The migration pattern of newly generated neurons, darkly stained (BrdU incorporation experiments), was normal in the neocortex of wild-type animals (left panel), but abnormal in the Glast-/-/Glt1-/- E16 neocortex (right panel). Images courtesy of T. R. Matsugami, Tokyo Medical and Dental University, Japan.
The involvement of glutamate in early brain development has been somewhat contentious: although a large body of in vitro evidence highlights signalling roles for glutamate during proliferation, migration, differentiation and survival, genetic disruption to glutamatergic activity has little or no effect on brain development. New work by Matsugami and colleagues goes some way to resolving this issue and provides compelling in vivo evidence that glutamatergic activity is vital for early developmental events.
The absence of an effect following genetic disruption to glutamate receptors or glutamate release might reflect the compensatory action of other neurotransmitters during development. To overcome this potential confound, Matsugami and co-workers adopted the opposite approach: they overstimulated glutamate receptors in mice by knocking out the glutamate transporters GLAST and GLT1, which normally maintain low levels of extracellular glutamate. These knockout mice had multiple brain defects in the cortex, hippocampus and olfactory bulb after embryonic day (E) 15 and died by E17–18.
By E16, the number of cells in the ventricular zone — a layer of mitotic cells that eventually give rise to all cell types in the mature brain — was decreased in mutant brains compared with wild-type brains. The proportion of proliferating cells in the ventricular zone (detected by BrdU labelling) was reduced in mutant brains, but the amount of cell death was unchanged, suggesting that extracellular glutamate concentration modulates neurogenesis at E16.
A laminar organization of the neocortex is usually seen by E16, but this pattern was severely disturbed in the brains of mutant mice, suggesting an impairment of cortical cell migration from the ventricular zone to other layers. BrdU labelling confirmed the presence of migration defects. Moreover, antibody staining revealed a disruption of radial glial fibres, which normally guide postmitotic neurons during migration. Neuronal tracing experiments with a fluorescent dye showed that these guidance defects resulted in severe disruptions to corticothalamic and thalamocortical pathways.
To confirm the involvement of excess glutamatergic signalling in these developmental defects, the authors administered antagonists of NMDA (N-methyl-D-aspartate receptors) and AMPA (
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors to mutant mice. Treatment resulted in a partial restoration of the neocortical laminar structure, suggesting that signalling through these receptors does indeed modulate early brain developmental processes, but also that other mechanisms are important for these processes.
It will be interesting to determine which other neurotransmitter systems have a role in early brain development and to unravel the separate contributions of neuronal activity and genetic programming. The use of excess stimulation, in addition to genetic ablation, of neurotransmitter pathways might be one way to answer these questions.