Dentate Gyrus Gating Function Captured by VSD

Voltage sensitive dye imaging (VSDI) monitors neuronal activity though use of voltage-sensitive fluorescent or non-fluorescent dye molecules that are inserted into the cell membrane. Changes in membrane potential are in essence changes in the electrical environment in which the voltage sensitive dye (VSD) resides (the membrane), and elicit voltage-dependent shifts in the absorption spectra and/or shifts in the fluorescence emission spectra of a VSD. One can monitor AP firing and synaptic transmission using this technique. Optical acquisition systems in VSDI frequently have high temporal resolution exceeding 1 kHz frame rates, a significant strength of this form of imaging. The most commonly used VSD imaging method is wide-field microscopy combined with image acquisition using a fast CCD camera. Due to this high temporal resolution, the CCD camera must be sensitive enough to detect low light levels since the amount of light captured during a 1 ms or shorter epoch (one frame) is limited. The CCD camera also has to have a high readout speed, a low dark noise and a low readout noise. In VSD experiments in our laboratory, we use a low noise back-thinned CCD camera that has a smaller number of pixels (80x80 pixels), achieving an image acquisition rate of 2 kHz. Conventional laser scanning confocal microscopy or multi-photon microscopy is not suitable for VSD imaging of neuronal circuit function due to slow image acquisition rate (typically 1-10 Hz). Alternatively, multi-beam scanning devices such as a spinning disk microscope, a swept-field microscope, and a multi-beam multi-photon microscope are, in principle, sufficiently fast to allow VSD imaging.

Most VSDs are lipophilic, and so can be bulk-loaded by applying a dye solution to the tissue. The bulk-loading method is suitable for monitoring network activity with a low magnification objective lens. However, in this form of recording, it is hard to identify single cell morphology or dendrites of a particular cell in bulk-loaded slice tissue. Many VSDs also suffer from a low signal to noise ratio. Because changes in VSD signal responses to neuronal activity could be as small as 0.1%-1%, event-averaging is often utilized to enhance the signal to noise ratio. Event averaging usually must be accompanied by triggered stimulation, such as a certain frequency of electrical pulse train, although in some cases, spontaneous events can be averaged post-hoc following an experiment based on event detection algorithms.

VSDs can also be internally loaded into neurons through a patch pipette in a conventional electrophysiology setup. With this loading method, one can study dynamic properties of synaptic transmission within a single neuron. In some cases, neuronal connectivity can be studied entirely using optical approaches by combining VSD imaging with a photolytic uncaging system. Recently genetically encoded voltage sensitive probes have been successfully developed. With this new technology, one would be able to characterize activity of a specific neuron at a high magnification and entire network activity at a low magnification within the same system.