Too Many Neurons: Naturally Occurring Cell Death

A chick embryo makes about 20,000 motor neurons for its legs. By about day 10, roughly 12,000 are left.

Nothing is wrong with that embryo. It has both legs, and it will hatch and walk. The loss is part of normal development, and it is not rare. As Chris Deppmann puts it once the axons have reached their targets: “Too many neurons were made, and connections aren’t yet refined. We have a problem with excess.” This lesson asks how anyone found out that neurons die on purpose, how you would prove it, and what the death is for.

Neural death and survival: how much, and how it happens

Across the developing vertebrate nervous system, 20% to 80% of neurons degenerate, depending on the population. Each number belongs to a system. In the chick lumbar lateral motor column, the group of motor neurons that serves the leg, about 40% of the peak population dies during incubation. In the rat retina, about half of the ganglion cells, the neurons whose axons form the optic nerve, are eliminated. In frog limb motor neurons roughly half to three quarters die, and in mouse cortical interneurons about 40% die after birth.

You will often hear that “half of all neurons die.” That rounds a range into a rule. The “roughly half” comes from Ronald Oppenheim’s 1991 review in the Annual Review of Neuroscience, which gathered direct counts in many vertebrate populations, each made inside a defined developmental window. Within those windows, around half was a common value, but some populations lost far less and some far more. The review concerned development. It says nothing about how many neurons an adult loses.

How do the cells die? Not by injury. Necrosis is death from trauma: the cell swells, bursts and spills its contents, and the tissue becomes inflamed. Developing neurons die by apoptosis instead, a programmed death run by the cell’s own machinery of Bcl-2 family proteins and caspases (see that lesson). The chromatin condenses into a small, dark nucleus, a state called pyknosis, and the cell shrinks and breaks into fragments. It also flips phosphatidylserine, a lipid normally kept on the inner face of the membrane, to the outside. Microglia, the resident phagocytes of the brain, read that lipid as an “eat me” signal and engulf the corpse. The clean-up is fast, which is one reason the death went unnoticed for so long: at any one moment only a few dying cells are visible. How the neuron’s death machinery is triggered is the subject of a later lesson.

Historical context: limb bud experiments

The story begins with a missing wing. In 1934 Viktor Hamburger removed one wing bud from young chick embryos, building on earlier limb-removal work by Marian Shorey. A wing bud is the small swelling on the embryo’s flank that becomes the wing. Because only one side was operated on, the other side of the same spinal cord served as the control. Days later the operated side had fewer motor neurons.

Hamburger explained the deficit with a signal from the limb. In his model the limb sends a message back along the nerves that reach it, and the message sets how many neurons the spinal cord produces to serve it, by acting on cell division or differentiation. Remove the limb early and fewer neurons form.

Think of a bakery that ends the day with half the usual loaves on its shelf. Maybe it baked half as many. Maybe it baked a full batch and sent half to another shop. Maybe some loaves came out as rolls and were not counted as bread. Or maybe it baked a full batch and threw half away. The empty shelf looks the same in every case.

A low neuron count is that shelf. It could mean the cord divided too few times; or that it made enough cells but sent too few to the place being counted; or that the cells arrived but too few became the cell type the count looked for. A count made only at the end cannot separate these from a fourth history: cells that were made and then lost.

a count can reveal a mechanism only if you also looked at the cells between the operation and the count

Design problem

You remove a wing bud from one side of a chick embryo and later find fewer motor neurons on that side. Design the experiment that tells whether the missing neurons were never made or were made and then died.

Made, then lost

Something close to that experiment was done during the war, first in Turin and then in the Piedmont countryside, where Rita Levi-Montalcini and Giuseppe Levi removed limb buds from chick embryos and studied silver-stained embryos at a series of stages after the operation. In the nerve centres whose limb was gone, they found neurons that had already differentiated, already become their final cell type, and were degenerating. The cells had been made. They published the work in 1942, 1943 and 1944. Their results suggested that the limb normally helps keep neurons alive after they form, which contradicted Hamburger’s reading.

Hamburger read the wartime papers and invited Levi-Montalcini to St. Louis in 1947 to settle the question. Their joint 1949 paper followed chick spinal ganglia, clusters of sensory neurons beside the cord, from proliferation through differentiation to degeneration, in normal embryos and after limb removal or limb grafting. Many neurons differentiated and sent out processes before the deficit appeared, and later degenerating cells and their removal by macrophages became conspicuous. The target was not calling neurons into existence. It was keeping them alive.

In 1975 Hamburger came back to the question in normal embryos with all their limbs, and counted the death itself. In the lumbar lateral motor column of chick embryos, the count peaked at just over 20,000 at days 5.5 to 6.5 of incubation and settled at a plateau of about 12,000 after day 9.5, a loss of about 40% in uncorrected counts, most of it between days 6.5 and 9.5.

The next year Margaret Hollyday and Hamburger tested the logic from the other direction by grafting an extra leg. At day 6, before the normal loss, the grafted side had no more motor neurons than the control. Later, in embryos whose grafts moved vigorously, it had more: about 11% to 28% more in their counts. More target did not mean more births. It meant fewer deaths.

Together these results led to the idea Deppmann’s lecture states as “Targets Govern PNS Development”: the target supplies something limited that neurons need to survive. The search for that something produced nerve growth factor, a story told in that lesson. The tests of whether NGF is the limiting factor are in the next lesson.

Why make neurons just to kill them?

That is the question Deppmann puts to his class, as a poll: why does the nervous system kill about half its neurons during development? His answer is two jobs at once: matching plus error correction.

Matching. The number of neurons a target needs is hard to specify in advance. Limbs, muscles and organs vary in size between individuals and change as the embryo grows. Overproduce neurons, let the target supply a limited amount of support, and the number that survives settles at what the target can sustain. The extra-limb experiment shows the dial turning. It is the bakery’s strategy: bake extra and remove what no one takes.

Error correction. Axons sometimes grow to the wrong place or make weak connections. If survival depends on support received at the right target, neurons that went wrong, or connected poorly, lose out. Activity adds a second check: in chick embryos, blocking transmission at the muscle with curare rescues many motor neurons that would otherwise die, which ties survival to working connections. How the competition plays out is the subject of a later lesson.

Where the target account stops

If the target set all death, every wave of neuron death should answer to the target. Two studies show that some does not.

In 1996 Hiroyuki Yaginuma, Ronald Oppenheim and colleagues described an early wave of motor-neuron death in the chick cervical cord, around days 4 to 5 of incubation, before the later death that follows target contact. Moving cervical tissue to a larger target field, paralysing the embryo with curare and supplying muscle extracts or a long list of trophic factors all failed to change the early wave, though they helped the later one. Drugs that block RNA or protein synthesis reduced it, so it is an active process. When cervical neural tube was transplanted to other segmental levels, its early death was not altered. The schedule was carried within the cervical neural tube, though whether by each doomed neuron or by its neighbours the grafts could not say.

In 2012 Derek Southwell, Arturo Alvarez-Buylla and colleagues transplanted precursors of cortical interneurons, the local inhibitory neurons of the cortex, into the cortex of host mice. The transplanted cells died when they reached the age at which the host’s own interneurons die, and about the same fraction died whatever the number transplanted. If they had been competing for a fixed supply from the host, more cells should have meant a larger fraction dying. Their death followed a schedule set within the interneuron population itself, either in each cell or through competition among the interneurons, which the study could not tell apart.

So target-dependent survival explains much developmental neuron death, especially in the peripheral nervous system and in motor neurons after they reach their muscles. It is one source of control among several, and some populations run on their own clocks.