A genetic analysis of glutamatergic function in Drosophila

Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity [3H]-glutamate binding sites are presented. We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). To begin to examine whether mutations in any of these functions are capable of affecting glutamatergic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.     

A genetic analysis of glutamaterigic function in Drosophila

Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. (1) To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity [³H]-glutamate binding sites are presented. (2) We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). (3) To begin to examine whether mutations in any of these functions are capable of affecting glutamaterigic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.     

Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors.     

Kinesin‐2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre‐ and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule‐dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin‐2 motor subunit Klp64D and separate siRNA‐mediated knock‐outs of all the three kinesin‐2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin‐2 subunits but not with each other. Altogether, these suggest that kinesin‐2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Acetylcholine and histamine are transmitter candidates in identifiable mechanosensitive neurons of the spider Cupiennius salei: an immunocytochemical study

Histochemical and indirect immunocytochemical techniques were used to search for neuroactive substances and transmitter candidates in identified sensory neurons of two types of cuticular mechanoreceptors in the spider Cupiennius salei Keys.: (1) in lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits each innervated by 2 bipolar neurons), and (2) in tactile hairs (each supplied by 3 bipolar sensory cells). All neurons are mechanosensitive. A polyclonal antibody against choline acetyltransferase (ChAT) strongly labeled all cell bodies and afferent fibers of both mechanoreceptor types. Western blot analysis using the same antibody against samples of spider sensory hypodermis and against samples from the central nervous system demonstrated a clear band at 65 kDa, corresponding to the molecular mass of ChAT in insects. Moreover, staining for acetylcholine esterase (AChE) revealed AChE activity in one neuron of each mechanoreceptor type. Incubation with a polyclonal antibody against histamine clearly labeled one neuron in each set of sensilla, whereas activity in the remaining one or two cells was near background. All mechanoreceptor preparations treated with a polyclonal antiserum against serotonin tested negative, whereas sections through the central nervous system of the same spiders were clearly labeled for serotonin. The presence of ChAT-like immunoreactivity and AChE implicates acetylcholine as a transmitter candidate in the two mechanoreceptive organs. We assume that histamine serves as a mechanosensory co-transmitter in the central nervous system and may also act at peripheral synapses that exist in these sensilla. Received: 15 July 1996 / Accepted: 26 August 1996     

Tissue specific expression of the acetylcholinesterase gene in Drosophila melanogaster

Summary Acetylcholinesterase (AChE) is mainly membrane bound in the central nervous system (CNS) of larvae and in the head and thorax of adults of Drosophila melanogaster; it is mostly soluble in the larval carcass, the adult abdomen, similar to that of the embryos (Zador et al. 1986). The enzyme shows the same number of isozymes (four or five) in larvae and adults as in the head of the fly or in embryos (Zador et al. 1986). In the Df(3R)GE26/MKRS stock both the membrane bound and the soluble enzyme are at about half normal levels while in the Df(3R)Ace ^HD1/MKRS stock this is true only for the membrane bound AChE. Therefore the effect of the above deficiencies in larvae and adults is consistent with that in embryos (Zador et al. 1986). In heat-sensitive combinations of certain Ace mutant alleles both the membrane bound and the soluble enzyme has reduced activity.Abbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - BAP 1,5-bis(allyldimethylammonium-phenyl)-pentan-3-one dibromide - CNS central nervous system     

Expression of acetylcholinesterase during visual system development in Drosophila

As in other insects acetylcholine (ACh) and acetylcholinesterase (AChE) function in synaptic transmission in the central nervous system of Drosophila. Studies on flies mutant for AChE indicate that in addition to its synaptic function of inactivating acetylcholine, this neural enzyme is required for normal development of the nervous system (J.C. Hall, S.N. Alahiotis, D.A. Strumpf, and K. White, 1980, Genetics 96, 939-965; R.J. Greenspan, J.A. Finn, and J.C. Hall, 1980, J. Comp. Neurol. 189, 741-774). In order to understand what role AChE may play in neural development, it is necessary to know, in detail, where and when the enzyme appears. The use of monoclonal antibodies to localize AChE in the developing visual system of wild type Drosophila has yielded the novel observation that AChE appears in photoreceptor (retinula) cells 4-6 hr after they differentiate and 3 to 4 days before they are functional. Three days later the staining in the cell body of these cells is reduced. Because retinula cells have no functional connections at the time when AChE is first detected, AChE can not be performing its standard synaptic function. Subsequent to the reduction of AChE in the retinula cells, midway through the pupal stage, the enzyme accumulates rapidly in the neuropils of the optic lobes of the brain. Thus, there is a biphasic accumulation of AChE in the developing visual system with the enzyme initially being expressed in the retinula cells and accumulating later in the optic lobes.     

