Serotonin, Netrins, and Brain Development: Why a Neurotransmitter Is Not Always a Neurotransmitter

In July's installment ("Microchimeras and Serotonin," page 20), I discussed the role of serotonin in the growth and differentiation of many systems within the developing embryo. From establishing left-right body axis to mediating eye morphogenesis, serotonin is involved in a bewildering number of developmental decisions in mammalian embryos--it appears to be a morphogen before it is a neurotransmitter.

Serotonin is also involved in the development of the CNS. Concerns about this neurotransmitter's effects on the brain actually held up the FDA's approval of fluoxetine (Prozac, Sarafem). There have been a number of recent developments regarding the molecular mechanisms that guide the relationship between serotonin and brain development, and I will address one of them here.

I start with a brief discussion on the attractive and repulsive guidance cues that affect nerve cells and axons as they travel to their final destinations in the developing brain, paying particular attention to a molecule called netrin-1 and the signal transduction mechanisms that are associated with it. Some interesting findings about the relationship between netrin-1, serotonin, and thalamic development will then be explored.

Feel free to skip to the "Data" section if thalamocortical neural pathfinding mechanisms and in utero electroporation are already working parts of your vocabulary.

Neural pathfinding

How embryonic neurons get their instructions to wire themselves into a functioning brain has fascinated developmental biologists for decades. Much energy has been spent in attempting to understand neural growth and the ex-ternal cues that guide neural migration patterns. Specialized structures at the tips of nerve fibers called axonal growth cones have been studied close-ly. Axonal growth cones assist in guiding fibers to their proper destinations during their development.

A growth cone is a surprisingly complex structure. It has finger-like projections called filopodia, which are highly dynamic structures whose internal molecular composition includes the protein actin. The proteins in the cone are constantly disassembling and reassembling in such a manner as to give the cone migratory capabilities. Between the finger-like projections are web-like structures called lamellipodia, which possess very few receptors.

Much effort has been devoted to understanding the extracellular cues that assist in the maintenance of this architecture and its relationship to axonal growth cone migration. The basic model involves receptors within the filopodia that respond to extracellular signals provided by previous cells. These interactions are communicated to the interiors of the cells that possess the cones. Decisions about migration and differentiation occur as a result, changing the gene expression. These changes are felt over the entire nerve fiber.

Three classes of molecules interact to produce the movement, and ultimately, the wiring patterns: ligands (the extracellular signals), receptors (lying on the exposed membranes of the cone), and the nerve fiber's internal signal transduction mechanisms (which are linked to the receptors and are capable of transferring information to the cell interior).

The major molecular players that are used in the experiments examined in this column are briefly summarized below. The nerve fibers examined throughout the experiments were thalamocortical cells or their direct progenitors.

The migration-promoting molecule

The molecule used to coax thalamic neurons to migrate in these experiments was netrin-1. Netrins are a family of secreted proteins that are deeply involved in neural development. They are bifunctional, meaning they can attract certain classes of axons and repel others. They serve as short-range cues for migrating neurons, acting close to the cell surfaces that secreted them. They can also act at a distance, far from the sources of their manufacture and attract cells that are not in the immediate vicinity of their manufacture.

Netrin-1 is expressed in both embryonic and adult cells and can generate attractive and repulsive signals. First characterized as mediating short-range interactions, it has also been shown to have long-term cue characteristics. In short, netrin-1 is a very versatile molecular gadget.

How a given axon responds to this flexible family of molecules is determined by 2 factors: the receptor proteins residing on the cell surface of the growth cone and the internal signal transduction mechanisms the cone possesses. Two types of receptors have been isolated. One family of receptors is known by the improbable name "deleted in colorectal carcinoma" (DCC). The others are certain homologs of the UNC-5 family, first characterized in the roundworm Caenorhabditis elegans but shown to exist in vertebrates.

If an axonal growth cone that is carrying a DCC receptor encounters netrin-1, it will interpret the interaction as attractive, and the axon will migrate toward the signal. If an axonal growth cone that is carrying a UNC-5 receptor encounters netrin-1, it will interpret the interaction as repulsive, and the axon will migrate away from the signal. This is why netrin-1 is termed a bifunctional developmental molecule. As will be shown, serotonin and its attendant receptors also have a developmental relationship with the netrin family.

It has taken much effort to understand how functional circuits can be constructed from the interactions of these classes of molecules. Even when the neurons "arrive" at their proper locations, the chore of finding the proper synaptic partners capable of establishing the circuit's function remains. Temporal, spatial, and even cell type issues must be addressed and precisely coordinated so that the cells not only arrive at their proper destination but hook up properly once available.

