The fastest calcium sensors ever are expanding the potential of neural imaging


• Genetically encoded calcium indicators (GECIs) are designer proteins that target intracellular calcium ions (Ca2+)Inflows – which occur during the activation of neurons – by the emission of light.

• GECIs have allowed researchers to gain countless insights into how the brain works, but until now, proteins have lacked the precision to record a basic unit of activity, called the action potential, in individual neurons.

• Registered mail NatureZhang et al.1 describe a GECI that represents a step towards solving this problem.

MICHAEL B RYAN & ANNE K CHURCHLAND: Lamps that illuminated the brain

Imagine rowing across still, dark waters on a summer night, when the water is suddenly transformed by the brilliant glow of myriad jellyfish, illuminating a hidden world of activity. In the 1960s, scientists at Friday Harbor Laboratories in Washington collected nearly 10,000 specimens Aequorea Jellyfish by hand with the aim of isolating the organic component responsible for this natural bioluminescence2. The scientists extracted and purified a calcium-sensitive protein they named aequorin along with a co-located green fluorescent protein (GFP). Soon after, the researchers loaded purified aequorin into living cells to monitor changes in Ca2+an ion that is central to neuronal activity3. This ability to measure neuronal activity vicariously – through flashes of light – revolutionized the field of neuroscience and led to the identification of previously unknown dynamics in the brain.

By injecting aequorin into neurons, the scientists discovered how changes in the concentrations of intracellular Ca2+that result from neuronal activation contribute to the process by which these cells release neurotransmitters to communicate with each other4. Several decades after aequorin was first isolated, another breakthrough came in the form of GECIs. While aequorin and other calcium-sensitive dyes must be manually loaded into cells, GECIs are engineered to become part of the organism’s DNA5,6. The most widely used GECIs, known as the GCaMP family7, consist of a calcium-binding protein called calmodulin and a attached peptide (a short chain of amino acids) fused to a modified form of GFP. As approx2+ When GFP binds to calmodulin, it triggers a conformational change in the fusion protein that causes GFP to emit light (Fig. 1a).

Figure 1 | Structure and response profile of GCaMP8 proteins. AZhang et al.1 have developed GCaMP8 – optimized forms of genetically engineered calcium indicators that respond to intracellular changes in calcium ion levels (Ca2+) by fluorescence. GCaMP8 proteins consist of a transmembrane calcium-binding domain (calmodulin) linked to a peptide (a fragment of endothelial nitric oxide synthase; ENOSP) that modifies the activity of calmodulin. Two linkers bind calmodulin to a modified form of green fluorescent protein (GFP). As approx2+ binds, a change in protein configuration leads to GFP fluorescence. BThe GCaMP8 sensors (8s and 8f) show several improved qualities compared to existing GCaMP sensors, including higher sensitivity (shown as a change in relative brightness from the protein’s baseline) and faster decay kinetics (a key response rate parameter, shown). as an inverse scale in 1/second).

Despite the potential of these calcium sensors, early versions of GCaMP were hampered by problems such as poor response kinetics, poor calcium sensitivity, and limited correlation with neuronal electrical activity. First versions of the sensors could only detect large changes in Ca2+ caused by dozens of action potentials with reaction kinetics on the order of several hundred milliseconds.

Since then, significant efforts have been made to improve the performance of GCaMP sensors through structure-guided design. Later versions of GCaMP were even optimized for sensitivity or speed, with “slow” variants maximizing signal strength and “fast” variants maximizing reaction kinetics. Fast variants of GCaMP can now detect changes in calcium in time windows of tens of milliseconds. Other GECIs have also been developed that emit different wavelengths of light in response to Ca2+whereby multiple populations of neurons can be imaged simultaneously.

