A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity.

A novel organotypic culture method of mouse retina explants is being introduced and characterized to evaluate its usefulness in studying glutamate excitotoxicity. Retinal whole-mounts were dissected from eyes of C57BL/6 mice aged P10-14 and transferred to poly-D-lysine/laminin coated round coverslips. After 7 days in vitro, retina explants were treated with varying concentrations of L-glutamate and cell death was accessed with TUNEL histochemistry. Neurofilament-68 kDa immunoreactivity was used to identify retinal ganglion cells (RGC) with immunohistochemistry. Additional cell markers were used to further characterize the cytoarchitecture of the organotypic retina cultures. Retina explants attached very well to the coated coverslips allowing for experimental manipulation and pharmacological access to the tissue. Hematoxylin-Eosin (HE) staining of vertical cryostat sections of retina explants demonstrated well preserved intact cytoarchitecture under organotypic culture conditions and PKCalpha, Calbindin, GABA, Rhodopsin, GFAP and neurofilament immunoreactivities identifying rod bipolar, horizontal, amacrine, photoreceptor, glial, and retinal ganglion cells, respectively, were not different from freshly isolated mouse retina. Dose dependent glutamate toxicity and accompanying RGC apoptotic cell death were determined by TUNEL histochemistry. In contrast to previously published methods using slice or floating whole-mount cultures, the ex vivo culture system presented here combines accessibility to experimental manipulation, and adherence of whole-mount cultures to a substrate with a significant preservation of retinal cell types, numbers and morphology. The described retina explant culture on glass coverslips allows for effective pharmacological manipulation including the study of neuronal cell death and RGC physiology.

Homer proteins control neuronal differentiation through IP(3) receptor signaling.

Neurons expand, sustain or prune their dendritic trees during ontogenesis [Cline, H.T. (2001). Dendritic arbor development and synaptogenesis. Curr. Opin. Neurobiol. 11, 118-126; Wong, W.T. and Wong, R.O.L. (2000) Rapid dendritic movements during synapse formation and rearrangement. Curr. Opin. Neurobiol. 10, 118-124] which critically depends on neuronal activity [Wong, W.T., Faulkner-Jones, B.E., Sanes, J.R. and Wong, R.O.L. (2000) Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho. J. Neurosci. 20, 5024-5036; Li, Z., Van Aelst, L. and Cline, H.T. (2000) Rho GTPases regulate distinct aspects of dendritic arbor growth in Xenopus central neurons in vivo. Nat. Neurosci. 3, 217-225; Wong, W.T. and Wong, R.O.L. (2001) Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat. Neurosci. 4, 351-352.] and sub-cellular Ca(2+) signals [Lohmann, C., Myhr, K.L. and Wong, R.O. (2002) Transmitter-evoked local calcium release stabilizes developing dendrites, Nature 418, 177-181.]. The role of synaptic clustering proteins connecting both processes is unclear. Here, we show that expression levels of Vesl-1/Homer 1 isoforms critically control properties of Ca(2+) release from intracellular stores and dendritic morphology of CNS neurons. Vesl-1L/Homer 1c, an isoform with a functional WH1 and coiled-coil domain, but not isoforms missing these features were capable of potentiating intracellular calcium signaling activity indicating that such regulatory interactions function as a general paradigm in cellular differentiation and are subject to changes in expression levels of Vesl/Homer isoforms.

Polycystin-2 accelerates Ca2+ release from intracellular stores in Caenorhabditis elegans.

Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that polycystin-2 is an intracellular Ca(2+) release channel that is required for the normal pattern of Ca(2+) responses involving IP(3) and ryanodine receptor-mediated Ca(2+) release from intracellular stores. Activity of polycystin-2 creates brief cytosolic Ca(2+) transients with increased amplitude and decreased duration. Polycystin-2, along with the IP(3) and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.