The K+ channel gene ether a go-go is required for the transduction of a subset of odorants in adult Drosophila melanogaster

The functional identity of an olfactory receptor neuron is determined in part by its repertoire of responses to odorants. As an approach toward understanding the contributions of particular conductances to olfactory neuron excitability and odor discrimination, we have investigated the role of the putative cyclic nucleotide-modulated K+ channel subunit encoded by the ether a go-go (eag) gene in odorant responsiveness in Drosophila melanogaster. Four independent mutant eag alleles exhibited reduced antennal sensitivity to a subset of nine odorants, all having short aliphatic side chains: ethyl butyrate (EB), propionic acid, 2-butanone, and ethyl acetate. Significantly fewer eag antennal neurons responded to EB compared with control neurons; the proportion sensitive to 2-heptanone was similar to controls. Two aspects of the character of EB-induced excitability were affected by mutations in eag. First, fewer EB-induced inhibitory responses were observed in eag mutants, and second, fewer excitatory odorant responses dependent on extracellular Ca2+ were observed. Furthermore, modulation of neuronal excitability by membrane-permeant cyclic nucleotide analogs was largely eag dependent. Focal application of high K+ saline to sensillae altered the excitability of the majority of neurons from wild-type but not eag antennae, suggesting that Eag may have a dendritic localization.     

OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans

Although cyclic nucleotide-gated channels mediate sensory transduction in olfaction and vision, other forms of sensory transduction are independent of these channels. Caenorhabditis elegans cyclic nucleotide-gated channel mutants respond normally to some olfactory stimuli and to osmotic stimuli, suggesting that these chemosensory responses use an alternative sensory transduction pathway. One gene that may act in this pathway is osm-9, which is required for each of these responses as well as a mechanosensory response to nose touch. osm-9 encodes a protein with ankyrin repeats and multiple predicted transmembrane domains that has limited similarity to the Drosophila phototransduction channels transient receptor potential (TRP) and TRP-like (TRPL). The sequence of OSM-9 and other TRP-like genes reveals a previously unsuspected diversity of mammalian and invertebrate genes in this family. osm-9 is required for the activity of the predicted G-protein-coupled odorant receptor ODR-10, which acts in the AWA olfactory neurons; its similarity to other G-protein-regulated transduction channels suggests that OSM-9 is involved in AWA signaling. osm-9:: GFP fusion genes are expressed in a subset of chemosensory, mechanosensory, and osmosensory neurons. osm-9 also affects olfactory adaptation within neurons that require the cyclic nucleotide-gated channel for olfaction; in these neurons, the gene has a regulatory function and not a primary role in sensory transduction.     

A modified coaxial compound micropipette for extracellular iontophoresis and intracellular recording: fabrication, performance and theory

Different olfactory cues elicit distinct behaviors such as attraction, avoidance, feeding, or mating. In the nematode C. elegans, these cues are sensed by a small number of olfactory neurons, each of which expresses several different odorant receptors. The type of behavioral response elicited by an odorant could be specified by the olfactory receptor or by the olfactory neuron in which the receptor is activated. The attractive odorant diacetyl is detected by the receptor protein ODR-10, which is normally expressed in the AWA olfactory neurons. The repulsive odorant 2-nonanone is detected by the AWB olfactory neurons. Transgenic animals that express ODR-10 in AWB rather than AWA avoid diacetyl, while maintaining qualitatively normal responses to other attractive and repulsive odorants. Animals that express ODR-10 simultaneously in AWA and AWB have a defective response to diacetyl, possibly because of conflicting olfactory inputs. Thus, an animal's preference for an odor is defined by the sensory neurons that express a given odorant receptor molecule.     

The Drosophila dgq gene encodes a G alpha protein that mediates phototransduction

We examined the roles of the Drosophila Gq alpha proteins (DGq) in the phototransduction pathway. The DGq proteins immunolocalized to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocked the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulated DGq. Dominantly active DGq1 mutants exhibited a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics compared with wild-type flies. Dominant DGq2 mutants exhibited a light-independent GTPase activity with normal electrophysiological light responses. Retinas of double mutants of DGq1, but not DGq2, with the light-dependent retinal degeneration mutant rdgB degenerated even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark was norpA-dependent. These results indicate that DGq1 mediates the stimulation by light-activated rhodopsin of the norpA-encoded phospholipase C in the visual transduction cascade.     

