But to truly confirm that the two brain areas didn’t evolve from the same ancestral source, Tosches and her team realized they needed to know more about how the neural cell types in mammals and reptiles might compare to the neurons in an ancient common ancestor.
They decided to look for clues in the brain of a salamander called the sharp-ribbed newt. (It takes its name from its ability to push its ribs out through its skin to poison and impale predators.) Salamanders are amphibians, which split away from the lineage they shared with mammals and reptiles about 30 million years after the first four-legged animals wandered onto land and millions of years before the mammals and reptiles split from each other. Like all vertebrates, salamanders have a structure called a pallium that sits near the front of the brain. If salamanders had neurons in their pallium that were similar to neurons in the mammalian neocortex or the reptilian DVR, then those neurons must have existed in an ancient ancestor that all three groups of animals shared.
Starting Over With the Neocortex
In their 2022 paper, Tosches’ lab performed single-cell RNA sequencing on thousands of salamander brain cells and compared the results to data collected previously from reptiles and mammals. Tiny salamander brains, each about one-fiftieth the volume of a mouse brain, were painstakingly prepared and labeled by the researchers. The brains were then put into a machine about the size of a shoebox that prepared all the samples for sequencing in about 20 minutes. (Tosches noted that before the recent technological improvements, it would have taken a year.)
After the researchers analyzed the sequencing data, the answer to the debate became clear. Some of the neurons in the salamander matched neurons in the reptilian DVR, but some did not. This suggested that at least parts of the DVR evolved from the pallium of an ancestor shared with amphibians. The unmatched cells in the DVR seemed to be innovations that appeared after the amphibian and reptile lineages diverged. The reptilian DVR was therefore a mix of inherited and novel types of neurons.
Mammals, however, were a different story. Salamander neurons didn’t match anything in the mammalian neocortex, although they did resemble cells in parts of the mammalian brain outside the neocortex.
Moreover, several kinds of cells in the neocortex — specifically, the types of pyramidal neurons that make up the majority of neurons in the structure — didn’t match with cells in the reptiles either. Tosches and her colleagues therefore suggested that these neurons evolved solely in mammals. They aren’t the first researchers to propose that origin for the cells, but they are the first to produce evidence for it using the powerful resolution of single-cell RNA sequencing.
Tosches and her team propose that essentially all of the mammalian neocortex is an evolutionary innovation. So while at least part of the reptilian DVR was adapted from the brain region of an ancestral creature, the mammalian neocortex evolved as a new brain region burgeoning with novel cell types. Their answer to the decades of debate is that the mammalian neocortex and the reptile DVR are not homologous because they don’t have a common origin.
Georg Striedter, a neuroscience researcher at the University of California, Irvine who studies comparative neurobiology and animal behavior, hailed these findings as exciting and surprising. “I felt like it was providing really good evidence for something that I had only speculated about,” he said.
The new answer from Tosches’ team doesn’t mean that the neocortex in mammals evolved to sit neatly atop older brain regions, as the triune brain theory proposed. Instead, as the neocortex expanded and new types of pyramidal neurons were born within it, other brain regions kept evolving in concert with it. They didn’t just hang on as an ancient “lizard brain” underneath. It’s even possible that the complexity emerging in the neocortex pushed other brain regions to evolve — or vice versa.