To investigate the role of Dicer and microRNAs in the mammalian CNS, we used mice in which the second RNase III domain of Dicer was conditionally floxed. Conditional Dicer mice were bred with mice expressing an ␣-calmodulin kinase II Cre to selectively inactivate Dicer in excitatory forebrain neurons in vivo. Inactivation of Dicer results in an array of phenotypes including microcephaly, reduced dendritic branch elaboration, and large increases in dendritic spine length with no concomitant change in spine density. Microcephaly is likely caused by a 5.5-fold increase in early postnatal apoptosis in these animals as determined by active caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) staining in the cortex. Loss of Dicer function had no measurable effect on cortical lamination as determined by in situ hybridization, suggesting that microcephaly is not caused by defects in neuronal migration. Together, these results illustrate the in vivo significance of Dicer and miRNAs in the mammalian CNS and provide additional support for previous in vitro studies indicating that misregulation of this pathway may result in gross abnormalities in cell number and function that may contribute to a variety of neurological disorders.

Voltage-gated Naϩ channel ␤1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that ␤1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that ␤1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. ␤1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on ␤1-mediated neurite extension. These results suggest that ␤1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that ␤1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that ␤1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for ␤1 in vivo. We conclude that voltage-gated Na ϩ channel ␤1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel ␤1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or ␤1(؊/؊) mice were plated on top of parental, mock-, or ␤1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on ␤1-transfected monolayers, whereas ␤1(؊/؊) neurons showed no increase compared with control conditions. ␤1-Mediated neurite extension was mimicked using a soluble ␤1 extracellular domain and was blocked by antibodies directed against the ␤1 extracellular domain. Immunohistochemical analysis suggests that the ␤1 and ␤4 subunits, but not ␤2 and ␤3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for ␤1 during neuronal development and are the first demonstration of a functional role for sodium channel ␤ subunit-mediated cell adhesive interactions.Intercellular communications mediate critical developmental events in neurons. Interactions between integrins, cadherins, and immunoglobulin superfamily cell adhesion molecules (IGSF CAMs) 1 on opposing cells result in events such as growth cone guidance and neurite extension. For example, NCAM-and L1-CAM-mediated cell adhesive interactions result in signal transduction pathways involving kinase activation, modulation of local, submembraneous calcium concentrations, gene transcription, and ultimately, neurite extension (1-4). Some IGSF CAMs such as myelin-associated glycoprotein and contactin balance the growth promoting activity of other molecules through inhibition of neuritogenesis (5, 6). Thus, it is the concerted effort of growth-promoting and growth-inhibitory molecules on neuronal and non-neuronal cells that act to influence the developing nervous system. Postnatal cerebellar development involves migration of cerebellar granule neurons from the external germinal layer to the rapidly developing granule cell layer. During migration, a granule neuron develops several neurites, two of which ultimately become parallel fibers of the cerebellar molecular layer (7). Whereas most cell migration and neuritogenesis in the cerebellum is complete within the second postnatal week, migration of granule neurons, growth of the granule cell layer, and extension of parallel and vertical fibers continues through postnatal day 21 (P21) (7,8).Voltage-gated sodium channels are composed of a central, pore forming ␣-subunit and one or two ␤ subunits (9). Whereas ␣ alone is sufficient to form the ion-conducting pore, current density, channel kinetics, gating mode, and channel cell surface density are influenced by ␤ subunit expression (9, 10). There are five known ␤ subunits: ␤1, ␤1A, ␤2, ␤3, and ␤4. ␤1, ␤1A, and ␤3 are non-covalently linked to the pore-forming ␣ subunit, while ␤2 and ␤4 are disulfide linked to ␣. Based on structur...

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Background: Voltage-gated Na ϩ channels are composed of ␣ and ␤ subunits. Results: We identified the cysteine residue in ␤2 responsible for disulfide linkage to ␣. Conclusion: ␣ and ␤2 associate through a single disulfide bridge to achieve proper subcellular targeting in neurons. Significance: Understanding how Na ϩ channel complexes are formed in neurons is crucial for understanding the development