Male-male courtship behavior induced by ectopic expression of the Drosophila white gene: role of sensory function and age

Male-male courtship behavior was recently reported to be induced in large populations of Drosophila (e.g., 600-1500 flies) by ectopic expression of the white (w) gene. Little is known about the basis of this behavior; in male-female courtship, sensory cues are believed to play an important role. Previous data are consistent with the possibility that misexpression of w causes abnormal reception or processing of sensory information. We show here that w-induced male-male courtship occurs in isolated pairs of flies. Thus the behavior does not depend on sensory cues found only among large populations of flies, or on cues produced only by a small subset of such populations. This finding enabled quantitative analysis of mechanisms that underlie the behavior. Specifically, male-male courtship does not depend on the reception of olfactory information, nor on the reception or generation of auditory cues, as determined by surgical ablation of antennae, maxillary palps, or wings. Although the rapid onset of the behavior following w induction suggested that its basis could lie in a modulation of sensory physiology, we found visual, olfactory, and gustatory function to be normal in physiological or behavioral tests. The only sensory deprivation to produce an effect on male-male courtship was testing under dim red light; the percentage of flies courting another male was reduced to one-fourth of control values. A striking age dependence of the behavior is also documented: courtship between paired male mini-w+ flies was not observed in tests of very young (1-day-old) flies, but occurs at high levels between the ages of 1 and 4 weeks.     

Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors

The lineage of olfactory neurons has been relatively well characterized at the cellular level, but the genes that regulate the proliferation and differentiation of their progenitors are currently unknown. In this study, we report the isolation of a novel murine gene, Math4C/neurogenin1, which is distantly related to the Drosophila proneural gene atonal. We show that Math4C/neurogenin1 and the basic helix-loop-helix gene Mash1 are expressed in the olfactory epithelium by different dividing progenitor populations, while another basic helix-loop-helix gene, NeuroD, is expressed at the onset of neuronal differentiation. These expression patterns suggest that each gene marks a distinct stage of olfactory neuron progenitor development, in the following sequence: Mash1>Math4C/neurogenin1>NeuroD. We have previously reported that inactivation of Mash1 function leads to a severe reduction in the number of olfactory neurons. We show here that most cells in the olfactory epithelium of Mash1 mutant embryos fail to express Math4C/neurogenin1 or NeuroD. Strikingly, a subset of progenitor cells in a ventrocaudal domain of Mash1 mutant olfactory epithelium still express Math4C/neurogenin1 and NeuroD and differentiate into neurons. Cells in this domain also express Math4A/neurogenin2, another member of the Math4/neurogenin gene family, and not Mash1. Our results demonstrate that Mash1 is required at an early stage in the olfactory neuron lineage to initiate a differentiation program involving Math4C/neurogenin1 and NeuroD. Another gene activates a similar program in a separate population of olfactory neuron progenitors.     

Functional and molecular characterization of individual olfactory neurons

To gain an understanding of the olfactory signal transduction process, individual chemosensory neurons have been assessed for odor-induced Ca2+ responses and the molecular elements of transduction cascades using Ca2+ imaging technique in combination with single-cell RT-PCR approaches. It has been demonstrated that responsiveness of cells to cyclic AMP or inositol trisphosphate odorants was blocked by specific adenylyl cyclase inhibitors or phospholipase C inhibitors, respectively. Using specific primers in single-cell RT-PCR analysis, olfactory marker protein, two G protein subtypes (G(olf) and G(o)), and adenylyl cyclase (subtype III) and a phospholipase C (phospholipase Cbeta2-related subtype) were identified. For a subpopulation of sensory neurons it was demonstrated that both transduction cascades coexist and are active in the same cell. These data support the notion that two second messenger pathways are active in olfactory sensory neurons and emphasize the concept of dual transduction cascades in olfaction.     

Genetic interactions between mei-S332 and ord in the control of sister-chromatid cohesion

The Drosophila mei-S332 and ord gene products are essential for proper sister-chromatid cohesion during meiosis in both males and females. We have constructed flies that contain null mutations for both genes. Double-mutant flies are viable and fertile. Therefore, the lack of an essential role for either gene in mitotic cohesion cannot be explained by compensatory activity of the two proteins during mitotic divisions. Analysis of sex chromosome segregation in the double mutant indicates that ord is epistatic to mei-S332. We demonstrate that ord is not required for MEI-S332 protein to localize to meiotic centromeres. Although overexpression of either protein in a wild-type background does not interfere with normal meiotic chromosome segregation, extra ORD+ protein in mei-S332 mutant males enhances nondisjunction at meiosis II. Our results suggest that a balance between the activity of mei-S332 and ord is required for proper regulation of meiotic cohesion and demonstrate that additional proteins must be functioning to ensure mitotic sister-chromatid cohesion.     

