20 April 2009. Migrating is a stressful endeavor, even at the microscopic level. In the developing mammalian brain, neurons have to wander through a variety of regions before settling down at their final destination. How do they know what route to take, how far to go, and how to integrate with the local community once they are there? In last week’s PNAS, papers from two independent laboratories outline interactions that are essential for the proper integration of two different cell types into the brain. Researchers led by Eva Anton at the University of North Carolina School of Medicine, Chapel Hill, report that a protein called netrin guides the migration of GABAergic interneurons in the brain during embryonic development, while a team led by Johannes van Hooft at the University of Amsterdam, The Netherlands, shows that by pruning the growth of dendrites in the postnatal brain, the protein reelin helps integrate pyramidal neurons into the cerebral cortex once they have migrated there. Interestingly, these pre- and postnatal processes both rely on α3β1 integrin, a cell surface protein. The findings may be of interest to schizophrenia researchers since pyramidal cells and GABAergic interneurons have been linked to the underlying pathogenesis of schizophrenia, and there is evidence to suggest that altered neurodevelopment in early life may lead to the emergence of schizophrenia in later years. Moreover, both netrin and reelin have been linked to schizophrenia in previous research.
Guiding GABAergic interneurons
Neurodevelopment is a complex affair. As the brain develops, neural precursors must move to fill the space that eventually becomes the cerebral cortex. Some cells do this by migrating outward, or radially, just like hair grows, while other cells creep tangentially at first, like the blood vessels along the scalp, before eventually moving up or down (or sometimes both) to their final destination (see simple explanation on Wikipedia). The process is even more complex because the cortex is formed in an inside-out fashion. The outermost layer I is formed last and the cells that end up there must migrate through the other layers to reach their resting place.
GABAergic interneurons begin life as precursors in the medial ganglionic eminences—transient structures that are essential for embryonic brain development. From there, the precursors must migrate tangentially at first through a mesh of glial cells. Interactions between the neurons and the glia are thought to be critical for this migration. Netrin-1 is highly expressed in ganglionic eminences and is a member of a family of proteins that act as guidance cues for migration of other neurons. Anton and colleagues wondered if it might guide migration of GABAergic interneurons, and because recent work suggested netrin-1 binds to α3β1 integrin, first author Amelia Stanco and colleagues looked to see if the two proteins might interact. Using a variety of immunoprecipitation (IP) experiments, the researchers found that they do. IPs of forebrain lysates, netrin-1- and α3β1 integrin-expressing cells, and solutions of the two proteins, all demonstrated they bind to each other. Using an antibody that only recognizes an active form of the integrin, the researchers also found that netrin-1 turns on β1 integrin receptors.
To test the functional significance of the interaction, Stanco and colleagues cultured medial ganglionic eminences (MGEs) extracted from mouse embryos with cells that do or do not express netrin-1. In the absence of netrin-producing cells, neurons from the MGEs migrated randomly in all directions, but when netrin 1-producing cells were present, neurons migrated toward them. To get a better view of the role of these two proteins in neural migration, the researchers developed a double knockout mouse. They crossed a conditional knockout—where the α3 integrin subunit is specifically knocked out in interneurons that emanate from the MGE—with netrin-1-negative animals. They followed neuronal migration by the expression of enhanced green fluorescent protein, which is expressed in interneurons in the conditional knockout.
Looking at cell distribution in these animals at embryonic day 13.5, the researchers found that distances migrated by interneurons in the double knockout were significantly shorter than in wild-type. By E16.5 there was a substantial reduction in the number of interneurons migrating through the cortical plate. Using real-time imaging, they also found that in the absence of netrin-1 and the α3 integrin subunit, fewer neurons migrated toward the cortex and the numbers of neurons migrating radially instead of tangentially almost tripled. There was also an almost sixfold increase in the number of neurons that spontaneously stopped and reversed direction. Finally, at birth, there was a dramatic 64 percent reduction in the number of interneurons that reached the upper cortical plate, a 57 percent reduction in GABA-positive interneurons throughout the cerebral cortex, and a significant loss of interneurons in the hippocampus. Not all interneurons were equally effected, however. Neuropeptide Y-expressing interneurons were unaffected and calretinin-positive interneuron effects appeared limited to the region of the ventricular surface and cerebral wall. “Thus, netrin-1-α3β1 integrin interactions guide the appropriate navigation of subsets of GABAergic interneuronal populations through the developing cerebral wall and are necessary for the proper placement of interneurons in the cerebral cortex,” conclude the authors.
