Gene Expression in Neurons Solves a Brain Evolution Puzzle

Our brain's neocortex is where our intelligence resides. According to recent research, mammals evolved new types of cells only after they diverged from reptiles in terms of evolution.

Many scientists believed that a similar brain area in reptiles and the neocortex in mammals may have originated from a common ancestor. However, recent research demonstrates that the structures originated independently and are composed of many cell types.

The astonishing feat of biological evolution represented by the neocortex stands out. The six layers of tightly packed neurons that make up the brain's covering are present in all animals, and they are responsible for the complex calculations and linkages that lead to cognitive brilliance. The neocortex is only seen in mammals, therefore scientists have pondered how such a sophisticated brain area developed.

Reptile brains appeared to include a hint. In addition to being the closest living relatives of mammals, reptiles also have a brain structure with three layers termed a dorsal ventricular ridge (DVR) that functions similarly to the neocortex. Some evolutionary neuroscientists have maintained for more than 50 years that the neocortex and the DVR both descended from a more rudimentary characteristic in an ancestor that both mammals and reptiles shared.

However, researchers have now disproved that theory by examining molecular features that are imperceptible to the human eye. Researchers at Columbia University have demonstrated that, despite their structural similarity, the neocortex in mammals and the DVR in reptiles are unrelated by examining patterns of gene expression in certain brain cells. The neocortex, on the other hand, appears to have originated in mammals as a totally new brain area, unrelated to anything that came before it. New varieties of neurons that appear to be unique to modern animals make up the neocortex.

The evolutionary and developmental scientist Maria Antonietta Tosches was the lead author on the Science publication that detailed this research.

The development of additional pieces is only one aspect of the brain's evolutionary creativity. In a different study published in the same issue of Science, Tosches and her coworkers demonstrated that even seemingly ancient brain areas are still evolving by being rewired with new types of cells. Researchers are redefining some brain areas and reevaluating whether some animals may have more sophisticated brains than previously believed in light of the revelation that gene expression might show these types of critical differences between neurons.

Single Neurons with Active Genes

The prominent neuroscientist Paul MacLean put out an incorrect but popular theory regarding brain evolution back in the 1960s. He proposed that a "lizard brain" that arose in reptiles and was in charge of survival instincts and behaviors was the source of the basal ganglia, a collection of structures near the base of the brain. Above the basal ganglia, early animals acquired a limbic system for the control of emotions. And a neocortex was later added by humans and other evolved animals, claims MacLean. It sat at the top of the stack and provided greater cognition, acting as a "thinking cap."

Carl Sagan wrote about the "triune brain" paradigm in The Dragons of Eden, which won the 1977 Pulitzer Prize, which captured the attention of the general public. Neuroevolutionary researchers weren't as impressed. Studies quickly disproved the hypothesis by demonstrating unequivocally that brain areas do not logically evolve one on top of the other. Instead, according to Paul Cisek, a cognitive neuroscientist at the University of Montreal, the brain evolves as a whole, with older sections undergoing adjustments to accommodate the arrival of new components. He said, "It's not like updating your iPhone, where you pull up a new app.

The best-supported theory for how new brain areas developed was that they did so primarily through the duplication and modification of established neuronal circuits and structures. Many evolutionary scientists, like Harvey Karten of the University of California, San Diego, believed that the mammalian neocortex and the reptilian DVR were analogous in terms of evolution and that they both descended from a structure that was shared by both reptiles and mammals.

However, some scientists, such as Luis Puelles of the University of Murcia in Spain, were not in agreement. They saw evidence that the neocortex and the DVR developed through very separate pathways in the development of mammals and reptiles. This suggested that the DVR and neocortex developed separately. If so, their similarities were most likely accidental coincidences caused by the structures' limits and functions rather than homology.

It took decades for the origins of the neocortex and DVR to be disputed. Now, though, a freshly discovered method is assisting in breaking the impasse. Scientists can find out which genes are being transcribed in a single cell using single-cell RNA sequencing. Evolutionary neuroscientists can deduce a richness of specific distinctions between individual neurons from these gene expression patterns. They may assess the neurons' evolutionary similarity using these differences.

Trygve Bakken, a molecular neuroscientist at the Allen Institute for Brain Science, noted that one benefit of examining gene expression is that it allows for apples-to-apples comparisons. "We know... that they are actually the same thing when you compare gene A in a lizard to gene A in a mammal since they have a same evolutionary origin," the statement reads.

The method is starting a new phase in the study of evolutionary neuroscience. Courtney Babbitt, a specialist in evolutionary genomics at the University of Massachusetts, Amherst, stated, "It's showed [us] new cell populations that we just didn't know existed." "Researching something you don't know exists is difficult."

The number of cells that could be employed for single-cell RNA sequencing in a sample grew by an order of magnitude in 2015 because to innovations in the field. Tosches was eager to apply the method to investigate the neocortex's earliest development because she was just starting her postdoc at Gilles Laurent's group at the Max Planck Institute for Brain Research in Germany at the time. We agreed to test it out, she recalled, saying, "OK."

Three years later, Tosches and her coworkers published their initial findings contrasting mouse and human neuron cell types with those found in turtle and lizard neurons. The variations in gene expression showed that the mammalian neocortex and the reptile DVR developed separately from several brain areas.

According to Bradley Colquitt, a molecular neuroscientist at the University of California, Santa Cruz, "the 2018 article was truly a milestone paper in that it was the first very complete molecular characterisation of neuronal types across mammals and reptiles."

Tosches and her colleagues recognized they needed to learn more about how the neural cell types in mammals and reptiles would compare to the neurons in an early common ancestor if they were to be able to conclusively prove that the two brain regions did not diverge from the same ancestral source.