Where serotonin fits

Growth cones respond to the information supplied by external molecular cues through internal interactions that are usually termed signal transduction mechanisms. Secondary messengers are molecules that are capable of carrying the information from the membrane and communicating it throughout the cytoplasm.

The canonical secondary messenger is cyclic adenosine monophosphate (cAMP), which is manufactured by the enzyme adenyl cyclase. The activity of this enzyme can be modulated, in turn, by another protein, cAMP-dependent kinase. If the kinase is inhibited, cAMP levels are reduced. If the kinase is stimulated, cAMP levels can be greatly elevated.

In recent years, it has become clear that serotonin plays a role in brain development. The mechanism of this role was made clear by the discovery that several serotonin receptor subtypes (5-HT_1B and 5-HT₁₁) were expressed by axonal growth cones in developing thalamic cells. Eventually, it was shown that serotonin exerts its effects via a signal transduction mechanism involving cAMP. That, in a nutshell, is the thrust of the new data presented next.

Data

Researchers used culture explants from the dorsal thalamus of mice. The axons in these tissues are usually attracted to target human embryonic kidney-293 (HEK293) cells that have been genetically engineered to overexpress netrin-1. They normally extend their filopodia and begin migrating in response to the secreted protein.

When these explants were soaked in serotonin, however, an extraordinary behavior was observed. Rather than normally migrating toward the overexpressing HEK293 cells, they actually reversed course and started migrating away from their targets.

The reason for this reversal appeared to be the inhibition of a critical step in the signal transduction process. Serotonin seemed to cause a decrease in cAMP levels in the thalamic cells, which was shown in 2 ways (Figure).

Blocking experiments. There are pharmacological reagents capable of inhibiting cAMP-dependent kinase, a reaction that artificially reduces cAMP levels. Such an inhibiting reagent was added to the thalamic cells, which were then exposed to the netrin-1 producing HEK293 cells. They responded to these overexpressing HEK293 cells as if they had been treated with serotonin, even though there was no serotonin in sight. The thalamic cells were repulsed, and they reversed their normal attractive migration patterns.

Restoring experiments. There are also pharmacological reagents capable of restoring kinase function. This treatment keeps cAMP levels artificially elevated. When pretreated thalamic cells were exposed to the HEK293 cells in the presence of serotonin, something extraordinary was observed. Rather than being repulsed, which is what serotonin should do, the cells were once again attracted.

Additional experiments were conducted with developing mouse embryos in vivo. In utero electroporation is a technique that introduces foreign DNA into intact embryonic mice via electrical fields. It is also possible to follow the fates of these cells after they are manipulated by adding fluorescent reporter molecules.

The researchers took advantage of these technologies to study the migratory patterns of thalamic progenitor cells in the developing mice embryos. They made 2 very powerful genetic constructs. One was capable of artificially depressing the expression of serotonin receptors 5-HT_1B and 5-HT₁₁ in their thalamic targets. This had the effect of reducing cAMP levels in these cell types. The other was capable of keeping the expression artificially elevated, thus artificially elevating cAMP in these cell types.

Each construct was injected into a different mouse embryo and the mice were then allowed to develop normally. After they had developed, the mi- gratory patterns in the thalamus were examined.

The results were astonishing but familiar. Mice that had their serotonin receptor levels artificially depressed (with cAMP levels reduced as a result) produced migratory patterns that were strongly repelled by known netrin-1 sites, which is not what normally happens. At these observed sites, netrin-1 typically attracts the progenitor cells. The results achieved in dishes using HEK293 cells were repeated in vivo.

Conversely, the mice that had their serotonin receptor levels artificially elevated (with cAMP levels elevated as a result) produced migratory patterns of strong attraction to known netrin-1 sites. The migrations were greatly enhanced compared with those of controls.

What was shown in dishes was also shown in mice--this is quite a finding.

Conclusion

As with any good research project, these results raise more questions than they answer. For a molecular biologist, understanding the molecular relays that link changes in cAMP levels to the decision to migrate--or not--is the next frontier. For a clinician, the chief concern lies in whether these findings, which have only been observed in laboratory animals, can be translated into human biology. Of particular interest are the effects of medications that are known to perturb serotonin biochemistry during pregnancy.

For all of us, the ability to translate findings obtained from experiments in a dish to the real world of brain development provides a powerful and exciting glimpse into the future of developmental biology. That one of its most surprising discoveries is to show that a neurotransmitter is not always a neu- rotransmitter only adds fuel to the promise of this most exciting area of research.