Affiliations

Optophysiology


We are taking advantage of several recent technical breakthroughs, which combine genetics and optical imaging. By inducing the expression of specific proteins, which respond to light activation in different manners (genetically-encoded optophysiological probes) we can monitor synaptic activity and/or control neuronal activity using light in a relatively non-invasive manner. We are also exploring new methods and uses for these tools. In the coming decade(s), there is little doubt the use of optogenetic tools will change the face of biology.

One probe synaptopHluorin (spH) permits the study of synaptic vesicle exo/endocytosis at individual synapses by reporting changes in pH. An illustration of the mechanisms by which spH reports synaptic vesicle exocytosis is shown below left. An image of a brain section from a thy-1-spH mouse and data traces reporting synaptic vesicle exocytosis obtained from neurons cultured from such a mouse are also shown below.

























































By clicking on the image at left a time-lapsed series of optical images will be played. The movie at illustrates a synaptopHluorin response obtained from a slice of hippocampus from a genetically-modified mouse, which expresses spH under control of a thy-1 promoter. The green channel corresponds to spH signals in response to Schaffer collateral stimulation while the red channel corresponds to a length of CA1 apical dendrite from a cell whole-cell voltage clamped and filled with Alexa-594.




Other genetically-encoded optophysiological probes are capable of reporting other aspects of neuronal activity. Some of these probes, such as G-CaMP report calcium transients. To view calcium transients in cultured neurons transfected with G-CaMP click on the image at right.




 
Even other genetically-encoded probes relying on interactions with light (often referred to as "optogenetics") confer millisecond control over neuronal activity with unprecedented spatial scales offering control of single synapses. One such probe known as Channelrhodopsin-2 is activated by "blue" light and is being employed in our lab towards efforts of stimulating ChR2 neurons using organic light emitting diodes (OLEDs). The basic aim is to conduct feasibility studies of engineering novel neural interfaces providing sensory feedback for smart prosthetics. Data from some initial feasibility experiments designed to determine if OLEDs can be used for neuronal stimulation of ChR2+ neurons in brain slices are shown at left. Optogenetic probes for controlling neuronal activity are emerging with different photo-excitation characteristics. For example NpHR (Halorhodopsin) enables the inhibition of neurons using "yellow" light. Even other light-gated ion channels and transporters are rapidly emerging, which are activated using multi-spectral wavelengths of light. Thus, we have begun to build custom brain slice chambers with OLEDs, which may offer the photo-dissection of brain circuits using these different probes simultaneously. Examples of such OLED photo-stimulation/inhibition chambers are shown below. We continue to evaluate a variety of OLED materials and designs in the optimization of our studies.