Serotonin-deficient mutants and male mating behavior in the nematode Caenorhabditis elegans

Defining a behavior that requires the function of specific neurons in the free-living nematode Caenorhabditis elegans can allow one to screen for mutations that disrupt the specification or function of those neurons. We identified serotonin-immunoreactive neurons required for tail curling or "turning" behavior exhibited by C. elegans males during mating. Males mutant in three different genes that reduce serotonin expression, cat-1, cat-4, and bas-1, exhibited defects in turning behavior similar to those of wild-type males in which these neurons were ablated. The turning defect of cat-4 males was rescued by exogenous serotonin, consistent with the idea that their behavioral defect is caused by a lack of serotonin. While the serotonin-deficient mutants we analyzed shared certain behavioral traits, they were blocked for serotonin synthesis at different steps. Analysis of these and additional serotonin-deficient mutants may help us understand how a neuron controls the expression of a serotonergic phenotype.     

The shaking-B2 mutation disrupts electrical synapses in a flight circuit in adult Drosophila

The shaking-B2 mutation was used to analyze synapses between haltere afferents and a flight motoneuron in adult Drosophila. We show that the electrical synapses among many neurons in the flight circuit are disrupted in shaking-B2 flies, suggesting that shaking-B expression is required for electrical synapses throughout the nervous system. In wild-type flies haltere afferents are dye-coupled to the first basalar motoneuron, and stimulation of these afferents evokes electromyograms from the first basalar muscle with short latencies. In shaking-B2 flies dye coupling between haltere afferents and the motoneuron is abolished, and afferent stimulation evokes electromyograms at abnormally long latencies. Intracellular recordings from the motoneuron confirm that the site of the defect in shaking-B2 flies is at the synapses between haltere afferents and the flight motoneuron. The nicotinic cholinergic antagonist mecamylamine blocks the haltere-to-flight motoneuron synapses in shaking-B2 flies but does not block those synapses in wild-type flies. Together, these results show that the haltere-to-flight motoneuron synapses comprise an electrical component that requires shaking-B and a chemical component that is likely to be cholinergic.     

Growth-related and antennular amputation-induced changes in the olfactory centers of crayfish brain

Freshwater crayfish increase in size throughout their lives, and this growth is accompanied by an increase in the length of the appendages and number of mechanoreceptive and chemoreceptive sensilla on them. We find that in the Australian freshwater crayfish Cherax destructor, neuropil volumes of the olfactory centers increase linearly with body size over the entire size range of animals found in their natural habitat. The number of cell somata of two groups of interneurons associated with the olfactory centers (projection neurons and small local neurons) also increases linearly with the size of the animals. In contrast, axon counts of interneurons that represent a nonolfactory input to the olfactory centers show that these reach a total number in the very early adult stages that then remains constant regardless of the size of the animal. Only the axon diameter of these interneurons increases linearly with body size. Amputation of the antennule and olfactory sensilla reduces the number of projection and local interneurons on the amputated side. No change in the size of the olfactory centers occurs on the unamputated side. Amputation of the olfactory receptor neurons in crayfish therefore leads not only to a degeneration of the receptor cell endings in the olfactory lobe but also to a trans-synaptic response in which the number of higher order neurons decreases. Reconstitution of the antennule and olfactory receptor neurons in small adult crayfish is accompanied by the reestablishment of the normal number of interneurons and neuropil volume in the olfactory centers.     

Selective loss of cholinergic neurons projecting to the olfactory system increases perceptual generalization between similar, but not dissimilar, odorants.

The neuromodulator acetylcholine is thought to modulate information processing in the olfactory system. The authors used 192 IgG-saporin, a lesioning agent selective for basal forebrain cholinergic neurons, to determine whether selective lesions of cholinergic neurons projecting to the olfactory bulb and cortex affect odor perception in rats. Lesioned and sham-operated rats were tested in an olfactory generalization paradigm with sets of chemically related odorants (n-aliphatic aldehydes, acids, and alcohols). Lesioned rats generalized more between chemically similar odorants but did not differ from controls in their response to chemically unrelated odorants or in acquisition of the conditioned odor. Results show that cholinergic inputs to the olfactory system influence perceptual qualities of odorants and confirm predictions made by computational models of this system. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Phosphorylation-dependent power output of transgenic flies: an integrated study

