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Cyclic nucleotide--gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409-417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric ([Alpha] subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability ([P.sub.o]), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition ora channel. In single-channel recordings, DAG decreased the [P.sub.o] but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state (s) of a CNG channel by binding directly to the channel and/or by altering bilayer-channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer. KEY WORDS: rod * olfactory receptor * channel modulation * lipid bilayer * tetracaine

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens. [ABSTRACT FROM AUTHOR]

Background: Insect metamorphosis relies on temporal and spatial cues that are precisely controlled. Previous studies in Drosophila have shown that untimely activation of genes that are essential to metamorphosis results in growth defects, developmental delay and death. Multiple factors exist that safeguard these genes against dysregulated expression. The list of identified negative regulators that play such a role in Drosophila development continues to expand. Results: By using RNAi transgene-induced gene silencing coupled to spatio/temporal assessment, we have unraveled an important role for the Drosophila dopamine 1-like receptor, Dop1R2, in development. We show that Dop1R2 knockdown leads to pre-adult lethality. In adults that escape death, abnormal wing expansion and/or melanization defects occur. Furthermore we show that salivary gland expression of this GPCR during the late larval/prepupal stage is essential for the flies to survive through adulthood. In addition to RNAi-induced effects, treatment of larvae with the high affinity D1-like receptor antagonist flupenthixol, also results in developmental arrest, and in morphological defects comparable to those seen in Dop1R2 RNAi flies. To examine the basis for pupal lethality in Dop1R2 RNAi flies, we carried out transcriptome analysis. These studies revealed up-regulation of genes that respond to ecdysone, regulate morphogenesis and/or modulate defense/immunity. Conclusion: Taken together our findings suggest a role for Dop1R2 in the repression of genes that coordinate metamorphosis. Premature release of this inhibition is not tolerated by the developing fly. [ABSTRACT FROM AUTHOR]

Synapses are surrounded by a layer of extracellular matrix (ECM), which is instrumental for their development and maintenance. ECM composition is dynamically controlled by proteases, but how the precise composition of the ECM affects synaptic morphology is largely unknown. Through an unbiased forward genetic screen, we found that Caenorhabditis elegans gon-1, a conserved extracellular ADAMTS protease, is required for maintaining proper synaptic morphology at the neuromuscular junction. In gon-1 mutants, once synapse formation is complete, motor neuron presynaptic varicosities develop into large bulbous protrusions that contain synaptic vesicles and active zone proteins. A concomitant overgrowth of postsynaptic muscle membrane is found in close apposition to presynaptic axonal protrusions. Mutations in the muscle-specific, actin-severing protein cofilin (unc-60) suppress the axon phenotype, suggesting that muscle outgrowth is necessary for presynaptic protrusions. gon-1 mutants can also be suppressed by loss of the ECM components collagen IV (EMB-9) and fibulin (FBL-1). We propose that GON-1 regulates a developmental switch out of an initial "pro-growth" phase during which muscle arms grow out and form synapses with motor neuron axons. We postulate that this switch involves degradation or reorganization of collagen IV (EMB-9), whereas FBL-1 opposes GON-1 by stabilizing EMB-9. Our results describe a mechanism for regulating synaptic ECM composition and reveal the importance of precise ECM composition for neuronal morphology and synapse integrity. [ABSTRACT FROM AUTHOR]

Synaptogenesis involves the transformation of a growth cone into synaptic boutons specialized for transmitter release. In Drosophila embryos lacking the α2δ-3 subunit of presynaptic, voltage-dependent Ca2+ channels, we found that motor neuron terminals failed to develop synaptic boutons and cytoskeletal abnormalities arose, including the loss of ankyrin2. Nevertheless, functional presynaptic specializations were present and apposed to clusters of postsynaptic glutamate receptors. The α2δ-3 protein has been thought to function strictly as an auxiliary subunit of the Ca2+ channel, but the phenotype of α2δ-3 (also known as stj) mutations cannot be explained by a channel defect; embryos lacking the pore-forming α1 subunit cacophony formed boutons. The synaptogenic function of α2δ-3 required only the α2 peptide, whose expression sufficed to rescue bouton formation. Our results indicate that α2δ proteins have functions that are independent of their roles in the biophysics and localization of Ca2+ channels and that synaptic architecture depends on these functions. [ABSTRACT FROM AUTHOR]

Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their α1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary α2δ, β, and γ subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. We have isolated mutations in the gene encoding an α2δ calcium channel subunit (dα2δ -3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of dα2δ-3 mutants resembles that of the calcium channel α1 mutant cacophony (cac), suggesting shared functions. We have examined in detail genotypes that survive to the third-instar stage. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the α2δ calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel α1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in dα2δ-3 mutants. This is the first characterization of an α2δ-3 mutant in any organism and indicates a necessary role for α2δ-3 in presynaptic vesicle release and calcium channel expression at active zones. [ABSTRACT FROM AUTHOR]

The morphological transition of growth cones to synaptic boutons characterizes synaptogenesis. Here we have isolated mutations in immaculate connections (imac; CG8566), a previously uncharacterized Drosophila gene encoding a member of the Kinesin-3 family. Whereas earlier studies in Drosophila implicated Kinesin-1 in transporting synaptic vesicle precursors, we find that Imac is essential for this transport. An unexpected feature of imac mutants is the failure of synaptic boutons to form. Motor neurons lacking imac properly target to muscles but remain within target fields as thin processes, a structure that is distinct from either growth cones or mature terminals. Few active zones form at these endings. We show that the arrest of synaptogenesis is not a secondary consequence of the absence of transmission. Our data thus indicate that Imac transports components required for synaptic maturation and provide insight into presynaptic maturation as a process that can be differentiated from axon outgrowth and targeting. [ABSTRACT FROM AUTHOR]

Synaptogenesis involves the transformation of a growth cone into synaptic boutons specialized for transmitter release. In Drosophila embryos lacking the alpha(2)delta-3 subunit of presynaptic, voltage-dependent Ca(2+) channels, we found that motor neuron terminals failed to develop synaptic boutons and cytoskeletal abnormalities arose, including the loss of ankyrin2. Nevertheless, functional presynaptic specializations were present and apposed to clusters of postsynaptic glutamate receptors. The alpha(2)delta-3 protein has been thought to function strictly as an auxiliary subunit of the Ca(2+) channel, but the phenotype of alpha(2)delta-3 (also known as stj) mutations cannot be explained by a channel defect; embryos lacking the pore-forming alpha(1) subunit cacophony formed boutons. The synaptogenic function of alpha(2)delta-3 required only the alpha(2) peptide, whose expression sufficed to rescue bouton formation. Our results indicate that alpha(2)delta proteins have functions that are independent of their roles in the biophysics and localization of Ca(2+) channels and that synaptic architecture depends on these functions.

Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their alpha1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary alpha2delta, beta, and gamma subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. We have isolated mutations in the gene encoding an alpha2delta calcium channel subunit (d alpha2delta-3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of d alpha2delta-3 mutants resembles that of the calcium channel alpha1 mutant cacophony (cac), suggesting shared functions. We have examined in detail genotypes that survive to the third-instar stage. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the alpha2delta calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel alpha1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in d alpha2delta-3 mutants. This is the first characterization of an alpha2delta-3 mutant in any organism and indicates a necessary role for alpha2delta-3 in presynaptic vesicle release and calcium channel expression at active zones.

The expression patterns of two dopamine receptor genes, Amdop1 and Amdop2, in the developing mushroom bodies of the honeybee brain were determined by using in situ hybridisation. Both genes were expressed throughout pupal development, but their patterns of expression in the three major divisions of mushroom body intrinsic neurons (outer compact cells, noncompact cells, and inner compact cells) were quite distinct. Amdop1 expression could be detected in all three mushroom body cell groups throughout development. Staining for Amdop1 mRNA was particularly intense in newly born Kenyon cells, suggesting that levels of Amdop1 expression are higher in newborn cells than in more mature mushroom body neurons. This was not the case for Amdop2. Amdop2 expression in the mushroom bodies was restricted to inner and outer compact cells during most of pupal development, appearing in noncompact cells only late in metamorphosis or at adult eclosion. In contrast to the case with Amdop1, staining for Amdop2 mRNA was observed in glial cells. Expression of Amdop2 in glial cells was detected only at early stages of glial cell development, when the cells are reported to be actively dividing. This study not only implicates dopamine in the development of honeybee mushroom bodies but also suggests different roles for the two dopamine receptors investigated., (Copyright 2003 Wiley-Liss, Inc.)