Now, in a study on mice conducted by the Neural Computation Unit at the Okinawa Institute of Science and Technology Graduate University (OIST), the authors, Dr. Katsuhiko Miyazaki and Dr. Kayoko Miyazaki, pinpoint specific areas of the brain that individually promote patience through the action of serotonin. Their findings were published 27th November in Science Advances.

“Serotonin is one of the most famous neuromodulators of behavior, helping to regulate mood, sleep-wake cycles and appetite,” said Dr. Katsuhiko Miyazaki. “Our research shows that release of this chemical messenger also plays a crucial role in promoting patience, increasing the time that mice are willing to wait for a food reward.”

Their most recent work draws heavily on previous research, where the unit used a powerful technique called optogenetics — using light to stimulate specific neurons in the brain — to establish a causal link between serotonin and patience.

The scientists bred genetically engineered mice which had serotonin-releasing neurons that expressed a light-sensitive protein. This meant that the researchers could stimulate these neurons to release serotonin at precise times by shining light, using an optical fiber implanted in the brain.

The researchers found that stimulating these neurons while the mice were waiting for food increased their waiting time, with the maximum effect seen when the probability of receiving a reward was high but when the timing of the reward was uncertain.

“In other words, for the serotonin to promote patience, the mice had to be confident that a reward would come but uncertain about when it would arrive,” said Dr. Miyazaki.

In the previous study, the scientists focused on an area of the brain called the dorsal raphe nucleus — the central hub of serotonin-releasing neurons. Neurons from the dorsal raphe nucleus reach out into other areas of the forebrain and in their most recent study, the scientists explored specifically which of these other brain areas contributed to regulating patience.

The team focused on three brain areas that had been shown to increase impulsive behaviors when they were damaged — a deep brain structure called the nucleus accumbens, and two parts of the frontal lobe called the orbitofrontal cortex and the medial prefrontal cortex.

“Impulse behaviors are intrinsically linked to patience — the more impulsive an individual is, the less patient — so these brain areas were prime candidates,” explained Dr. Miyazaki.

Good things come to those who wait (or not…)

In the study, the scientists implanted optical fibers into the dorsal raphe nucleus and also one of either the nucleus accumbens, the orbitofrontal cortex, or the medial prefrontal cortex.

The researchers trained mice to perform a waiting task where the mice held with their nose inside a hole, called a “nose poke,” until a food pellet was delivered. The scientists rewarded the mice in 75% of trials. In some test conditions, the timing of the reward was fixed at six or ten seconds after the mice started the nose poke and in other test conditions, the timing of the reward varied.

In the remaining 25% of trials, called the omission trials, the scientists did not provide a food reward to the mice. They measured how long the mice continued performing the nose poke during omission trials — in other words, how patient they were — when serotonin-releasing neurons were and were not stimulated.

When the researchers stimulated serotonin-releasing neural fibers that reached into the nucleus accumbens, they found no increase in waiting time, suggesting that serotonin in this area of the brain has no role in regulating patience.

But when the scientists stimulated serotonin release in the orbitofrontal cortex and the medial prefrontal cortex while the mice were holding the nose poke, they found the mice waited longer, with a few crucial differences.

In the orbitofrontal cortex, release of serotonin promoted patience as effectively as serotonin activation in the dorsal raphe nucleus; both when reward timing was fixed and when reward timing was uncertain, with stronger effects in the latter.

But in the medial prefrontal cortex, the scientists only saw an increase in patience when the timing of the reward was varied, with no effect observed when the timing was fixed.

“The differences seen in how each area of the brain responded to serotonin suggests that each brain area contributes to the overall waiting behavior of the mice in separate ways,” said Dr. Miyazaki.

Modelling patience

To investigate this further, the scientists constructed a computational model to explain the waiting behavior of the mice.

The model assumes that the mice have an internal model of the timing of reward delivery and keep estimating the probability that a reward will be delivered. They can therefore judge over time whether they are in a reward or non-reward trial and decide whether or not to keep waiting. The model also assumes that the orbitofrontal cortex and the medial prefrontal cortex use different internal models of reward timing, with the latter being more sensitive to variations in timing, to calculate reward probabilities individually.

The researchers found that the model best fitted the experimental data of waiting time by increasing the expected reward probability from 75% to 94% under serotonin stimulation. Put more simply, serotonin increased the mice’s belief that they were in a reward trial, and so they waited longer.

