ABOVE: Marking adult mouse hippocampal stem cells that express the protein marker GFAP+ and thymidine kinase allows researchers to kill the cells and observe the animals’ behavior when their brains no longer undergo neurogenesis. FLICKR, JASON SNYDER

In March 2018, researchers reported evidence suggesting that adult humans do not generate new neurons in the hippocampus—the brain’s epicenter of learning and memory. The result contradicted two decades of work that said human adults actually do grow new neurons there, and revealed a need for new and better tools to study neurogenesis, Salk Institute President Fred Gage, who generated foundational evidence for adult human neurogenesis, told The Scientist at the time. 

See “Study Finds No Neurogenesis in Adult Humans’ Hippocampi

Since that study was published, several other teams have used similar techniques—but have come to different conclusions, publishing evidence that adult humans do indeed grow new hippocampal neurons,...

Neurogenesis is “fundamentally important for the brain to react to all sorts of different insults and prevent neurological and psychiatric problems,” Boldrini says. Because of its role in brain function, researchers want to learn how neurogenesis works to potentially use it to treat brain trauma, neurodegeneration, psychiatric disorders, such as depression, and possibly even the ill effects of aging.

See “Abundant Neurogenesis Found in Adult Humans’ Hippocampi” and “More Evidence that Humans Do Appear to Create New Neurons in Old Age

The growth of new neurons is well studied in newborn and adult animals, especially rodents. There’s prolific neurogenesis as the brain develops, which then drops off and plateaus in adulthood, only occurring in particular areas of the brain. Examinations of human postmortem tissue suggest that the process is similar in people, based on antibody markers that label neural progenitors and young neurons. But those signals can be hard to detect in preserved cells, and the gap in time between the death of a donor and when her tissue is fixed and analyzed can affect the reliability of the markers, scientists say, which might explain the disparities in findings between different studies. 

To get a reliable picture of the extent of neurogenesis in adults, scientists are pursuing a variety of new tools. Combining the direct detection techniques, such as RNA sequencing, with indirect ones, such as fMRI, Boldrini says, will indicate “what’s actually real” when it comes to the human brain’s ability to make new neurons.

Probing the living brain

In a recent study, Boldrini and colleagues found that the dentate gyrus, a region of the hippocampus where neurogenesis occurs, is bigger in people who were more resilient to early life stresses, such as abuse or separation from their parents. “They have more cells in the region, more neurons, and probably more neurogenesis,” she says.

Of course, Boldrini notes, the study has limitations. She and colleagues were working with tissue from deceased patients’ brains, which brings with it the challenges of preservation and the limitations of studying dead cells. Studies in postmortem tissue have made it extremely difficult to assess whether treatments, especially in psychiatric disorders, are effective, Boldrini explains. That’s why colleagues in her department and in other labs around the world have been working to develop fMRI as a way to track neural changes that correlate with neurogenesis-related network activity in living patients. 

Neurogenesis is “fundamentally important for the brain to react to all sorts of different insults and prevent neurological and psychiatric problems.”

—Maura Boldrini, Columbia University

She and colleagues, for example, are tracking how different regions of the hippocampus in patients with depression connect with other brain regions before and after antidepressant treatment. The measurements, though, are indirect, so if the team sees increased connectivity, it cannot immediately conclude there is increased neurogenesis. “You can say there is increased plasticity,” Boldrini explains, which could be formed by dendrite sprouting or the making of new neurons. The same is true if the region grows in volume, which could be caused by an increase in blood capillaries or, again, the growth of new neurons. What’s generating the change can’t be teased out of the results, she explains. 

Studies in adult rodents have used MRI to visualize the migration of neural stem cells in the brain, but those need to be labeled with MRI contrast agents that are directly injected into neurogenic regions, a technique not suitable to use in humans.

