Like a typical cell membrane peppered with transmembrane proteins of the voltage, ligand, stretch or g-coupled varieties, pond scum possess a unique light gated channel.This photo-sensitive, g-coupled receptor present in the single-celled green algae is thought to serve a motile purpose; to move algae closer to light. After their discovery, scientists hypothesized introducing these proteins into membranes of various cell types. But, what benefit would this bring to biological research and neuroscience specifically?

A fundamental obstacle in current neuroscience research is providing a single stimulus to a specific neuron or neuronal population. Traditionally, the image of Wilder Penfield electrically probing a conscious patient to observe their response may come to mind. Likewise in Deep Brain Stimulation, the precise surgical implantation of a battery-powered neurostimulator is shown to reduce the involuntary movements of neurodegenerative diseases by electrically stimulating targeted areas of the brain.

Unfortunately, this level of stimuli specificity still fails to meet the needs of modern research. Photo-sensitiveproteins introduced into a cell membrane via genetic engineering may come close to filling this need. Similar to the functions of other gated channels, the photo-sensitive proteins on even a single cell can control the inherent functions of the cell. The significance of this is extensive, as control of nonlight-sensitive channels for research is limited. With a stimulus as simple as a specific wavelength of light, a cell of a variety of types may be activated, or even, inactivated. In neuroscience, this level of control allows for explicit regulation of a specific cell type, at a specific time and during specific biochemical events.

      

Blue light-activated channelrhodopsin and yellow light-activated halorhodopsin.

Our focus is to optogenetically activate or inhibit particular neurons implanted with the light-sensitive proteins in an attempt to further navigate the neural circuits, specifically, those relevant to pain and itch.

Further use of optogenetics in neuroscience is applicable across a diverse number of research fields. Use in controlling channels moving ions in both directions would be expected to play an immense role in physiological effects. In situations of disease, an understanding of the particular neural circuits would provide insight into the pathological mechanisms and allow for a type of “reverse engineering”. Integration of these proteins with mechanisms such as DHP/Ca2+ may also be used as a “measurement” device.

With optogenetics, a shift from a neurotransmitter based understanding of the brain to a circuit based understanding is plausible.

Resources:

• Optogenetics Wikipedia
• Stanford Optogenetics Resource Center
• Nature Method of the Year 2101 Video
• OpenOptogenetics
• Ed Boyden Optogenetics TED Talk
• Gero Miesenboeck Optogenetics TED Talk

Live imaging of slice cultures using the spectral detection to do FRET, courtesy of Zak Wills.

Coming soon, we will use the ultra-fast confocal to do calcium imaging of optogenetically modified spinal neurons.

To learn more about confocal microscopy, visit:
Fundamental Concepts in Confocal Microscopy
Fluorescence Resonance Energy Transfer (FRET) Microscopy
Nikon Confocal Microscopy Image Gallery

Over the last 10-weeks, Kathryn engaged in two research projects. The first project involved testing the involvement of kappa opioids in the regulation of itch, and Kathryn mastered doing some behavioral experiments in mice. The second project stemmed from an observation Kathryn made while in the lab. She discovered that residues that are phosphorylated on Olig1 are conserved on Bhlhb5. This similarity suggested that Bhlhb5 might be likewise regulated by phosphorylation, and Kathryn designed and executed experiments to test this hypothesis using site-directed mutagenesis and immunohistochemistry. Way to go Kathryn!

Kathryn hard at work.

As a fellowship recipient, Kathryn was awarded a $3,500 stipend from the Center for Neuroscience at the University of Pittsburgh to participate in 10-weeks of intensive and challenging laboratory-based research. More information about this program, as well as how to apply, can be found at: CNUP Summer Research Program