New Neurons in the Adult Brain

The neurons at the back of your cerebral cortex are as old as you are. Some neurons in your hippocampus may be younger, and part of the evidence for that came from nuclear bomb tests.

For most of the twentieth century the working rule was simple: the neurons you are born with are the neurons you keep, and those you lose are not replaced. This lesson is about the exceptions. Where are new neurons made in adult animals? How can anyone tell that a neuron is new? And why is neurogenesis in human beings, as adults, still argued over?

The idea: make a dividing cell sign its DNA

To find a newborn cell you need a mark that only newborn cells carry. A cell copies its DNA once per division, in S phase, and a neuron that has stopped dividing never copies its genome again. Give an animal a labelled form of thymidine, the DNA building block used only in DNA, and every cell in S phase at that moment builds the label into its chromosomes. Any labelled neuron found weeks later descends from a cell that divided after the injection.

Think of it as a stamp put on every page printed on one day: a stamped page was printed that day.

Joseph Altman did this in rats with tritiated thymidine, thymidine carrying radioactive hydrogen. A photographic emulsion laid over a thin brain section shows labelled nuclei as black silver grains, a method called autoradiography. In 1965, with Gopal Das, he reported labelled granule cells in the dentate gyrus of the hippocampus in rats after birth, and in 1969 he described dividing cells in the wall of the lateral ventricle that moved forward into the olfactory bulb. The claims were largely set aside for years. With a light microscope it is hard to prove that a labelled cell is a neuron and not a glial cell lying against one. And in 1985 Pasko Rakic reported that thymidine given to rhesus monkeys after puberty, examined from 3 days to 6 years later, labelled glial cells but no neurons.

Songbirds reopened the question. Adult female canaries given testosterone start to sing, and a song-control nucleus called HVC grows. In 1983 Steven Goldman and Fernando Nottebohm injected tritiated thymidine into adult female canaries and found that 1.8 to 4.0 percent of HVC neurons were labelled, with or without the hormone. Electron microscopy confirmed they were neurons. In birds killed soon after the injection the label was in the ventricular zone above HVC, not in HVC, so the neurons were born at the ventricle and migrated in.

Then the label changed. Bromodeoxyuridine (BrdU) is a thymidine look-alike that an antibody can detect, so the same cell can be stained for BrdU and for a neuron-only protein such as NeuN. That answered the old objection. In 1999 two groups using BrdU reported new dentate gyrus neurons in adult monkeys (Old World monkeys in Gould and colleagues; macaques in Kornack and Rakic), with Kornack and Rakic estimating a rate about ten times lower than in rodents.

Where adult stem cells live in rodents

Adult rodents make neurons all their lives in two places.

The first is the subventricular zone (SVZ), a thin layer in the wall of the lateral ventricles. Neuroblasts, young neurons that can still migrate, line up in chains and travel forward along the rostral migratory stream to the olfactory bulb, where they become interneurons. Carlos Lois and Arturo Alvarez-Buylla showed this long-distance migration in adult mice in 1994; the stream returns as a case of tangential migration in that lesson.

Which cell in the SVZ is the stem cell? In 1999 Fiona Doetsch, Alvarez-Buylla and colleagues described four cell types there: ependymal cells lining the ventricle, astrocytes (B cells), rapidly dividing immature precursors (C cells, the transit-amplifying cells), and migrating neuroblasts (A cells). They infused an antimitotic drug that killed the C and A cells. The astrocytes survived, began dividing, and regenerated C cells and then neuroblasts. In untreated mice, a retrovirus that could enter only astrocytes labelled new olfactory bulb neurons. So the lineage runs:

B cell (astrocyte-like stem cell) → C cell (transit-amplifying) → A cell (neuroblast) → olfactory bulb interneuron

The adult stem cell looks like a glial cell, which fits development: the embryonic progenitors of the brain, the radial glia, are glial in character too (that lesson).

The second niche is the subgranular zone (SGZ) at the inner edge of the dentate gyrus. Its stem cells are also astrocyte-like, and their progeny become dentate granule neurons that grow dendrites and send axons into the hippocampal circuit over several weeks. Local signals set the balance between resting and dividing. Notch signalling helps keep stem cells in reserve (that lesson), and in rodents Wnt3 in the hippocampal niche increases neuron production while blocking Wnt signalling nearly abolishes it (that lesson).

Experience changes the output: mice with a running wheel had more new dentate gyrus neurons than mice in standard cages (van Praag, Kempermann and Gage, 1999), and mice housed in larger cages with toys and companions had more hippocampal neurons (Kempermann, Kuhn and Gage, 1997).

Neurogenesis in adults: dating human neurons with the bomb

BrdU cannot be given to healthy people for research. Jonas Frisén’s group at the Karolinska Institute found a label already in the atmosphere.