These advances have allowed scientists to benefit from the versatility, specificity, and longevity of GECIs. The fact that they are genetically encoded allows them to be continuously expressed in many cells simultaneously. This made it easier in vivo Measurements of large cell populations in flies, rodents and primates. The cell type in which GECIs are expressed can also be genetically controlled, which has helped researchers understand the variability of neural circuits. For example, GECIs have been used to describe subpopulations of neurons in the cerebral cortex that have different calcium dynamics and roles in decision making8th. They have also been used to measure Ca2+ in non-neuronal cells in the brain. For example, calcium imaging of astrocytes — the most abundant non-neuronal cell type in the brain — has revealed how neurotransmitters such as dopamine shape astrocyte responses to neural activity9,10.

Nevertheless, the latest GCaMP iterations are not able to reliably identify individual action potentials under the “noisy” conditions in living brains. Several research groups have endeavored to identify GCaMP variants that can detect these changes – which occur within a few milliseconds – without affecting the brightness. The work of Zhang and colleagues introduces a competitor to this race and points to a bright future for calcium imaging.

YIYANG GONG & CASEY BAKER: Time for the next generation

Single action potentials are the basic unit of neural communication. The development of Zhang and colleagues is therefore a great step forward. The authors’ GCaMP8 sensors have significantly faster kinetics than existing latest-generation GECIs (Fig. 1b), such as the GCaMP7 and XCaMP series11,12. The GCaMP8 sensors also avoid the typical tradeoff between sensitivity and speed, allowing slower sensor kinetics to produce larger optical responses.

Zhang and colleagues based their sensors on GCaMP6. The authors optimized this protein by constructing different versions of several of its modules, including the two linkers connecting GFP to the bound peptide and the calmodulin domain, which attacks the peptide when Ca2+ binds. They paid particular attention to the interface between the bound peptide (which in GCaMP8 is a fragment of endothelial nitric oxide synthase) and the calmodulin domain, since this region of the sensor plays a crucial role in its response and kinetics12,13. The team then used an optimization process that involved swapping out many amino acids and running multiple rounds of testing to identify the best-performing sensors. They validated the performance of these indicators in flies and mice. The strong performance of the GCaMP8 sensors suggests that the way Ca2+ Fluorescence induced differs in this series from that in other GCaMPs.

The fast kinetics and high accuracy of the GCaMP8 sensors will enable researchers to analyze phenomena in living animals that previously could only be studied by electrical measurements or genetically encoded voltage indicators (GEVIs) that respond to voltage changes by fluorescence (similar to how GECIs responsive to calcium). Previously, it was thought that GECIs and GEVIs report different types of neuronal activity—GEVIs determine the timing of action potentials, and GECIs reveal key activity in neuronal compartments (e.g., by measuring Ca2+ Dynamics in processes called dendrites that receive signals from other neurons)14.

However, with the development of GCaMP8, the information that can be gleaned from these tools has converged. Through computational analysis of the light bursts triggered by GCaMP8, Zhang and colleagues show that their sensors can detect action potentials almost as accurately as voltage imaging15,16. Similarly, GCaMP8 could reliably detect when fly neurons, which normally fire, often become transiently inactive, and provide results similar to GEVI-based measurements17,18.

If both voltage and calcium imaging can be used to record many neurons simultaneously, practical experimental considerations could persuade future researchers to choose calcium imaging over voltage imaging. GCaMP8 is compatible with existing microscopy configurations and preparation methods. The series can be used to study the activity of broad neural networks on a larger scale than is possible with voltage imaging. One could imagine that the GCaMP8 series will soon be used to interpret detailed, millisecond-precise sequences of activity flowing through many neurons in a specific area of ​​the brain.

The fast kinetics associated with this generation of GCaMPs, along with existing GEVIs, will motivate the development of fast optical microscopy and imaging technologies. Such microscopes would be able to detect transient flashes of light emitted by many neurons simultaneously. But for now, the fact that calcium sensor kinetics are no longer the bottleneck in interpreting rapid neuronal activity is welcomed by many neuroscientists.

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