented. “This suggests that the signals that people are measuring with regular GCaMP could include crosstalk. There’s the possibility of artifactual synchrony between the cells.”

Using a new calcium indicator that accumulates in the cell bodies of neurons (boxes at right), MIT neuroscientists are able to more accurately image neuron activity. Traditional calcium indicators (boxes at left) can generate crosstalk that blurs the images. [Howard Gritton, Boston University]
The authors noted, “We observed decreased crosstalk, as reflected by lower numbers of artifactual (e.g., not detectable via patch pipette) spikes, and reduced artifactual correlation between neurons that are nearby in both zebrafish and mouse brains.”

The animal studies all indicated that the artificial, coiled-coil protein produced a brighter signal than the naturally occurring peptide tested. Boyden acknowledges that it is not clear why the coiled-coil proteins work so well, but one possibility is that they bind to each other, making them less likely to travel very far within the cell.

“… we explored de novo designed coiled-coil proteins that self-assemble into complexes, hypothesizing that their mutual binding could potentially slow their diffusion from the cell body,” the scientists wrote. “In intact brain circuits of larval zebrafish and mice, such soma-targeted GCaMPs greatly reduced the number of neuropil contamination spikes mistakenly attributed to a given neural cell body and reduced artifactual correlations between nearby neurons.”

Boyden hopes to use the new molecules to try to image the entire brains of small animals such as worms and fish, and his lab is also making the new indicators available to any researchers who want to use them. “It should be very easy to implement, and in fact, many groups are already using it,” Boyden stated. “They can just use the regular microscopes that they already are using for calcium imaging, but instead of using the regular GCaMP molecule, they can substitute our new version.”

“Having fewer artifactual spikes will increase the accuracy of the assessment of neural codes in the living brain,” the authors stated. “Reducing artifactual correlation may also help with studies of functional connectivity.” They concluded: “The advantage of SomaGCaMP in performing single-photon imaging in these model systems is that they may enable separation of bonafide physiological correlation from non-physiological correlation, something that post hoc computational methods cannot guarantee.”

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