Papers by Wendi Neckameyer
Elsevier eBooks, 2002
The biogenic amines include the phenolamines such as octopamine, dopamine, and the trace amine ty... more The biogenic amines include the phenolamines such as octopamine, dopamine, and the trace amine tyramine, as well as the indolamine serotonin. They are ancient, evolutionarily conserved molecules that have been adapted for use in several physiological contexts, with critical and nonoverlapping functions in development beyond their roles as signaling molecules in the nervous system. These molecules interact with hormone-signaling pathways to elicit distinct behavioral and developmental responses. Both dopamine and serotonin are vital for life; insects lacking dopamine or serotonin die as embryonic lethals. Octopamine and tyramine, while not vital, play diverse critical roles in behavior and development. While the number of octopamine-, tyramine-, dopamine-, and serotonin-containing neurons is relatively small in most insect species (approximately 100 or fewer throughout the central nervous system), their processes extend throughout the body to target numerous peripheral organs, strong evidence for a neurohormonal role for these molecules. These numerous functions are key to maintaining the homeostasis of the organism, and current data suggest that several of these pathways have been conserved throughout evolution.
Elsevier eBooks, 2009
The biogenic amines include the phenolamines such as octopamine, dopamine, and the trace amine ty... more The biogenic amines include the phenolamines such as octopamine, dopamine, and the trace amine tyramine, as well as the indolamine serotonin. They are ancient, evolutionarily conserved molecules that have been adapted for use in several physiological contexts, with critical and nonoverlapping functions in development beyond their roles as signaling molecules in the nervous system. These molecules interact with hormone-signaling pathways to elicit distinct behavioral and developmental responses. Both dopamine and serotonin are vital for life; insects lacking dopamine or serotonin die as embryonic lethals. Octopamine and tyramine, while not vital, play diverse critical roles in behavior and development. While the number of octopamine-, tyramine-, dopamine-, and serotonin-containing neurons is relatively small in most insect species (approximately 100 or fewer throughout the central nervous system), their processes extend throughout the body to target numerous peripheral organs, strong evidence for a neurohormonal role for these molecules. These numerous functions are key to maintaining the homeostasis of the organism, and current data suggest that several of these pathways have been conserved throughout evolution.
Conservation of the viral and cellular ros genes PossIble mechanism of transduction of e-ros Expr... more Conservation of the viral and cellular ros genes PossIble mechanism of transduction of e-ros Expression of c-ros FunctIon of cellular ros In normal cells -vi i- The two differ only by a 9 bp duplication, a single base change not resulting in an amino acid change, and the divergence of their 3' ends. e-ros and v-ros abruptly diverge 36 bp upstream of the v-ros termination codon. The open read ing frame of e-ros continues after this divergence and may terminate 34 amino acids downstream, or, more likely, the reading frame is spliced to further 3' cod ing sequences using a splice donor site 24 nucleotides downstream of the diver gence. The v-ros sequence 3' to the divergence was not found in the 3' e-ros sequences in the lambda clone or in helper virus-related sequences. Comparison of the nucleotide sequences of viral and cellular ros suggests the viral ros and � gag junction in lJR2 was formed by splicing. A 3.1 kb e-ros transcript has been detected in adult muscle tissue and in kid ney from chickens only after long exposure of the Northern filters. It appears that the e-ros transcript in kidney is preferentially degraded relative to src, and this may account for the seeming lack of expression in this tissue. In all other tissues examined, a.nd in several cell lines, e-ros transcripts are not detectable. Although the exact function of the cellular ros protein is not known, evidence based on the sequence of the viral and structural genes has enabled us to detect its structural similarity with the EGF and insulin receptors and postulate that ros is a mem ber of a family of growth factor receptors.
Psychology & Neuroscience, 2018
Disruptions in neuronal function resulting from improper central nervous system development and m... more Disruptions in neuronal function resulting from improper central nervous system development and maintenance have been implicated in a wide array of neurological diseases, including Parkinson’s disease. Oxidative stress has been shown to damage neural fibers in model systems that simulate Parkinson’s disease via an increase in reactive oxygen species; paraquat (PQ) is the canonical method by which to induce oxidative stress in many models, including the fruit fly, Drosophila melanogaster. The present study examined the stomatogastric feeding circuit in Drosophila larvae to correlate its functional output (i.e., feeding) with changes in the axonal architecture innervating the foregut. Second instar larvae were exposed to PQ (i.e., a viologen) for 24 hr to induce oxidative stress, and the effects on both feeding and neurite architecture of the foregut examined. At PQ exposures of 30 mM and below, a depression in feeding without other obvious physiological impairments, as well as a significant increase in the number and size of serotonergic presynaptic vesicles along the neurite length, was observed. The results from this study show that acute oxidative stress, induced by PQ exposure, directly affects the size and number of serotonergic presynaptic vesicles within this neural circuit, correlating with functional behavioral consequences.
