The maintenance of spine and synapse number during development is critical for neuronal circuit formation and function. Here we show that ␦-catenin, a component of the cadherin-catenin cell adhesion complex, regulates spine and synapse morphogenesis during development. Genetic ablation or acute knockdown of ␦-catenin leads to increases in spine and synapse density, accompanied by a decrease in tetrodotoxin induced spine plasticity. Our results indicate that ␦-catenin may mediate conversion of activity-dependent signals to morphological spine plasticity. The functional role of ␦-catenin in regulating spine density does not require binding to cadherins, but does require interactions with PDZ domain-containing proteins. We propose that the perturbations in spine and synaptic structure and function observed after depletion of ␦-catenin during development may contribute to functional alterations in neural circuitry, the cognitive deficits observed in mutant mice, and the mental retardation pathology of Cri-du-chat syndrome.

The cacophony (cac) locus in Drosophila encodes a Ca 2ϩ channel ␣ subunit, but little is known about properties of cac-mediated currents and functional consequences of cac mutations in central neurons. We found that, in Drosophila cultured neurons, Ca 2ϩ currents were mediated predominantly by the cac channels. The cac channels contribute to low-and high-threshold, fast-and slow-inactivating types of Ca 2ϩ currents, take part in membrane depolarization, and strongly activate Ca 2ϩ -activated K ϩ current [I K(Ca) ]. In cac neurons, unexpectedly, voltage-activated transient K ϩ current I A is upregulated to a level that matches I K(Ca) reduction, implicating a homeostatic regulation that was mimicked by chronic pharmacological blockade of Ca 2ϩ currents in wild-type neurons. Among K ϩ channel transcripts, Shaker mRNA levels were preferentially increased in cac flies. However, Ca 2ϩ current expression levels remained unaltered in several K ϩ channel mutants, illustrating a key role of cac in developmental regulation of Drosophila neuronal excitability.

Different K+ currents participate in generating neuronal firing patterns. The Drosophila embryonic “giant” neuron culture system has facilitated current- and voltage-clamp recordings to correlate distinct excitability patterns with the underlying K+ currents and to delineate the mutational effects of identified K+ channels. Mutations of Sh and Shab K+ channels removed part of inactivating IA and sustained IK, respectively, and the remaining IA and IK revealed the properties of their counterparts, e.g., Shal and Shaw channels. Neuronal subsets displaying the delayed, tonic, adaptive, and damping spike patterns were characterized by different profiles of K+ current voltage dependence and kinetics and by differential mutational effects. Shab channels regulated membrane repolarization and repetitive firing over hundreds of milliseconds, and Shab neurons showed a gradual decline in repolarization during current injection and their spike activities became limited to high-frequency, damping firing. In contrast, Sh channels acted on events within tens of milliseconds, and Sh mutations broadened spikes and reduced firing rates without eliminating any categories of firing patterns. However, removing both Sh and Shal IA by 4-aminopyridine converted the delayed to damping firing pattern, demonstrating their actions in regulating spike initiation. Specific blockade of Shab IK by quinidine mimicked the Shab phenotypes and converted tonic firing to a damping pattern. These conversions suggest a hierarchy of complexity in K+ current interactions underlying different firing patterns. Different lineage-defined neuronal subsets, identifiable by employing the GAL4-UAS system, displayed different profiles of spike properties and K+ current compositions, providing opportunities for mutational analysis in functionally specialized neurons.

Dact1 (Dapper/Frodo), an intracellular phosphoprotein that binds Dishevelled, catenins, and other signaling proteins, is expressed in the developing and mature mammalian CNS, but its function there is unknown. Dact1 colocalized with synaptic markers and partitioned to postsynaptic fractions from cultured mouse forebrain neurons. Hippocampal neurons from Dact1 knock-out mice had simpler dendritic arbors and fewer spines than hippocampal neurons from wild-type littermates. This correlated with reductions in excitatory synapses and miniature EPSCs, whereas inhibitory synapses were not affected. Loss of Dact1 resulted in a decrease in activated Rac, and recombinant expression of either Dact1 or constitutively active Rac, but not Rho or Cdc42, rescued dendrite and spine phenotypes in Dact1 mutant neurons. Our findings suggest that, during neuronal differentiation, Dact1 plays a critical role in a molecular pathway promoting Rac activity underlying the elaboration of dendrites and the establishment of spines and excitatory synapses.

Environmental temperature is an important factor exerting pervasive influence on neuronal morphology and synaptic physiology. In the Drosophila brain, axonal arborization of mushroom body Kenyon cells was enhanced when flies were raised at high temperature (30°C rather than 22°C) for several days. Isolated embryonic neurons in culture that lacked cell-cell contacts also displayed a robust temperature-induced neurite outgrowth. This cell-autonomous effect was reflected by significantly increased high-order branching and enlarged growth cones. The temperature-induced morphological alterations were blocked by the Na ϩ channel blocker tetrodotoxin and a Ca 2ϩ channel mutation but could be mimicked by raising cultures at room temperature with suppressed K ϩ channel activity. Physiological analyses revealed increased inward Ca 2ϩ currents and decreased outward K ϩ currents, in conjunction with a distal shift in the site of action potential initiation and increased prevalence of TTX-sensitive spontaneous Ca 2ϩ transients. Importantly, the overgrowth caused by both temperature and hyperexcitability K ϩ channel mutations were sensitive to genetic perturbations of cAMP metabolism. Thus, temperature acts in a cell-autonomous manner to regulate neuronal excitability and spontaneous activity. Presumably, activitydependent Ca 2ϩ accumulation triggers the cAMP cascade to confer the activity-dependent plasticity of neuronal excitability and growth.

Localization of presynaptic components to synaptic sites is critical for hippocampal synapse formation. Cell adhesion–regulated signaling is important for synaptic development and function, but little is known about differentiation of the presynaptic compartment. In this study, we describe a pathway that promotes presynaptic development involving p120catenin (p120ctn), the cytoplasmic tyrosine kinase Fer, the protein phosphatase SHP-2, and β-catenin. Presynaptic Fer depletion prevents localization of active zone constituents and synaptic vesicles and inhibits excitatory synapse formation and synaptic transmission. Depletion of p120ctn or SHP-2 similarly disrupts synaptic vesicle localization with active SHP-2, restoring synapse formation in the absence of Fer. Fer or SHP-2 depletion results in elevated tyrosine phosphorylation of β-catenin. β-Catenin overexpression restores normal synaptic vesicle localization in the absence of Fer or SHP-2. Our results indicate that a presynaptic signaling pathway through p120ctn, Fer, SHP-2, and β-catenin promotes excitatory synapse development and function.

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