No new cortical neurons in humans, BrdU and C14 analysis

I have talked before about evidence that there is no new neurogenesis in the adult cortex, but that paper used stereological techniques.

A new paper in PNAS shows a more direct method to demonstrate that there are no newly created neurons in the adult cortex -- and their technique for this is so clever that I have to talk about it. They use a spike in the atmospheric levels of Carbon 14 isotopes after nuclear testing -- before the Nuclear Test Ban Treaty of 1963 -- to carbon date the creation of new cells in the human brain.

First some background: If you want to start a fight among neuroscientists, try arguing that new neurons are created in the human brain. For years and years, the dogma in neuroscience was that you are born with all the neurons that you will ever have. And this is for the most part true, but there has been some evidence created over the last decade that neurons are indeed created in adults -- even if the areas of the brain in which they are created are relatively limited. The most notable of these areas are the olfactory bulb -- where you get your sense of smell -- and the hippocampus -- a structure that is involved in memory. However, it is has been speculated that neurons may also be formed in other areas at low rates.

This paper suggests that this is not the case. Here is there description of their technique:

We have recently developed a new method to retrospectively determine the age of cells in humans by measuring 14C in DNA. The entry of cosmic rays into the atmosphere results in de novo generation of 14C, which is matched by radioactive decay (t1/2 = 5,730 years), resulting in stable steady-state atmospheric levels. A striking exception was caused by above-ground nuclear bomb tests during the Cold War, which produced an approximate doubling of 14C levels in the atmosphere from 1955 to 1963 that rapidly distributed around the globe. After the 1963 Test Ban Treaty, there have been no significant above-ground high-yield nuclear detonations, and 14C levels have decreased nearly exponentially, not because of radioactive decay, but because of equilibration with the oceans and uptake in the biotope. 14C in the atmosphere reacts with oxygen to form CO2 and is taken up by plants in photosynthesis. Our consumption of plants and animals that live off plants results in 14C levels in the human body mirroring those in the atmosphere at any given time. Because DNA is stable after a cell has gone through its last cell division, the 14C level in DNA serves as a date mark for when a cell was born and can be used to retrospectively birth date cells in humans.

Here we present a systematic analysis of cell turnover in the major areas of the human neocortex. We have retrospectively birth-dated neurons by measuring the level of 14C and have analyzed the brains of individuals that received BrdU. We failed to detect BrdU-labeled neurons and report that neocortical neurons have 14C levels corresponding to the atmospheric levels at the time of birth of the individual. (Emphasis mine. Citations removed.)

Typically if I wanted to measure the production of any new cell, I would use a chemical called BrdU. BrdU, or 5-bromo-2-deoxyuridine, is a thymidine analog that can be incorporated into cells when they are replicating their DNA during S phase of the cell cycle. Later on if you wanted to know whether that cell had divided, you can stain for the BrdU using specific antibodies for it. Therefore, you could read any BrdU incorporated cell as having definitely replicated its DNA and therefore divided during the period when you treated the cell with BrdU. For more information about how this would work, check out this swanky video from Wellesley.

Unfortunately BrdU is not something that you really want to use on humans. 1) It's a carcinogen, and we as a profession try to avoid adminstering carcinogens to our test subjects. 2) It does funky things to cells much less to whole organisms. We sometimes do administer BrdU to individuals, to for example test for the level of proliferation in someone with metastatic cancer. However, it would be better if there were some simpler and safer way.

In this study they compare the C14 incorporation into cells in the human cortex to BrdU incorporation. The samples of human brain -- you would be SHOCKED how difficult it is to get a human brain these days -- were obtained by the consent of families of individuals who had died and had their bodies submitted to autopsy. The samples of the human brain from an individual who was treated with BrdU to measure the proliferation rate of squamous cell carcinoma were also obtained prior to death.

These samples were compared -- the logic being that you want to prove that your new C14 test is just as good as the old test.

