Canonical BMP Signaling Is Required For Allodynia In Drosophila Melanogaster

In the United States alone over 100 million people suffer from chronic pain and unfortunately, even still, there is a lack in scientific understanding for the mechanisms of abnormal pain sensitivity. The present study utilized a candidate gene approach to identify novel components required for modulation of the tissue damage induced pain sensitization pathway in Drosophila melanogaster. We have shown that RNAi silencing of decapentaplegic (dpp), a member of the Bone Morphogenetic Protein (BMP) signaling pathway, specifically in the class IV multidendritic nociceptor neurons significantly attenuated UV-induced nociceptive sensitization. Furthermore, overexpression of dpp in nociceptor neurons was sufficient to induce sensitization in the absence of tissue damage. We then show that the dpp receptors are required on the nociceptor neuron in order to produce allodynia, demonstrating that dpp is signaling to the very neuron that produced it. Lastly, we show that this BMP pathway is utilizing the canonical signaling SMAD factors to induce allodynia. We show that the effects of BMP signaling were largely specific to the sensitization pathway and not to normal nociception or dendritic morphology. Thus, we have shown that dpp plays a crucial and novel role in sensitization. Because the BMP family is so strongly conserved between vertebrates and invertebrates it seems likely that the genes we have analyzed represent potential therapeutic targets applicable to humans

Steroid Receptor Isoform Expression in <i>Drosophila</i> Nociceptor Neurons Is Required for Normal Dendritic Arbor and Sensitivity

<div><p>Steroid hormones organize many aspects of development, including that of the nervous system. Steroids also play neuromodulatory and other activational roles, including regulation of sensitivity to painful stimuli in mammals. In <i>Drosophila</i>, ecdysteroids are the only steroid hormones, and therefore the fly represents a simplified model system in which to explore mechanisms of steroid neuromodulation of nociception. In this report, we present evidence that ecdysteroids, acting through two isoforms of their nuclear ecdysone receptor (EcR), modulate sensitivity to noxious thermal and mechanical stimuli in the fly larva. We show that EcRA and EcRB1 are expressed by third instar larvae in the primary nociceptor neurons, known as the class IV multidendritic neurons. Suppression of EcRA by RNA interference in these cells leads to hyposensitivity to noxious stimulation. Suppression of EcRB1 leads to reduction of dendritic branching and length of nociceptor neurons. We show that specific isoforms of the ecdysone receptor play critical cell autonomous roles in modulating the sensitivity of nociceptor neurons and may indicate human orthologs that represent targets for novel analgesic drugs.</p></div

EcRA and EcRB1 are expressed in the class IV primary nociceptor neurons.

<p>(A) <i>ppk-eGFP</i> larva stained with anti-EcRA. (B) The class IV neuron is identified by eGFP expression in <i>ppk-eGFP</i>. (C) Merge of (A and B) indicates the presence of EcRA in the nociceptor neurons. (D) Sensory cluster of <i>ppk-eGFP</i> larva stained with anti-EcRB1. (E) Nociceptor neuron of same cluster is identified by eGFP expression in <i>ppk-eGFP</i>. (F) Merge of (D and E) indicates the presence of EcRB1 in the class IV nociceptor neurons.</p

Morphometric analysis of class IV neurons in EcR, EcRA, EcRB1 and USP mutant flies.

<p>All neurons in the analysis were class IV ddaC neurons from third instar larvae. Experimental animals were compared to control animals for number of dendritic terminals (left), total dendritic length (center) and the ratio of dendritic terminals/total dendritic length as a measure of complexity (right). (A) <i>EcR</i> mutants have significantly fewer dendritic terminals and a reduced dendritic length, but maintain the same complexity. Control n = 20, <i>EcR</i> mutant n = 19. (B) <i>EcRA</i> mutants show reduced dendritic length, but both the number of dendritic terminals and complexity remain the same. Control n = 18, <i>EcRA</i> mutant n = 19. (C) <i>EcRB1</i> mutants show a reduced number of dendritic terminals and reduced dendritic length, but are similar in complexity. Control n = 18, <i>EcRB1</i> mutant n = 17. (D) <i>USP</i> mutant animals show no difference in number of dendritic terminals, total dendritic length or complexity. Control n = 18 <i>USP</i> mutant n = 16. *** indicates p < 0.001.</p

Summary of consequences of nociceptor-specific RNAi suppression of EcR isoforms and USP.

<p>Summary of consequences of nociceptor-specific RNAi suppression of EcR isoforms and USP.</p

Analysis of ecdysone receptor mutant locomotion.

<p>Third instar larvae were assayed for spontaneous locomotor activity using the <i>Drosophila</i> activity monitor (DAM). Data were collected as moves per minute for each larva and averaged over a twenty-minute period. A Students t-test was used to determine statistical significance (** indicates p<0.01, *** indicates p<0.001), n = 32 per genotype. (A) Mutants bearing one null allele of <i>EcR</i> (<i>EcR</i>^{<i>M554fs</i>}) and one temperature sensitive allele (<i>EcR</i>^<i>A483T</i>) were significantly less motile (p<0.011) than controls comprising the genetic backgrounds of each mutation (+ = Canton-S). (B) Locomotor activity was increased in larvae in which all EcR isoforms were suppressed specifically in nociceptor neurons. (C-E) The spontaneous locomotion of various other EcR and a USP RNAi driven by <i>ppk-Gal4</i> were not significantly different from both controls.</p

Ecdysone receptor mutants are less sensitive to noxious mechanical stimulation.

<p>Foraging third instar larvae were stimulated on their dorsal surface with a von Frey filament calibrated to deliver 45 mN. Larvae responding with a nocifensive roll were classified as responders. Distributions were compared with Fisher’s exact test. Asterisks indicate statistically different groups (* is p<0.05, *** is p<0.001). N > 90. (A) Mutants bearing one null allele of EcR (EcR^M554fs) and one temperature sensitive allele (EcR^A483T) were significantly less sensitive than controls comprising the genetic backgrounds of each mutation (+ = Canton-S). (B) EcR RNAi is driven by the mifepristone-inducible neuron-specific elav-Geneswitch. (C-F) Various EcR and a USP RNAi are driven by ppk1.9-Gal4, specific to the nociceptive class IV multidendritic neurons.</p

Ecdysone receptor mutants are less sensitive to noxious thermal stimulation.

<p>Foraging third instar larvae were gently touched on their dorsal surface with a probe at 45°C. Larvae responding with a nocifensive roll were classified as fast (<6 seconds) or slow (between 6 and 20 seconds), or nonresponders if they did not respond within 20 seconds. Distributions were compared using Fisher’s exact test. Asterisks indicate statistically different results (* is p<0.05, *** is p<0.001). N > 90. (A) Mutants bearing one null allele (<i>EcR</i>^{<i>M554fs</i>}) and one temperature sensitive allele (<i>EcR</i>^<i>A483T</i>) were significantly less sensitive than controls (+ = Canton-S) comprising the genetic backgrounds of each mutation. (B) <i>EcR</i> RNAi was driven by the mifepristone-inducible neuron-specific <i>elav-Geneswitch</i>. (C-F) Various <i>EcR</i> and a <i>USP</i> RNAi were driven by <i>ppk1</i>.<i>9-Gal4</i>, specific to the nociceptive class IV multidendritic neurons.</p