Importantly, the model showed that stimulation of the dorsal raphe nucleus increased the probability from 75% to 94% in both the orbital frontal cortex and the medial prefrontal cortex, whereas stimulation of the brain areas separately only increased the probability in that particular area.

“This confirmed the idea that these two brain areas are calculating the probability of a reward independently from each other, and that these independent calculations are then combined to ultimately determine how long the mice will wait,” explained Dr. Miyazaki. “This sort of complementary system allows animals to behave more flexibly to changing environments.”

Ultimately, increasing our knowledge of how different areas of the brain are more or less affected by serotonin could have vital implications in future development of drugs. For example, selective serotonin reuptake inhibitors (SSRIs) are drugs that boost levels of serotonin in the brain and are used to treat depression.

“This is an area we are keen to explore in the future, by using depression models of mice,” said Dr. Miyazaki. “We may find under certain genetic or environmental conditions that some of these identified brain areas have altered functions. By pinning down these regions, this could open avenues to provide more targeted treatments that act on specific areas of the brain, rather than the whole brain.”

From 1997 to 2014, the U.S. Fish and Wildlife Service moved more than 9,100 Mojave desert tortoises to the 100-square-kilometer (about 39 miles square) Large Scale Translocation Site. The newcomers, many of which were abandoned pets or had been displaced by development, joined nearly 1,500 desert tortoises already living there.

Conventional wisdom would suggest that tortoises from areas closest to the translocation site would fare best. But a new UCLA study, published today in Science, found no connection between the tortoises’ place of origin and their chances of survival. It did, however, uncover a far better predictor.

Tortoises with lots of genetic variation were much more likely to survive after their relocation, said UCLA conservation ecologist Brad Shaffer, the study’s senior author. Like most organisms, tortoises have two copies of their entire genome, with one copy from each parent. The more those copies differ from each other, the higher the organism’s heterozygosity.

The researchers compared translocated tortoises that lived or died over the same time period after being relocated to the site. They found that survivors averaged 23% greater heterozygosity than those that perished. Simply put, tortoises with more genetic variation had higher survival rates.

“It flies in the face of what we know from other translocation studies, but lots of genetic variation was hands-down the best predictor of whether a tortoise lived or died,” said Shaffer, a professor of ecology and evolutionary biology and director of the UCLA La Kretz Center for California Conservation Science. “Relocating endangered plants and animals is increasingly necessary to counteract the effects of climate change, and this gives us a new tool to increase survival rates.”

Although the relationship between heterozygosity and survival was well supported by the study, it’s unclear why greater genetic variation is linked to survival rates, said former UCLA postdoctoral scholar Peter Scott, the study’s lead author.

“Potentially, individuals with higher heterozygosity have more genomic flexibility,” said Scott, who is now an assistant professor at West Texas A&M University. “It’s likely that tortoises with more variation have a better chance of having one copy of a gene that works really well in stressful or new environments compared to those individuals with two identical copies that only work really well in their environment of origin.”

The researchers wanted to make tortoise conservation efforts more effective, and uncover trends that would help other species as well, Scott said.

“Oftentimes, the chances of success for relocating plants or animals is pretty dismal,” he said. “We wanted to understand why, and use that understanding to increase survival.”

Over the years, tortoises that were given up as pets, or removed from places like developments in suburban Las Vegas and solar farms in the desert, were surrendered to the U.S. Fish and Wildlife Service.

The agency took blood samples to screen for diseases and marked each animal before releasing them into the Ivanpah Valley site, which enabled the animals to be tracked in later surveys. The UCLA researchers sequenced blood samples drawn from 79 tortoises that were released to the site and were known to be alive in 2015, and from another 87 known to have died after they were released at the site.

Although the Large Scale Translocation Site provided an intriguing dataset, it’s not the same as a controlled experiment. Additional studies would be needed to understand why more heterozygous tortoises had a higher survival probability and precisely how much of an increase in genetic variation improves a tortoise’s odds of surviving.

“The only reason we could do this study was because the U.S. Fish and Wildlife Service was incredibly forward-thinking when they set up the translocation site and tracked who lived and died,” Shaffer said. “Many died, and no one was happy about that. But we can learn a lot from that unfortunate result to help conservation management improve.