Here is an example of the C14 data (click to enlarge):

(The bar on the x-axis of the graph represents the birth date of the individual.) They sort the cells using a process called Flourescent Activated Cell Sorting (FACS) -- a technique for counting the number of cells in a population that express one or more marker proteins. In the graph they measure the C14 emission of the cells versus the expression of a neuronal marker NeuN. For the cells that express NeuN -- the neurons -- the C14 emission suggests that they are as old as the individual. For cells that do not express NeuN -- the other cells in the brain like astrocytes and oligodendrocytes -- the C14 emission suggests that they are younger or newly created.

This data suggests that new neurons in the cortex are created only at or around birth, whereas other cell types are created throughout life.

Here is there description of the comparable data for BrdU:

BrdU-positive cells were disproportionately distributed through the depth of the motor cortex; 46% of the BrdU-positive cells were located in the white matter and <1-17% in the specific lamina...In total, in all patients studied, 515 BrdU-positive cells were identified in 205-mm3 tissue. We analyzed the identity of labeled cells in the frontal and motor cortexes by immunohistochemistry by using antibodies against cell type-specific markers. Less than 1% of the BrdU-positive cells were glia-like satellite cells, and a small subpopulation constituted GFAP-immunoreactive astrocytes...Most importantly, none of the BrdU-labeled cells had neuronal morphology or were immunoreactive to the neuronal markers NeuN or neurofilament. In cases where a BrdU-positive nucleus was located in close proximity to a NeuN- or neurofilament-immunoreactive neuron, 3D confocal reconstruction was performed to establish whether the labels coexisted in the same cell, but this was never the case... (Emphasis mine.)

For the individual treated with BrdU, there was no evidence that the cells who incorporated BrdU also express neuronal markers. This suggests that whatever cells were dividing, they were not becoming neurons.

Here is there take home:

Several studies have demonstrated the presence of cells with in vitro neural stem cell potential in the human cortex, including in subcortical white matter. Our results do not exclude the possibility that neocortical neurogenesis may occur in certain pathologies, or that it may be possible to induce it, as has been suggested in the rodent cortex. There is no or minimal neurogenesis in the rodent striatum under normal conditions, but large numbers of neurons are generated in response to growth-factor administration or stroke. Although our current study indicates that neocortical neurogenesis does not take place in humans under normal conditions, it will be important to analyze whether there is a latent potential that results in neurogenesis in pathological situations.

There are clear species differences with regard to the extent of adult neurogenesis in vertebrates. Large numbers of neurons may be added throughout life in fish. However, fish often continue to grow, which could be viewed as a continuation of development. Substantial numbers of new neurons, including both interneurons and projection neurons, are added to several regions in birds such as zebra finches and canaries. In rodents, interneurons are added to the dentate gyrus of the hippocampus and to the olfactory bulb in mature animals. There are many reports indicating more low-grade neurogenesis in other areas of the rodent brain, but many of these studies await confirmation. The number of neurons that are added in the rodent hippocampus and olfactory bulb decreases substantially with age, although neurogenesis continues at low levels throughout life. 3H-thymidine studies originally indicated that there is less adult neurogenesis in the primate brain, and later studies using BrdU have demonstrated relatively lower levels of neurogenesis in the dentate gyrus and olfactory bulb compared to rodents. One study demonstrated neurogenesis in the adult human dentate gyrus, but it remains controversial whether neurons are added to the adult human olfactory bulb. Thus the distribution of adult neurogenesis appears to have been gradually more restricted with evolution, although there is still limited information available regarding the extent and distribution of neurogenesis in the adult human brain. (Emphasis mine. Citations removed.)

There are a couple things that I think are important about this study:

1) The way that they look at adult neurogenesis is absolutely cool. I would have never thought to do it that way.

2) There has been a lot of hype of adult neurogenesis in a variety of circumstances. This study does not rule out the possibility of neurogenesis in places like the hippocampus, but it does begin to suggest that whatever neurogenesis exists in humans it is decidedly more restricted than that which we are viewing in experimental animals. We need to start viewing experimental evidence in rats with a grain of salt.

Hat-tip: Faculty of 1000.