In vivo electroporation of calcium dyes

19 03 2007

Hot on the heels of the recent report on optimized in vivo imaging of calcium using genetically-encoded calcium indicators, Nagayama et al. report a novel neurotechnique for calcium imaging with dyes in vivo. See also Helmchen and Nevian’s preview. Previously, there were three major methods of imaging intracellular calcium dynamics in vivo.

Single cell loading of calcium dye through a patch or sharp electrode
This method results in high dye concentrations and vivid contrast. However, this limits the imaging to single cells and it can be difficult to hold the cell for sufficient time to fully fill the cell with dye.

Bulk loading of AM-ester forms of organic calcium dyes by perfusion
This produces a stain which includes many cells, both neurons and astrocytes. Although this is effective for observing population activity, the breadth of this staining prevents resolution of individual neuronal processes.

Genetically-encoded calcium indicators

These provide the greatest level of cell-type specificity. However, the best GECIs still have reduced response magnitude and poorer bleach resistance than the best calcium dyes. Furthermore, generation of mouse lines is time consuming.

In the current study, the authors report a clever method of loading calcium dyes in vivo. They insert a thin pipette filled with dye into the brain region of interest and then perform whole animal electroporation by repeatedly applying brief, very weak current pulses to the anesthetized mouse’s tail. The current pulses drive the dye out of the pipette and also transiently disrupt the cell membranes around the pipette tip. After 10 minutes of stimulation and an hour of diffusion, sufficient dye has passed into the neighboring neurons to fill them with ~20uM dye. Field stimulation before and after electroporation indicates there is little to no damage to the brain microcircuitry. The labeling is selective to neurons and produces a sparse distribution with a spatial extent dependent on circuit morphology. It appears that cells with fine processes within 10-20um of the pipette tip take up dye which then diffuses into cell bodies distal to the loading zone.

The authors demonstrate that this sparse labeling is sufficient to resolve clear calcium responses to electrical stimulation in single identified cells. They even show responses, albeit noisy ones, in some individual synaptic boutons with trial based averaging (Fig. 6). In the mitral cells of the olfactory bulb, they see cell specific calcium transients to odor presentation (Fig 7, 8). However, their claim to detect “sharpening of odor-tuning curve from the glomerular layer down to the mitral cell soma layer” seems a bit premature given the challenging signal to noise ratio that could be obscuring slightly smaller calcium transients in the soma (Fig 7B). When loaded into the barrel cortex, they impressively resolve calcium transients in pyramidal cell bodies from a presumably 1 or 2 AP natural stimulus of whisker deflection (Fig 8A).

Electroporation of calcium dyes has a number of clear advantages over other in vivo calcium imaging techniques and might be the best solution available today. The sparse, high contrast labeling allows tracing of each neuron’s processes, imaging of subcellular compartments, and network level analysis. The technique allows the use of a large number of extant dyes with a variety of colors and kinetic properties, and the response levels approach that of intracellular injection. However, the technique does not allow genetic targeting of the indicator to specific cell types or intracellular compartments, although Nevian and Helmchen also recently reported in vivo electroporation of single targeted neurons. It remains to be seen whether improved GECIs, electroporation or a yet undeveloped technique will become the preferred solution for optical recording of neuronal activity in in vivo mammalian systems.


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