Elsevier eBooks, 2017
The biogenic amines discussed in this chapter include tyramine (TA), octopamine (OA), dopamine (D... more The biogenic amines discussed in this chapter include tyramine (TA), octopamine (OA), dopamine (DA), and serotonin (5-HT). These ancient, evolutionarily conserved molecules have been adapted for use in several physiological contexts, including behavior, reproduction, and the development of neuronal and nonneuronal tissues. Additionally, they interact with hormone signaling pathways to elicit distinct behavioral and developmental responses. While the number of tyramine-, octopamine-, dopamine-, and serotonin-containing neurons is relatively small in most insect species (∼100 or fewer throughout the central nervous system for each population), their processes extend throughout the body to target numerous peripheral organs. Thus, they serve multiple roles beyond those classically defined as centrally mediated neurotransmitters.
Journal of Biological Chemistry, Nov 1, 2008
The signaling functions of dopamine require a finely tuned regulatory network for rapid induction... more The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from endproduct feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. V max is elevated, with no change in K m. These results demonstrate that these enzymes engage in mutual positive regulation.
Journal of Neurobiology, 2001
Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in ... more Dopamine is an important signaling molecule in the nervous system; it also plays a vital role in the development of diverse non-neuronal tissues in the fruit fly Drosophila melanogaster. The current study demonstrates that males depleted of dopamine as third instar larvae (via inhibition of the biosynthetic enzyme tyrosine hydroxylase) demonstrated abnormalities in courtship behavior as adults. These defects were suggestive of abnormalities in sensory perception and/or processing. Electroretinograms (ERGs) of eyes from adults depleted of dopamine for 1 day as third instar larvae revealed diminished or absent on- and off-transients. These sensory defects were rescued by the addition of L-DOPA in conjunction with tyrosine hydroxylase inhibition during the larval stage. Depletion of dopamine in the first or second larval instar was lethal, but this was not due to a general inhibition of proliferative cells. To establish that dopamine was synthesized in tissues destined to become part of the adult sensory apparatus, transgenic lines were generated containing 1 or 4 kb of 5' upstream sequences from the Drosophila tyrosine hydroxylase gene (DTH) fused to the E. coli beta-galactosidase reporter. The DTH promoters directed expression of the reporter gene in discrete and consistent patterns within the imaginal discs, in addition to the expected expression in gonadal, brain, and cuticular tissues. The beta-galactosidase expression colocalized with tyrosine hydroxylase protein. These results are consistent with a developmental requirement for dopamine in the normal physiology of adult sensory tissues.
Journal of Biological Chemistry, 2008
The signaling functions of dopamine require a finely tuned regulatory network for rapid induction... more The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from endproduct feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. V max is elevated, with no change in K m. These results demonstrate that these enzymes engage in mutual positive regulation.
Psychology & Neuroscience
The neurotransmitters dopamine (DA) and serotonin (5-HT) exhibit trophic actions on developing ne... more The neurotransmitters dopamine (DA) and serotonin (5-HT) exhibit trophic actions on developing neural circuits in addition to their roles as signaling molecules in the nervous system. Improper signaling of these trophic factors during development may underlie the etiology of anxiety, depression, attention-deficit/hyperactivity disorder (ADHD), addiction, obsessive–compulsive disorder, and autism spectrum disorders (ASD). Many neuropsychiatric disorders display sexual dimorphism in their manifestations. Disorders such as schizophrenia, ADHD, and ASD are disproportionately diagnosed in males, whereas anorexia nervosa, addiction, and depression tend to manifest more in females. However, the mechanisms by which this occurs are still unknown. We have previously described the effects of DA and 5-HT on the development on the serotonergic feeding circuit in Drosophila larvae and demonstrated that perturbations in DA and 5-HT levels during central nervous system development affected both the axonal architecture of the serotonergic projections extending from the brain to the foregut and feeding behavior, the functional output of the circuit. Using transgenic lines to reduce synthesis of neuronal DA and 5-HT, we observed differences in feeding behaviors between male and female larvae. In the presence of reduced DA levels, females displayed decreased feeding. Conversely, reduced levels of 5-HT decreased feeding in males to a greater extent than females. The results of these experiments illuminate the sexually dimorphic actions of neurotrophic factors in the development of the central nervous system.
