D45 Imaging of neural activity

Topic

This neural activity imaging—specifically applied to the insect serving as the model system—generally employs a genetically encoded fluorescent indicator sensitive to intracellular calcium within each specific neuron; this is coupled with two-photon or wide-field microscopy, enabling the observation of, for instance, a specialized neuron in the lobula system of a fly (another distinct model in this set).

When the insect's neuron fires an action potential—as measured by direct electrophysiology—free calcium enters through the neuron's voltage-gated channels; the resulting fluorescence of the indicator changes in proportion to this calcium influx, thereby providing an indirect readout of the neuron's electrical activity, in contrast to the direct electrophysiology mentioned earlier.

Normalizing the final fluorescence signal against the baseline fluorescence eliminates variations caused by external illumination or differences in indicator expression levels between neurons.

The indicator's final calibration curve converts the fluorescence signal into a concentration of free calcium, following the indicator's binding kinetics as described by the Michaelis-Menten equation. As the concentration of free calcium within the neuron rises further, the indicator's fluorescence intensity also increases, eventually approaching saturation. The temporal resolution of this imaging technique is considerably slower than the actual electrical activity of the neuron—specifically, the action potential—meaning the influx of calcium into the neuron is much slower than the action potential itself; this limits the imaging's temporal discrimination relative to the cutoff frequency associated with a receptor's temporal response. Consequently, the use of a calcium-sensitive fluorescent indicator means that the system's Abbe spatial resolution—defined by diffraction limits—determines how many individual neurons within the fly's lobula system can be resolved. Independent component analysis (ICA) separates the spectrally overlapping activity of individual neurons, thereby extracting the specific activity of each independent population from the raw imaging signal. Achieving finer spatial resolution allows the independent component analysis to successfully disentangle an even greater number of these overlapping neurons.

ID:2592