They made the decision to search the brain of a salamander known as the sharp-ribbed newt for hints. (It gets its name from the capacity to poison and impale predators by pushing its ribs through its skin.) About 30 million years after the first four-legged animals stumbled onto the planet's surface and millions of years before the mammals and reptiles broke apart, salamanders, which are amphibians, diverged from the lineage they shared with those two groups. Salamanders, like other vertebrates, have a pallium, which is a structure located close to the front of the brain. If salamanders have neurons in their pallium that resembled those in the mammalian neocortex or the reptile DVR, then such neurons must have existed in a distant ancestor that all three animal species shared.

Neocortex: Restarting the Process

Tosches' team used single-cell RNA sequencing on hundreds of salamander brain cells in their 2022 study, comparing the results to information previously gathered from reptiles and humans. The researchers meticulously constructed and labeled tiny salamander brains, each of which was approximately one-fiftieth the volume of a mouse brain. Then, the brains were placed into a shoebox-sized machine that took around 20 minutes to prepare all the samples for sequencing. (Tosches pointed out that it would have taken a year before the most current technical advancements.)

The debate's resolution was evident once the researchers examined the sequencing data. Some of the salamander's neurons resembled those in the reptile DVR, but not all of them. This implied that at least some of the DVR descended from an ancestor that shared a pallium with amphibians. The DVR's mismatched cells appeared to be novelties that emerged after the divergence of the amphibian and reptile lineages. Thus, a combination of inherited and new kinds of neurons made up the reptile DVR.

But things were different with mammals. Salamander neurons resembled cells in regions of the mammalian brain outside the neocortex, but they did not match anything in the neocortex of mammals.

Numerous neocortical cell types, particularly the pyramidal neurons that make up the majority of the structure's neurons, also did not match those seen in reptiles. Therefore, Tosches and her associates proposed that these neurons only appeared in mammals. They are not the first scientists to suggest that origin for cells, but they are the first to provide proof for it by employing single-cell RNA sequencing's potent resolution.

The majority of the mammalian neocortex, according to Tosches and her team, is an evolutionary novelty. Therefore, the mammalian neocortex originated as a new brain area, booming with unique cell types, whereas at least a portion of the reptile DVR was adapted from the brain region of an ancient organism. They claim that the mammalian neocortex and the reptile DVR are not homologous since they did not share a common ancestor, in response to the decades-long controversy.

These results have been praised as fascinating and surprising by Georg Striedter, a neuroscience researcher at the University of California, Irvine who specializes in comparative neurobiology and animal behavior. He remarked, "I felt like it was presenting pretty excellent proof for something that I had just conjectured about.

The triune brain theory stated that the neocortex in animals developed to neatly sit atop earlier brain areas, however the current result from Tosches' team refutes this idea. Instead, additional brain areas continued to develop alongside the neocortex as it grew and new varieties of pyramidal neurons were created there. They did not only remain as the subterranean "lizard brain" of antiquity. It's even conceivable that other brain areas evolved as a result of the neocortex's complexity or vice versa.

In a second publication that published in the September 2022 edition of Science, Tosches and her colleagues recently found evidence that brain areas that appear to be very old are continually changing. In a comparison of a lizard brain to a mouse brain, she collaborated with Laurent, her postdoc mentor, to discover what single-cell RNA sequencing may disclose about new and ancient cell types. In order to identify the brain cell types that each species shared and that had to have descended from a common ancestor, they first compared the whole range of neural cell types in each species. Then they sought for distinct species-specific brain cell types.

Their findings demonstrated that both novel and conserved neural cell types may be found across the whole brain, not just in areas of the brain that have recently developed. Justus Kebschull, an evolutionary neuroscientist at Johns Hopkins University, said that the whole brain is a "mosaic" of both ancient and modern cell types.

Definitions Reconsidered

But some scientists claim that concluding the argument is not so simple. Barbara Finlay, an evolutionary neuroscientist at Cornell University, believes that rather than just comparing where neurons end up in adult amphibian, reptilian, and mammalian brains, it is still important to examine how neurons are generated, how they migrate, and how they connect up during development. If all of those discoveries could be combined, Finlay thinks it would be "terrific." I believe we will in due course, she added.

According to Tosches, the complexity that existed in the brains of an earlier common ancestor may have been lost in amphibian brains. Tosches stated that in order to be certain, scientists will need to perform single-cell RNA sequencing on extinct amphibian species or other bony fish species. If any of the neuronal subtypes seen in mammals have ancestors in animals before amphibians, that experiment may show it.

The research by Tosches and her associates has also sparked fresh debates about whether the profession has to reevaluate what a cerebral cortex is and which animals possess one. Tosches views the requirement that a cerebral cortex include visible neuronal layers, such as the neocortex or DVR, as "baggage" from conventional neuroanatomy. Her team discovered indications of layers in the salamander's brain when they applied the new sequencing technologies.

According to Tosches, "there is no reason to argue that salamanders or amphibians don't have a cortex." At this time, the salamander pallium should also be referred to as a cortex if we are going to refer to the reptile brain as a cortex.

Tosches, in Babbitt's opinion, has a point. Using the tools we have now, Babbitt stated, "How these things were characterized with classical morphology is probably just not going to stand up."

The answer to this query affects how neuroscientists should see birds. According to experts, birds have amazing cognitive capacities that can be on par with or even superior than many animals. Birds have a DVR since they are derived from reptiles, but for some reason neither it nor any of their other "cortex-like" brain parts are arranged into distinct layers. These areas appear to have supported sophisticated actions and skills despite the lack of observable layers. However, the existence of a cortex in birds is still debated.

A significant emphasis on appearances might be misguiding scientists. According to Striedter, "appearance may be misleading when it comes to homology" as the new single-cell evidence from Tosches' research demonstrates.