Disruptions in interneuron development have been tied to epilepsy (see Powell et al., 2003) and there are suggestions that schizophrenia and autism may be similarly affected (see Lewis and Levitt, 2002). “This emerging evidence for interneuronal subtype specific dysfunction in a variety of neurodevelopmental disorders highlights the importance of understanding the mechanisms underlying interneuron subtype development,” write the authors.
Reeling in pyramidal dendrites
Van Hooft and colleagues looked at neurodevelopment from a slightly different perspective. They examined if Cajal-Retzius cells, which produce reelin, a protein crucial for embryonic development, might also be important for postnatal maturation of the cortex. First author Pascal Chameau and colleagues addressed this question using transgenic mice expressing a green fluorescent protein (GFP) driven by a 5-HT3A (serotonin receptor) promoter. Serotonin is known to regulate neuronal proliferation, migration, and differentiation, and the Cajal-Retzius cells in layer I of the cerebral cortex are known to receive inputs from serotonergic cells, but it was not known if they actually had receptors for, and responded to, the neurotransmitter.
The researchers found that cortical layer I Cajal-Retzius cells in the transgenic mice clearly expressed GFP, suggesting that the 5-HT3A receptor gene was active, and they were also able to show that action potentials in the cells could be induced or inhibited by activating or blocking, respectively, 5-HT3A receptors. To test if this activity was driving neuronal development, the researchers cultured brain tissue from newborn mice and tested whether serotonergic input or reelin produced from these Cajal-Retzius cells influences the maturation of cortical pyramidal neurons. They found that both the 5-HT3 receptor antagonist tropisetron and the reelin specific antibody G10 caused a dramatic increase in the complexity of the dendritic tree in cortical layer II/III pyramidal neurons. The complexity of only the apical (top) and not the basal (bottom) dendrites were affected, which is in keeping with their relative proximity to the Cajal-Retzius cells. The researchers saw a similar increase in apical dendrite complexity in adult 5-HT3A receptor knockout mice and in tissue cultured from newborns with the same knockout. The morphological disturbance in these knockouts was rescued by adding reelin, suggesting that serotonin and reelin act in the same developmental pathway. In keeping with this idea, they found that reelin also prevented increased apical dendrite complexity elicited by tropisetron.
The work suggests that serotonin somehow causes a release of reelin from Cajal-Retzius cells, which then acts to prevent overdevelopment of pyramidal cell dendritic arbors. “Considering the prominent role of both serotonin and Cajal-Retzius cells in the development of the cortex, the results of this study suggest that fast serotonergic signaling to Cajal-Retzius cells in early postnatal life is important for shaping the cortical microcircuitry,” write the authors. In fact, it has previously been suggested that reelin acts as a stop signal for the growth and branching of apical dendrites in vertical columnar structures in mouse cortex (see Nishikawa et al., 2002).
Exactly how reelin reigns over dendritic ambitions is not clear, but it does not seem to work the same way as when regulating neuronal migration in embryonic development. In the latter scenario, the central fragment of the protein, R3-6, interacts with two different apolipoprotein receptors, but Chameau and colleagues found that blocking these interactions had no effect on postnatal pyramidal cell arborization. Instead, the reelin N-terminus (N-R2) seems to be the business end in this context. N-R2 binds to the α3β1 integrin, and the researchers found that blocking this interaction led to increased apical dendrite complexity, just as when they simply blocked reelin itself. “It is yet unclear how the activation of integrin receptors by reelin leads to changes in dendritic morphology,” write the authors.
The role of reelin in pyramidal cell arborization may be of particular interest to schizophrenia researchers since brain levels of this protein are lower in some people with the disease (see Fatemi et al., 2000).
Given the potential genetic links between schizophrenia and both reelin and netrin, these two proteins, and respective relationships with α3β1 integrin, may deserve some closer scrutiny.—Tom Fagan.
Stanco A, Szekeres C, Patel N, Rao S, Campbell K, Kreidberg JA, Polleux F, Anton ES. Netrin-1—alpha3beta1 integrin interactions regulate the migration of interneurons through the cortical marginal zone. PNAS Early Edition April 6 2009.
Chameau P, Inta D, Vitalis T, Monyer H, Wadman WJ, van Hooft JA. N-terminal region of reelin regulates postnatal dendritic maturation of cortical pyramidal neurons. PNAS Early Edition April 6 2009. Abstract