Caltech Uses Fluorescent Protein to Visualize the Work of Living Neurons
Erin M. Schuman, an associate professor of biology at Caltech and an assistant investigator with the Howard Hughes Medical Institute, along with colleagues Girish Aakalu, Bryan Smith, Nhien Nguyen, and Changan Jiang, published their findings last month in the journal Neuron. Proving that protein synthesis does indeed occur in intact dendrites suggests the dendrites may also have the capacity to adjust the strength of connections between neurons. That in turn implies they may influence vital neural activities such as learning and memory.
Schuman and colleagues constructed a so-called "reporter" molecule that, when introduced into neurons, emits a telltale glow if protein synthesis is occurring. "There was early evidence that protein-synthesis machinery was present in dendrites," says Schuman. "Those findings were intriguing because they implied that dendrites had the capacity to make their own proteins."
The idea that dendrites should be able to synthesize proteins made sense to Schuman and others because it was more economical and efficient. "It's like the difference between centralized and distributed freight shipping," she says. "With central shipping, you need a huge number of trucks that drive all over town, moving freight from a central factory. But with distributed shipping, you have multiple distribution centers that serve local populations, with far less transport involved."
Previous studies had indicated that, in test tubes, tiny fragments of dendrites still had the capacity to synthesize proteins. Schuman and her colleagues believed that visualizing local protein synthesis in living neurons would provide a more compelling picture than was currently available.
The scientists began their efforts to create a reporter molecule by flanking a gene for a green fluorescent protein with two segments of another gene for a particular enzyme. Doing this ensured that the researchers would target the messenger RNA (mRNA) for their reporter molecule to dendrites.
Next, in a series of experiments, the group inserted the reporter molecule into rat neurons in culture, and then triggered protein synthesis using a growth factor called BDNF. By imaging the neurons over time, the investigators showed that the green fluorescent protein was expressed in the dendrites following BDNF treatment—proof that protein synthesis was taking place. Going a step further, the researchers showed they could cause the fluorescence to disappear by treating the neurons with a drug that blocked protein synthesis.
Schuman and her colleagues also addressed whether proteins synthesized in the main cell body, called the soma, could have diffused to the dendrites, rather than the dendrites themselves performing the protein synthesis. The researchers proved the proteins weren't coming from the soma by simply snipping the dendrites from the neurons, while maintaining their connection to their synaptic partners. Sure enough, the isolated dendrites still exhibited protein synthesis.
Intriguingly, says Schuman, hot spots of protein synthesis were observed within the dendrites. By tracking the location of the fluorescent signal over time, the researchers could see that these hotspots waxed and waned consistently in the same place. "The main attraction of local protein synthesis is that it could endow synapses with the capacity to make synapse-specific changes, which is a key property of information-storing systems," says Schuman. "The observation of such hot spots suggests there are localized areas of protein synthesis near synapses that may provide new proteins to synapses nearby."
Schuman and her colleagues are now applying their reporter molecule system to more complex brain slices and whole mice. "In the whole animals, we're exploring the role of dendritic protein synthesis in information processing and animal learning and behavior," says Schuman.