Methods in Molecular Biology, 2016
Drosophila melanogaster is an incredibly versatile organism capable of both innate and higher-ord... more Drosophila melanogaster is an incredibly versatile organism capable of both innate and higher-order behaviors. These behaviors offer not only a way to assay whether or not the animal is physiologically compromised (e.g., feeding, locomotion), but also serve to assess changes in centrally mediated functions. Here we describe several high throughput, reproducible, yet inexpensive and facile behavioral assays for both larval and adult Drosophila. The larval assays all employ an agar substrate in a petri dish; the adult assays are grouped into "vial-based" and "arena-based" paradigms. While these protocols are largely designed to assess individual animals, they are sufficiently rapid that ample numbers can be tested to determine behavioral significance. Importantly, this also allows for one to control for reproductive status, age, and sex, since these factors all have a significant impact on adult behaviors. In general, it is best to designate a dedicated area for any assay, so that lighting conditions are consistent, and all animals should be tested at roughly the same time each day to minimize circadian fluctuations. Temperature and humidity should also be maintained at a constant level to minimize variability in the assays.
Advances in experimental medicine and biology, 1993
We have reintroduced an 8 kb genomic fragment from the Drosophila tyrosine hydroxylase (DTH) locu... more We have reintroduced an 8 kb genomic fragment from the Drosophila tyrosine hydroxylase (DTH) locus into the genome of mutant pale (ple) flies. ple was first recovered as a recessive embryonic lethal by Jurgens et al. (1984) and maps to the same chromosomal region as DTH (65A-E). Mutant ple alleles affect pigmentation of the cuticle (L-DOPA, the product of the reaction catalyzed by TH, is an intermediate in the cuticular sclerotization and pigmentation pathways) and catecholamine biosynthesis. In this report we demonstrate that ple does encode the structural gene for TH, since the reintroduced sequences rescue ple flies from lethality to viable adults. Morphological, immunocytochemical, and behavioral characterization of three transformant lines suggests that the reintroduced sequences contain the necessary elements for correct temporal and spatial expression of the gene, but may not contain all the sequences essential for quantitative expression.
Comp Biochem Physiol Pt B, 2001
Molecular and cellular biology, 1986
A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-... more A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic re...
Journal of virology, 1987
Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previous... more Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing...
American journal of physiology. Regulatory, integrative and comparative physiology, 2014
The stress response in Drosophila melanogaster reveals sex differences in behavior, similar to wh... more The stress response in Drosophila melanogaster reveals sex differences in behavior, similar to what has been observed in mammals. However, unlike mammals, the sex determination pathway in Drosophila is well established, making this an ideal system to identify factors involved in the modulation of sex-specific responses to stress. In this study, we show that the Drosophila fat body, which has been shown to be important for energy homeostasis and sex determination, is a dynamic tissue that is altered in response to stress in a sex and time-dependent manner. We manipulated the sex determination pathway in the fat body via targeted expression of transformer and transformer-2 and analyzed these animals for changes in their response to stress. In the majority of cases, manipulation of transformer or transformer-2 was able to change the physiological output in response to starvation and oxidative stress to that of the opposite sex. Our data also uncover the possibility of additional downst...
Journal of visualized experiments : JoVE, 2013
The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neu... more The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitati...
Learning & memory (Cold Spring Harbor, N.Y.)
Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic intro... more Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminishes with time. Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation. One kilobase of 5' upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli beta-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies. Ablation of mushroom bodies by treatment of newly hatched larva with h...
Advances in experimental medicine and biology, 1993
Stress, 2015
All living organisms must maintain equilibrium in response to internal and external challenges wi... more All living organisms must maintain equilibrium in response to internal and external challenges within their environment. Changes in neural plasticity (alterations in neuronal populations, dendritic remodeling, and synaptic turnover) are critical components of the homeostatic response to stress, which has been strongly implicated in the onset of affective disorders. However, stress is differentially perceived depending on the type of stress and its context, as well as genetic background, age and sex; therefore, an individual's maintenance of neuronal homeostasis must differ depending upon these variables. We established Drosophila as a model to analyze homeostatic responses to stress. Sexually immature and mature females and males from an isogenic wild-type strain raised under controlled environmental conditions were exposed to four reproducible and high-throughput translatable stressors to facilitate the analysis of a large number of animals for direct comparisons. These animals were assessed in an open-field arena, in a light-dark box, and in a forced swim test, as well as for sensitivity to the sedative effects of ethanol. These studies establish that immature and mature females and males represent behaviorally distinct populations under control conditions as well as after exposure to different stressors. Therefore, the neural substrates mediating the stress response must be differentially expressed depending upon the hormonal status of the brain. In addition, an adaptive response to a given stressor in one paradigm was not predictive for outcomes in other paradigms.
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Papers by Wendi Neckameyer