Granule neurons, presynaptic afferents of Purkinje cells, are potent regulators of Purkinje cell development. Purified Purkinje cells survive and differentiate poorly, whereas coculture with granule neurons enhances their survival and dendritic development. Here we investigate the role of neurotrophins in granule-Purkinje cell interactions. BDNF or NT-4 improves, but NT-3 or CNTF reduces, survival of isolated Purkinje cells. When granule neurons are present, however, BDNF or NT-4 treatment leads to Purkinje cell loss. This decrease is overcome by anti-BDNF or TrkB-IgG-blocking reagents or by CNQX, a non-NMDA glutamate receptor antagonist. Furthermore, BDNF increases the spine density on the surviving Purkinje cells. These results suggest that Purkinje cell survival and differentiation are context-dependent and require a balance between neurotrophin- and activity-dependent signaling.

Neurotrophins cooperate with neural activity to modulate CNS neuronal survival and dendritic differentiation. In a previous study, we demonstrated that a critical balance of neurotrophin and neural activity is required for Purkinje cell survival in cocultures of purified granule and Purkinje cells (Morrison and Mason, 1998). Here we investigate whether TrkB signaling regulates dendrite and spine development of Purkinje cells. BDNF treatment of purified Purkinje cells cultured alone did not elicit formation of mature dendrites or spines. In cocultures of granule and Purkinje cells, however, continuous treatment with BDNF over a 2 week postnatal culture period increased the density of Purkinje cell dendritic spines relative to controls without causing a shift in the proportions of headed and filopodia-like spines. The increase in spine number was blocked by adding TrkB-IgG to the medium together with BDNF. Although BDNF alone did not consistently modify the morphology of dendritic spines, treatment with TrkB-IgG alone yielded spines with longer necks than those in control cultures. None of these treatments altered Purkinje cell dendritic complexity. These analyses reveal a role for TrkB signaling in modulating spine development, consistent with recently reported effects of neurotrophins on synaptic function. Moreover, spine development can be uncoupled from dendrite outgrowth in this reductionist system of purified presynaptic and postsynaptic neurons.

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal-low nasal and high ventral-low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.

The poliovirus receptor (Pvr) is a member of the immunoglobulin superfamily of proteins, but its function in the cell is not known. Southern blot hybridization analysis indicated that the murine genome contains a sequence homolog of pvr. As a first step toward using the murine pvr homolog (mph) to study the function of Pvr, murine genomic and cDNA clones encoding mph were isolated. mph encodes a polypeptide with extensive sequence similarity to the extracellular domains of the human PVR. mph mRNAs of 2.0 and 3.0 kb are transcribed in the adult mouse brain, the spinal cord, the spleen, the kidney, the heart, and the liver. The Mph protein does not function as a receptor for poliovirus. However, substitution of domain 1 of the Mph protein with the corresponding sequence from pvr produced a chimeric receptor that could bind poliovirus and lead to productive infection. By constructing pvr-mph chimeras, it will be possible to identify the contact points of poliovirus within domain 1 of Pvr. Identification of the ligand and the cellular function of the Mph protein may help us understand the role of Pvr in the cell.

Poliovirus initiates infection of primate cells by binding to the poliovirus receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr homolog, Mph, that does not function as a poliovirus receptor. Previous work has shown that the first immunoglobulin-like domain of the Pvr protein contains the virus binding site. To further identify sequences of Pvr important for its interaction with poliovirus, stable cell lines expressing mutated Pvr molecules were examined for their abilities to bind virus and support virus replication. Substitution of the amino-terminal domain of Mph with that of Pvr yields a molecule that can function as a poliovirus receptor. Cells expressing this chimeric receptor have normal binding affinity for poliovirus, yet the kinetics of virus replication are delayed. Results of virus alteration assays indicate that this chimeric receptor is defective in converting native virus to 135S altered particles. This defect is not observed with cells expressing receptor recombinants that include Pvr domains 1 and 2. Because altered particles are believed to be an intermediate in poliovirus entry, these findings suggest that Pvr domains 2 and 3 participate in early stages of infection. Additional mutants were made by substituting variant Mph residues for the corresponding residues in Pvr. The results were interpreted by using a model of Pvr predicted from the known structures of other

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