The Rab5 guanylate exchange factor Rin1 regulates endocytosis of the EphA4 receptor in mature excitatory neurons.

Ephrin signaling through Eph receptor tyrosine kinases regulates important morphogenetic events during development and synaptic plasticity in the adult brain. Although Eph-ephrin endocytosis is required for repulsive axon guidance, its role in postnatal brain and synaptic plasticity is unknown. Here, we show that Rin1, a postnatal brain-specific Rab5-GEF, is coexpressed with EphA4 in excitatory neurons and interacts with EphA4 in synaptosomal fractions. The interaction of Rin1 and EphA4 requires Rin1's SH2 domain, consistent with the view that Rin1 targets tyrosine phosphorylated receptors to Rab5 compartments. We find that Rin1 mediates EphA4 endocytosis in postnatal amygdala neurons after engagement of EphA4 with its cognate ligand ephrinB3. Rin1 was shown to suppress synaptic plasticity in the amygdala, a forebrain structure important for fear learning, possibly by internalizing synaptic receptors. We find that the EphA4 receptor is required for synaptic plasticity in the amygdala, raising the possibility that an underlying mechanism of Rin1 function in amygdala is to down-regulate EphA4 signaling by promoting its endocytosis.

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin.

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.

Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.

Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.