“When thinking about moving animals or plants out of danger, or repopulating an area emptied by wildfire, now we can easily and economically measure genetic variability to better gauge the survival probability of those translocated individuals. It’s not the only criteria, but it’s an important piece of the puzzle.”

The finding concerns a key process essential to life: the transcription phase of gene expression, which enables cells to live and do their jobs.

During transcription, an enzyme called RNA polymerase wraps itself around the double helix of DNA, using one strand to match nucleotides to make a copy of genetic material — resulting in a newly synthesized strand of RNA that breaks off when transcription is complete. That RNA enables production of proteins, which are essential to all life and perform most of the work inside cells.

Just as with any coherent message, RNA needs to start and stop in the right place to make sense. A bacterial protein called Rho was discovered more than 50 years ago because of its ability to stop, or terminate, transcription. In every textbook, Rho is used as a model terminator that, using its very strong motor force, binds to the RNA and pulls it out of RNA polymerase. But a closer look by these scientists showed that Rho wouldn’t be able to find the RNAs it needs to release using the textbook mechanism.

“We started studying Rho, and realized it cannot possibly work in ways people tell us it works,” said Irina Artsimovitch, co-lead author of the study and professor of microbiology at The Ohio State University.

The research, published online by the journal Science today, Nov. 26, 2020, determined that instead of attaching to a specific piece of RNA near the end of transcription and helping it unwind from DNA, Rho actually “hitchhikes” on RNA polymerase for the duration of transcription. Rho cooperates with other proteins to eventually coax the enzyme through a series of structural changes that end with an inactive state enabling release of the RNA.

The team used sophisticated microscopes to reveal how Rho acts on a complete transcription complex composed of RNA polymerase and two accessory proteins that travel with it throughout transcription.

“This is the first structure of a termination complex in any system, and was supposed to be impossible to obtain because it falls apart too quickly,” Artsimovitch said.

“It answers a fundamental question — transcription is fundamental to life, but if it were not controlled, nothing would work. RNA polymerase by itself has to be completely neutral. It has to be able to make any RNA, including those that are damaged or could harm the cell. While traveling with RNA polymerase, Rho can tell if the synthesized RNA is worth making — and if not, Rho releases it.”

Artsimovitch has made many important discoveries about how RNA polymerase so successfully completes transcription. She didn’t set out to counter years of understanding about Rho’s role in termination until an undergraduate student in her lab identified surprising mutations in Rho while working on a genetics project.

Rho is known to silence the expression of virulence genes in bacteria, essentially keeping them dormant until they’re needed to cause infection. But these genes do not have any RNA sequences that Rho is known to preferentially bind. Because of that, Artsimovitch said, it has never made sense that Rho looks only for specific RNA sequences, without even knowing if they are still attached to RNA polymerase.

In fact, the scientific understanding of the Rho mechanism was established using simplified biochemical experiments that frequently left out RNA polymerase — in essence, defining how a process ends without factoring in the process itself.

In this work, the researchers used cryo-electron microscopy to capture images of RNA polymerase operating on a DNA template in Escherichia coli, their model system. This high-resolution visualization, combined with high-end computation, made accurate modeling of transcription termination possible.

“RNA polymerase moves along, matching hundreds of thousands of nucleotides in bacteria. The complex is extremely stable because it has to be — if the RNA is released, it is lost,” Artsimovitch said. “Yet Rho is able to make the complex fall apart in a matter of minutes, if not seconds. You can look at it, but you can’t get a stable complex to analyze.”

Using a clever method to trap complexes just before they fall apart enabled the scientists to visualize seven complexes that represent sequential steps in the termination pathway, starting from Rho’s engagement with RNA polymerase and ending with a completely inactive RNA polymerase. The team created models based on what they saw, and then made sure that these models were correct using genetic and biochemical methods.

Though the study was conducted in bacteria, Artsimovitch said this termination process is likely to occur in other forms of life.

“It appears to be common,” she said. “In general, cells use similar working mechanisms from a common ancestor. They all learned the same tricks as long as these tricks were useful.”

Artsimovitch, working with an international research team of collaborators, co-led the study with Markus Wahl, a former Ohio State graduate student now at Freie Universität Berlin.

This work was supported by grants from the German Research Foundation; the German Federal Ministry of Education and Research; the Indian Council of Medical Research; the Department of Biotechnology, Government of India; the National Institutes of Health; and the Sigrid Jusélius Foundation.