Magnetic resonance spectroscopy, however, is non-invasive and measures biochemical changes in the body and brain. Scientists say they think it could give them a clue to how neurogenesis works in living humans, if they could identify a biomarker specific to neural stem cells or neural progenitor cells. In 2007, a team announced it had identified a metabolic biomarker that they could detect in living animals, and possibly in living humans, to track neurogenesis in vivo. “That would certainly be very attractive to follow how the extent of neurogenesis is affected in an individual over time or for example in response to disease or medication,” Jonas Frisén, a molecular biologist and stem cell scientist at the Karolinska Institute in Sweden, writes in an email to The Scientist. However, he says, “that study has been difficult to reproduce, and that field has not taken off at all yet, unfortunately.”

Another option in the works is PET imaging, a technique Yosky Kataoka’s team at the RIKEN Institute has been working on to identify new neuronal growth in living people. Three years ago, he and colleagues reported successfully tracking the proliferation of new cells in the neurogenic regions of rat brains using the PET tracer 3′-deoxy-3′-[18F]fluoro-l-thymidine and a drug called probenecid. The drug is a treatment for gout that appears to enhance the ability of the tracer to cross the blood-brain barrier. The tracer and drug together allowed the researchers to image the dentate gyrus and the subventricular zone, the two regions in adult rodents’ brains where neurogenesis takes place, and quantitatively visualize the neurogenic activity in the animals. The team says it is now testing the technique in adult non-human primates, with the intent to eventually use it in humans.

See “Advancing Techniques Reveal the Brain’s Impressive Diversity

With PET, “the challenge is to find a tracer small enough that it can be injected in the blood, pass the blood-brain barrier, and get to the brain to attach to some specific molecule that is stem-cell specific,” Boldrini says. “We are still trying to find markers that are stem-cell specific.”

Back to postmortem brains

Identifying such specificity requires a more in-depth investigation of neural stem cells. “The brain has tremendous heterogeneity, many, many different cell types. And if you don’t look at every single cell type, you can’t appreciate the complexity and heterogeneity of the brain,” says Hongjun Song, a neuroscientist at the University of Pennsylvania’s Perelman School of Medicine. Even the same cell type, he notes, can be in different states, so, for example, neural stem cells can be in an active state, proliferating rapidly and developing into new neurons, or a dormant state, rarely dividing and when they do, remaining as stem cells. Despite their distinct activities, cells in these different states may still express the same marker proteins, making them difficult to differentiate without single-cell analysis, such as single-cell RNA sequencing.

A three-dimensional reconstruction of nine cubic millimeters of mouse hippocampus, a part of the brain involved in memory, profiled with Slide-seq. Different cell types are shown in red, green, and blue.
Chen and Macosko labs, courtesy of Broad Institute of MIT and Harvard

“The question I think we’re all interested in in the human brain is, do we really have cells with stem cell properties or immature neurons? I think there’s probably less of a debate about whether we have those cells or not,” Song says. “The question is . . . are they the same as in rodents or are they very different than in rodents? Single-cell sequencing will allow us to get that kind of unbiased view.”

Isolating neuronal precursor cells in the human brain isn’t easy. It’s much different than doing it in rodents, Song explains. In animals’ brains, researchers can label neuronal stem cells when the rodent is alive, and later extract and study those cells with RNA sequencing, which he and colleagues did in 2015, revealing the transcriptomes of neural stems and the cells they mature into in the adult mouse hippocampus. In humans, however, researchers again have to work with postmortem brain tissue and can’t label the cells while a patient is alive. Instead, scientists have to go cell-by-cell looking for neural progenitors. The human brain, Song adds, is much larger than the mouse brain, so the cells are sparser and farther apart. “You have to go through many, many cells to find them” in humans, Song says.

His team and others, including Boldrini’s and Frisén’s, have been working on RNA sequencing in postmortem human brains for several years now, and Boldrini says a new technique developed by Harvard University and MIT scientists in March might help with sorting human hippocampal nerve cells. Called Slide-seq, the technique uses genetic sequencing to draw 3-D tissue maps that identify a cell’s type, function, and location in tissue samples. So far, it’s only been tested on mouse tissue, but may hold promise for studying neural stem cells and newly made neurons, Boldrini says.

Ashley Yeager is an associate editor at The Scientist. Email her at ayeager@the-scientist.com. 

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