We examine how the structure and function of indirect flight muscle (IFM) and the entire flight system of Drosophila melanogaster are affected by phosphorylation of the myosin regulatory light chain (MLC2). This integrated study uses site-directed mutagenesis to examine the relationship between removal of the myosin light chain kinase (MLCK) phosphorylation site, in vivo function of the flight system (flight tests, wing kinematics, metabolism, power output), isolated IFM fiber mechanics, MLC2 isoform pattern, and sarcomeric ultrastructure. The MLC2 mutants exhibit graded impairment of flight ability that correlates with a reduction in both IFM and flight system power output and a reduction in the constitutive level of MLC2 phosphorylation. The MLC2 mutants have wild-type IFM sarcomere and cross-bridge structures, ruling out obvious changes in the ultrastructure as the cause of the reduced performance. We describe a viscoelastic model of cross-bridge dynamics based on sinusoidal length perturbation analysis (Nyquist plots) of skinned IFM fibers. The sinusoidal analysis suggests the high power output of Drosophila IFM required for flight results from a phosphorylation-dependent recruitment of power-generating cross-bridges rather than a change in kinetics of the power generating step. The reduction in cross-bridge number appears to affect the way mutant flies generate flight forces of sufficient magnitude to keep them airborne. In two MLC2 mutant strains that exhibit a reduced IFM power output, flies appear to compensate by lowering wingbeat frequency and by elevating wingstroke amplitude (and presumably muscle strain). This behavioral alteration is not seen in another mutant strain in which the power output and estimated number of recruited cross-bridges is similar to that of wild type.     

Zebrafish touch-insensitive mutants reveal an essential role for the developmental regulation of sodium current

Developmental changes in neuronal connectivity and membrane properties underlie the stage-specific appearance of embryonic behaviors. The behavioral response of embryonic zebrafish to tactile stimulation first appears at 27 hr postfertilization. Because the touch response requires the activation of mechanosensory Rohon-Beard neurons, we have used whole-cell recordings in semi-intact preparations to characterize Rohon-Beard cell electrical membrane properties in several touch-insensitive mutants and then to correlate the development of excitability in these cells with changes in wild-type behavior. Electrophysiological analysis of mechanosensory neurons of touch-insensitive zebrafish mutants indicates that in three mutant lines that have been examined the sodium current amplitudes are reduced, and action potentials either have diminished overshoots or are not generated. In macho mutants the action potential never overshoots, and the sodium current remains small; alligator and steifftier show similar but weaker effects. The effects are specific to sodium channel function; resting membrane potentials are unaffected, and outward currents of normal amplitude are present. Developmental analysis of sodium current expression in mechanosensory neurons of wild-type embryos indicates that, during the transition from a touch-insensitive to a touch-sensitive embryo, action potentials acquire larger overshoots and briefer durations as both sodium and potassium currents increase in amplitude. However, in macho touch-insensitive mutants, developmental changes in action potential overshoot and sodium current are absent despite the normal regulation of action potential duration and potassium current. Thus, the maturation of a voltage-dependent sodium current promotes a behavioral response to touch. A study of these mutants will allow insight into the genes controlling the maturation of the affected sodium current.     

Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene

We have used gene targeting to examine the role of the G alpha subunit, G(olf), in olfactory signal transduction. Mice homozygous for a null mutation in G(olf) show a striking reduction in the electrophysiological response of primary olfactory sensory neurons to a wide variety of odors. Despite this profound diminution in response to odors, the topographic map of primary sensory projections to the olfactory bulb remains unaltered in G(olf) mutants. Greater than 75% of the G(olf) mutant mice are unable to nurse and die within 2 days after birth. Rare surviving homozygotes mate and are fertile, but mutant females exhibit inadequate maternal behaviors. Surviving homozygous mutant mice also exhibit hyperactive behaviors. These behavioral phenotypes, taken together with the patterns of G(olf) expression, suggest that G(olf) is required for olfactory signal transduction and may also function as an essential signaling molecule more centrally in the brain.     

Modulation of olfactory bulb neuron potassium current by tyrosine phosphorylation

Insulin causes a suppression of whole-cell voltage-dependent outward current in cultured neurons from the rat olfactory bulb. This suppression is time-dependent; it is mimicked by application of Src tyrosine kinase inside the cell via the whole-cell patch electrode or by treatment of the olfactory bulb neurons with the tyrosine phosphatase inhibitor pervanadate. The C-type inactivation properties of the outward current in olfactory bulb neurons resemble those of the cloned Kv1.3 potassium channel. In addition, at picomolar concentrations at which it is specific for Kv1.3, the scorpion toxin margatoxin blocks most of the olfactory bulb neuron outward current. Immunocytochemical analysis demonstrates that Kv1.3 is prominent in the cultured olfactory bulb neurons. To identify specific amino acid residues that might be important for potassium current modulation, we examined the effects of pervanadate and insulin on wild-type and mutant Kv1.3 channels expressed in human embryonic kidney (HEK 293) cells. As shown previously, treatment with either pervanadate or insulin suppresses Kv1.3 current in these cells. Mutational analysis demonstrates that at least two distinct tyrosine residues are required for current suppression by pervanadate. Insulin treatment stimulates the tyrosine phosphorylation of Kv1.3 in HEK 293 cells, and a different combination of tyrosine residues is required for the current suppression by insulin. The results suggest that complex patterns of phosphorylation may be involved in the modulation of neuronal potassium current by receptor and nonreceptor tyrosine kinases.