In his 1941 book Man on His Nature, the Nobel Prize-winning physiologist Sir Charles Sherrington described the brain as “an enchanted loom where millions of flashing shuttles weave a dissolving pattern.” Little could he have known that within 50 years neuroscientists would have at their disposal techniques for visualizing this pattern.
These techniques are collectively known as calcium imaging. Developed in the 1980s, they use synthetic chemical dyes or genetic constructs whose spectral properties change when they bind calcium ions, leading to a change in fluoresence which can be detected under the microscope. These calcium sensors enable researchers to detect the tiny and transient increases in calcium ion concentration which are characteristic of neuronal activity.
Calcium imaging techniques are now used routinely but, although powerful, they have limitations. Now an international team of researchers have developed a genetically encoded calcium sensor which can be used to detect the transient calcium currents associated with a single nervous impulse, in live behaving animals and in real time. The experiments are reported in the September issue of the journal Nature Methods.
Calcium signalling is ubiquitous in nerve cells and mediates a large number of processes. An influx of calcium ions occurs in response to the arrival of a nervous impulse at the nerve terminal. This triggers the fusion of synaptic vesicles to the presynaptic membrane, so that they can release their cargo of neurotransmitter molecules. Within nerve cells, dozens of neuronal proteins are sensitive to discrete and highly localized “microdomains” of calcium, which either enters the cell from the chemical milieu outide, or is released from a number of intracellular calcium stores.
Traditionally, neuronal activity was recorded by impaling neurons with microelectrodes which detect the movements of ions across the membrane. This method was used in the 1950s by Alan Hodgkin and Andrew Huxley for their classical experiments which determined the ionic fluxes underlying the action potential, and is still widely used today. It is limited by the small number of cells from which recordings can be taken, and its highly invasive nature, which invariably damages the delicate nervous tissue being investigated.
Calcium indicators have their advantages over electrophysiology, but they too have their limitations. The first dyes to be developed were small synthetic molecules which were loaded into individual cells one at a time, a laborious and time-consuming process. These dyes could detect the calcium currents triggered by single action potentials, but could not be targeted to specific cells, and could only be used to monitor neuronal activity for periods of about a day. The first generation of genetically encoded dyes overcame some of these problems, as they could be delivered to target cells using cell-type specific genetic elements and could be expressed within those cells for longer periods of time. However, they were less sensitive than the synthetic dyes – they emit less fluoresscence and could not detect single action potentials.
The new calcium sensor exhibits the properties of both types, and so enables researchers to monitor the activity of specific cells for periods of weeks or months. It is based on a genetically encoded fluorescent calcium indicator protein developed recently by Amy Palmer and her colleagues at the University of Colorado, in collaboration with Roger Tsien‘s laboratory at the University of California in San Diego. The sensor, called D3cpv, consists of a calcium binding protein called calmodulin fused to yellow and cyan fluorescent proteins (YFP and CFP, respectively). Binding of calcium to calmodulin causes a conformational change which brings the two fluorescent proteins closer together, so that they are able to transfer small amounts of energy between each other. As a result of this energy transfer, the fluorescence emitted by YFP increases, wherease that emitted by CFP decreases.
D3cpv was generated by slight modifications to existing genetically encoded calcium indicators. The main advance in the new method is the way in which the D3cpv DNA construct was delivered into cells. Mazahir Hasan of the Max Planck Institute for Medical Reseach in Heidelberg, Germany inserted the construct into an engineered form of the adeno-associated virus, then injected it directly into the brains of live mice through a hole in the skull. The viral vector allowed for stable, long-term expression of the calcium sensor in infected cells. Hazan’s group targeted the somatosensory cortex his experimental animals. In rodents, a large proportion of the somatosensory cortex is devoted to processing tactile information detected by the whiskers. The cells are organized into structures called barrels, each of which receives inputs from a single whisker and is activated by mechanical stimulation of that whisker.
Hasan’s method allowed for simultaneous electrophysiological and optical detection of the increases in calcium ion concentration triggered by a single action potential. Individual whiskers in immobilzed were mechanically stimulated with brief puffs of air, leading to activity in somatosensory cortical cells in the corresponding whisker. The colour changes were observed using a type of two-photon laser scanning microscopy called fluorescence resonance energy transfer (FRET). This detects the coincidence of the two fluorescence changes at a very high spatial and temporal resolution, and was performed through the hole in the skull. At the same time, electrophysiological recordings from somatosensory cortical cells showed that the colour changes corresponded to individual action potentials in somatosensorycortical cells evoked by whisker deflection (above right).
Importantly, the viral vector led to calcium sensor expression that remained stable for months, and could potentially remain stable for the remainder of the animals’ lives. The method should therefore prove to be very useful for researchers investigating development of the nervous system and the changes that occur in neurodegenerative diseases. However, the sensor could only be used to detect activity at or below a frequency of 1 Hz (or 1 action potential per second). This is much lower than the brain’s “clock speed”: an individual neuron can fire up to 1,000 times per second. Furthermore, neuronal calcium signalling is highly promiscuous; at any moment in time, there are likely to be many transient calcium microdomains at different locations in the cell, each mediating a different molecular mechanism.
Current calcium imaging techniques, including this one, do not differentiate between these microdomains, but instead monitor global changes in calcium ion concentration. Future work is likely to lead to indicators which are sensitive to higher frequencies of neuronal activity, and possibly to indicators which are sensitive to small ranges of calcium concentration. Combinations of such ultra-sensitive indicators may one day be used to differentiate between multiple parallel microdomains. This would enable researchers to visualize more fully the dissolving pattern of neuronal activity and to make real progress in deciphering the calcium code.
Update: The Royal Swedish Academy of Sciences has just announced that Roger Tsien is to receive the 2008 Nobel Prize in Chemistry, for his contribution to “the discovery and development of the green fluorescent protein, GFP.” Tsien will share the Prize with Osamu Shimomura of the Marine Biological Laboratory in Woods Hole, Massachusetts, who first isolated GFP from the bioluminescent jellyfish Aequorea victoria in 1962, and Martin Chalfie of Columbia University, who was the first to demonstrate that GFP can be used as a genetic tag in biological systems.
I’ve written about numerous studies which use GFP in various ways. See, for example, yesterday’s post about how the prion protein enters cells, and this post from last year about the ingenious Brainbow technique, which uses a combination of four differently coloured fluorescent proteins to generate a palette of more than 100 colours to label cells and illuminate entire neuronal circuits. Tsien’s work on the properties of GFP was instrumental for the development of this and similar techniques. For more information, see this comprehensive document about the scientific background to the 2008 Nobel Prize in Chemistry, produced by the Nobel Foundation.
Wallace, D. J. et al (2008). Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat. Meth. 5: 797-804. DOI: 10.1038/NMETH.1242