Investigating Deep Brain Stimulation (Continued)
So the second thing the researches wanted to test was whether the electrical stimulation was stimulating glial cells to secrete inhibitory chemicals. To do this they introduced channel rhodopsin (this protein activates cells wheras the halorhodopsin inhibits them) using viruses but put it under a glia-specific promoter so instead of excitatory cells expressing the protein it was the surrounding glial cells. Here are the results:
With all studies like this the researchers must include histological verification that the proteins they’re trying to express are, indeed, being expressed, and in the right places. Here we see a stain for GFAP (a glial-specific protein) in green and the channel rhdopsin in red. In the overlay it is clear that there is strong colocalization. In short, the protein is where it should be. It’s very important to check but fairly repetitive and uninteresting so I’ll neglect it in the next few figures.
The proteins are activating proteins so you might expect to see an increase in spiking, but… we don’t. We don’t because the cells creating those large spikes are the neurons, and the neurons were not being activated by the light. Instead the light was activating the glia which secreted chemicals that inhibited the surrounding neurons resulting in an overall decrease in firing.
This treatment had no effect on parkinson’s symptoms.
The researchers found that causing the excitatory cells in the STN to fire at normal rhythms (by supplying light pulses to transfected cells at those normal rhythms) also had no therapeutic effect.
At this point the researchers take a moment to prove that the light they’re supplying is really hitting the places they think it is. They take small samples of brain tissue and simply shine their lasers through it, measuring the intensity at various distances. The light-activated proteins being used require a light intensity of about one mW/ square mm.
The top graph shows that the two colors of light being used each are sufficiently intense after passing through up to 1.5 mm of tissue. Below shows what cells have been activated by the light. Neurons produce the protein c-fos when they are activated which can later be stained for. By measuring how far from the epicenter of the light application c-fos appears the researchers can determine what volume of tissue could have its light-sensitive proteins activated. It turned out that about 1 cubic millimeter of tissue received bright enough light. The viral injections that introduce the DNA for these proteins affect about one cubic millimeter, so any infected cell can be successfully activated by this light application technique.
Up next: an optogenetic interference that had therapeutic effect. Stay tuned!