Replacement of the glycoinositol phospholipid anchor of Drosophila acetylcholinesterase with a transmembrane domain does not alter sorting in neurons and epithelia but results in behavioral defects.

Drosophila has a single glycoinositol phospholipid (GPI)-anchored form of acetylcholinesterase (AChE) encoded by the Ace locus. To assess the role that GPI plays in the physiology, of AChE, we have replaced the wild-type GPI-AChE with a chimeric transmembrane form (TM-AChE) in the nervous system of the fly. Ace null alleles provided a genetic background completely lacking in endogenous GPI-AChE, and Ace minigene P transposon constructs were used to express both GPI- and TM-AChE forms in the tissues where AChE is normally expressed. Control experiments with the GPI-AChE minigene demonstrated a threshold between 9 and 12% of normal AChE activity for adult viability. Ace mutant flies were rescued by GPI-AChE minigene lines that expressed 12-40% of normal activity and were essentially unchanged from wild-type flies in behavior. TM-AChE minigene lines were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Although rescued flies expressing GPI-AChE at a level of 12% of normal activity were viable, flies expressing 13-16% of normal activity from the TM-AChE transgene died shortly after eclosion. Flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild-type flies in gross behavior but had a reduced lifespan secondary to subtle coordination defects. These flies also showed reduced locomotor activity and performed poorly in a grooming assay. However, light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, endogenous and ectopic-induced expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically. Most epithelial cells sorted GPI- and TM-AChE to the apical surface, but cuticle-secreting epithelia sorted both proteins basolaterally. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor or AChE plays some other more subtle cellular role in neuronal physiology.     

The east gene of Drosophila melanogaster is expressed in the developing embryonic nervous system and is required for normal olfactory and gustatory responses of the adult.

Drosophila melanogaster larvae and adults respond to a wide range of chemosensory stimuli. We describe the genetics and developmental expression of the east gene, mutations which result in adult-specific chemosensory defects. The original isolate of east is semidominant for the behavioral phenotype. Several mutations have been generated, some of which are recessive lethals and others that are viable alleles that show a recessive, adult-specific, chemosensory defect. No larval chemosensory defects were observed. The east gene is expressed in the neurogenic region at the time of neuroblast segregation and in cells in the peripheral and central nervous system. Our results suggest that east+ expression in the nervous system is required for a normal adult chemosensory response and both increases and decreases in levels of the gene product result in a mutant phenotype.     

The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages

The locus elav (ella-vee) of Drosophila melanogaster, which is necessary for the proper development of the embryonic and adult nervous systems, has been characterized both genetically and molecularly. This locus has been shown to be transcribed exclusively within, and ubiquitously throughout, the developing nervous system during Hours 6 to 12 of embryogenesis. We present in situ RNA localization data which demonstrate that elav is expressed in the central nervous system as well as the peripheral nervous system of embryos, larvae, pupae, and adults. We also demonstrate that elav is not transcribed in embryonic or larval neuroblasts (the neuronal progenitor cells), or in at least one type of glial cell. These data provide evidence that the requirement for elav function is not limited to the 6- to 12-hr embryonic nervous system and the adult eye and developing optic lobe, but that its function is required for the development and continued maintenance of all neurons of the organism.     

Localization of choline acetyltransferase-expresing neurons in the larval vissual system of Drosophila melanogaster

Choline acetyltransferease (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5 flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.This work was supported by a grant from the National Institute of Neurological Disorders and Stroke.     

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.     

Some properties of frog vestibular choline acetyltransferase and acetylcholinesterase

The amount and some properties of choline acetyltransferase (ChAT) and of acetylcholinesterase (AchE) were investigated in the frog vestibule. Enzyme activities were found to be of the same order of magnitude as in frog nervous tissue and various properties of vestibular ChAT (dependence on pH, chloride and Triton X-100 activation, phosphate sensitivity) and AchE (inhibition by eserine but not by Tetraisopropylpyrophosphoramide) were also similar as those of the homologous central nervous system enzymes. Although the precise localization of ChAT and AchE is not yet certain the efferent neurotransmitter in the vertebrate vestibular sensory periphery is believed to be acetylcholine and thus the enzymes responsible for its synthesis and degradation may participate in regulating inner ear function.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.