Carbon-14 is made at a nearly steady rate in the upper atmosphere. Above-ground nuclear weapons tests, at their height between 1955 and 1963, nearly doubled the level in the northern hemisphere air. After the 1963 treaty that banned tests in the atmosphere, the level fell year by year as the extra carbon-14 moved into the oceans and the biosphere. Plants fix that carbon dioxide, people eat the plants or animals that ate them, and the carbon ends up in the DNA of every cell that divides. Carbon-14 decays slowly (half-life about 5,730 years), so over a lifetime the decay is negligible. The DNA of a cell that never divides again keeps the carbon-14 of its birth year.

carbon-14 in the DNA of a cell = carbon-14 in the air in the year that DNA was made

In 2005 Kirsty Spalding, Frisén and colleagues showed that DNA carbon-14 tracks the atmospheric curve. They sorted nuclei from post-mortem brain into neuronal and non-neuronal with an antibody to NeuN, measured the carbon-14 by accelerator mass spectrometry, and found that occipital cortex neurons were as old as the person while non-neuronal cells were younger.

Take one example: “This person is born in 1972.” Their neuronal DNA holds the carbon-14 of 1976 on the falling side of the curve. Because a sample holds many nuclei, the value is an average:

measured carbon-14 = (fraction born at birth × level at birth) + (fraction born later × average level when they were born)

So the reading means the DNA was made, on average, four years after birth. It cannot say whether all neurons are a little younger or a few are much younger.

Olfactory bulb neurons matched the person’s birth year, showing very limited, if any, postnatal addition in humans (Bergmann and colleagues, 2012), unlike rodents. In the hippocampus, Spalding and colleagues (2013) estimated that about one third of neurons are subject to exchange, with about 700 new neurons added per hippocampus per day, a turnover of 1.75 percent a year within the renewing fraction. Ernst and colleagues (2014) reported new interneurons in the adult striatum, beside the ventricle wall.

Design problem

Two people donate hippocampus and cortex: one born in 1950, before the bomb peak, and one born in 1972, after it. In the person born in 1950, the carbon-14 of cortical neuron DNA matches the air of 1950, but the hippocampal neuron DNA holds more. What does each result mean, and why is the person born before the peak the more decisive test?

”Is that so?” Two answers about the adult human hippocampus

Is that so? For a paper discussion, start with March 2018. Sorrells, Alvarez-Buylla and colleagues stained human dentate gyrus for dividing progenitors and for young neurons, using doublecortin (DCX) and PSA-NCAM, proteins found on immature neurons. The young neurons fell sharply in the first year of life, a few remained at 7 and 13 years, and none were detected in adults aged 18 to 77, either in post-mortem controls or in tissue removed during epilepsy surgery. They saw a similar decline in juvenile macaques and concluded that adult human hippocampal neurogenesis does not continue or is extremely rare.

The next month, Boldrini and colleagues reported the opposite from whole post-mortem hippocampi of healthy people aged 14 to 79. They found similar numbers of intermediate progenitors and thousands of immature neurons at every age, with a smaller pool of resting stem cells in the front of the dentate gyrus in older people. In 2019 Moreno-Jiménez, Llorens-Martín and colleagues, working with tissue collected and fixed under tightly controlled conditions, reported thousands of DCX-positive neurons in healthy people into their ninth decade, and fewer as Alzheimer’s disease advanced.

Why would careful laboratories disagree? The arguments are about method.

• Tissue handling. DCX staining weakens with time after death and with long fixation, so a negative result may mean the antigen was lost.
• Markers. In humans it is debated whether DCX and PSA-NCAM mark only newborn neurons.
• Donors. Age, cause of death, epilepsy and medication all differ between collections.
• Carbon-14 assumptions. The 2013 estimate rests on a turnover model, on pure neuronal sorting, and on DNA repair adding little carbon.

Single-cell RNA sequencing studies published since then have also disagreed. A fair statement is that the evidence does not yet settle whether adult humans add hippocampal neurons, or at what rate.

What new neurons might be for

In rodents the answers are clearer. Young dentate granule neurons are thought to help with pattern separation, telling apart similar places or events: mice in which adult neurogenesis was blocked did worse at distinguishing nearby locations (Clelland and colleagues, 2009). In the rodent olfactory bulb, the steady supply of new interneurons has been linked to odour learning.

None of this transfers to people automatically. The human olfactory bulb receives few or no new neurons after birth, and the infant stream toward it declines after about 18 months and is nearly gone by adulthood. Whatever adult neurons humans make, the carbon-14 data point to the hippocampus and striatum, not the smell centre where rodents put most of theirs.