Neuroblastoma Research, 1967-1976

Marshall Nirenberg's curiosity was piqued by neuroblastomas, which are malignant tumors composed of developing neurons. Fully developed neurons differentiate into specific cells to perform specific tasks, so scientists cannot use the adult neurons to study neural development because they no longer divide. Nirenberg became interested in neuroblastomas because he thought the tumor neurons might retain the properties of undifferentiated neurons. He could then use them as a model system to study the development of neurons. The neuroblastoma system also offered Nirenberg an opportunity to study neurotransmitters in the brain. Neural information is communicated between neurons by way of chemical neurotransmitters such as norepinephrine and dopamine. Studying neurotransmitter synthesis with the neuroblastoma system enabled Nirenberg to investigate the details of information processing in the nervous system in much the same way that the study of protein synthesis enabled him to explore the details of information processing in the genetic system.

In the 1960s, scientists typically studied neuroblastomas only to fight the cancerous cell growths, which most often afflict infants and young children. Nirenberg made an innovative move by using the tumor cells towards a different end--he wanted to study the formation of neurons in tissue culture. Tissue cultures are small amounts of undifferentiated tissues or single cells grown in an artificial environment such as a petri dish. Nirenberg's study of neuroblastoma was one of the first times neurobiologists used tissue cultures, an experimental method that now pervades the field. The innovative use of tissue cultures forced Nirenberg to learn new experimental methods. "I had never done any tissue culture research before," Nirenberg remembered in a 2001 interview, "I just jumped in with feet, hands, and everything." Nirenberg enlisted the help of a fellow colleague at the NIH, Dr. Phillip Nelson, an expert neurophysiologist who taught Nirenberg the new methods needed to analyze the neuroblastoma tissue cultures. In turn, Nirenberg taught Nelson the biology needed to study the molecular components of the research.

Nirenberg and Nelson's combined approach to tissue cultures allowed for the neuroblastoma cells to develop in vitro. With the in vitro method neurobiologists could remove the neuroblastoma cells from the original tumor, grow the cells on petri dishes, and then study them with a microscope while they were still developing. The neurons could then be observed and studied in this isolated state. As Nirenberg and Nelson mastered the neuroblastoma system, they were able to clone and grow tumor cells based on the neural properties of the neuroblastomas, thereby creating cell lines of tissue cultures with desired characteristics. Nirenberg could develop specific cell lines based on characteristic traits such as how fast the neurons grew, how receptive the neurons were to morphine, or how the neurons synthesized a given neurotransmitter. Nirenberg and Nelson farmed a variety of different cell lines and even created a cell bank to store the various strains. For decades, Nirenberg's lab furnished samples of these cell lines to scientists all over the world.

The cell lines were used for multiple projects. For example, Nirenberg, with the help of post-doctoral fellow Xandra O. Breakefield, developed an experimental method that could distinguish neurons from the neuroblastoma system based on their abilities to synthesize different neurotransmitters. This method let Nirenberg and Breakefield classify the various cell lines formed from neuroblastomas even more precisely. Although Breakefield worked with Nirenberg at the NIH in the 1970s, this formal collaboration was not their first encounter. Breakefield initially contacted Nirenberg in 1962 when she was a sophomore at Wilson College in Chambersburg, Pennsylvania. Interested in biology, Breakefield sought career advice from the biochemist who had just deciphered the first word of the genetic code. Breakefield even visited Nirenberg's laboratory in 1962 to watch science in action. A decade later, Breakefield joined Nirenberg's laboratory as a post-doctoral fellow.

In the early 1970s, Nirenberg also utilized the neuroblastoma system to study the effects of morphine on the nervous system. Werner Klee, a biochemist at the National Institute of Mental Health at NIH, was interested in the chemical effects of morphine on the brain. Nirenberg and Klee developed a neuroblastoma cell line that possessed an unusually high percentage of morphine receptors. They found that morphine inhibits the production of an enzyme in the brain called adenylate cyclase, which promotes the construction of complex molecules vital to neural transmission. To counterbalance the reduction of adenylate cyclase, Nirenberg and Klee discovered, the brain then overproduces the chemical. If the morphine stimulation is removed, the increased level of adenylate cyclase production causes the brain to be flooded with this chemical. Nirenberg and Klee found that an individual who experiences this chemical imbalance goes through a withdrawal period as the nervous system slowly returns the adenylate cyclase production to its proper level.

Nirenberg studied neuroblastomas for more than a decade. The model system offered him a versatile tool to explore the intricate details of the nervous system. Most importantly, the neuroblastoma system allowed Nirenberg to use many of the conceptual and methodological practices he developed from biochemistry and molecular biology in a completely new field of research.