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. 2012;8(12):e1003071.
doi: 10.1371/journal.pgen.1003071. Epub 2012 Dec 6.

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

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Free PMC article

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

G Gregory Neely et al. PLoS Genet. .
Free PMC article

Abstract

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A global network map of thermal nociception.
The systems network includes data from significantly enriched Drosophila KEGG pathways and GO processes, mouse and human KEGG pathways and C2 gene sets. Pathways, processes and gene sets that share a role in a biological process were pooled into functional classes while the underlying genes that constitute them are depicted with a connection to their respective functional class. Functional classes (gold), genes representing direct hits with a thermal nociception phenotype (red), their first degree binding partners (green), and developmental lethal genes (blue) represent the nodes in the network. Only select KEGG pathways, biological processes and C2 gene sets were used to build systems map. For the entire list of individual pathways, gene sets, and processes see Tables S5, S6, S7.
Figure 2
Figure 2. PI5Kα signaling controls thermal and capsaicin nociception in vivo.
(A) Thermal pain thresholds of wild type (WT) and PIP5Kα −/− (KO) littermates in response to radiant heat (Hargreaves; n = 6 for WT; n = 6 for KO mice). (B) PIP5Kα KO mice also show enhanced thermal sensitivity in the hot plate assay (n = 12 for WT; n = 12 for KO mice) and (C) an exaggerated capsaicin-evoked behavioral response (n = 12 for WT; n = 9 for KO mice). (D) PIP5Kα KO mice exhibited normal mechanical pain (force threshold latency) as assessed by the von Frey test (n = 9 for WT; n = 9 for KO mice). All data are presented as mean +/− sem. *p<0.05; **p<0.01 (t-test).
Figure 3
Figure 3. PI3Kγ signaling controls thermal and capsaicin nociception in vivo.
(A) PI3Kγ −/− (KO) mice show enhanced thermal pain thresholds in response to radiant heat (Hargreaves test) as compared to wild type (WT) littermates (n = 22 for WT; n = 27 for KO mice). (B) PI3Kγ KO mice exhibit enhanced thermal sensitivity in the hot plate assay (n = 13 for WT; n = 9 for KO mice) and (C) an exaggerated capsaicin-evoked behavioral response (n = 9 for WT; n = 13 for KO mice). (D) Normal mechanical pain (force threshold latency) in PI3Kγ KO mice as assessed by the von Frey test (n = 21 for WT; n = 16 for KO mice). (E) PI3Kγ KO mice exhibit comparable cold sensitivity as assessed by acetone application (n = 21 for WT; n = 16 for KO mice). (F) Thermal nociception in PI3Kγ KO mice reconstituted with wild type (WT→KO) or PI3Kγ −/− (KO→KO) bone marrow. WT mice reconstituted with WT bone marrow are shown as controls. Mice were assayed using the hot plate assay at 54°C. n>6 for each group. (G) Thermal nociception thresholds in response to radiant heat (Hargreaves test) in littermate control and PI3Kγ kinase-dead (KD) knock-in mice (n = 19 for WT; n = 23 for KD mice). (H) Control and PI3Kγ KO mice exhibit similar coordination in the Rotarod test (n = 11 for WT and n = 12 for KO mice). All data are presented as mean +/− sem. *p<0.05; **p<0.01 (t-test).
Figure 4
Figure 4. PI3Kγ acts in DRG neurons as a negative regulator of thermal and TRPV1 responses.
(A) Representative temperature response ramps and Arrhenius plots for heat-activated currents measured in single DRG neurons isolated from wild type (WT) and PI3Kγ mutant (KO) mice. For temperature response ramps, red lines depict temperature ramps and black lines depict inward current. (B) Q10 as a measure of the rate of inward current changes in response to temperature. n = 37 for isolated WT; n = 9 for PI3Kγ KO DRG neurons. (C,D) Capsaicin sensitivity of DRG neurons isolated from WT and PI3Kγ KO mice. (C) Representative capsaicin responses from a single DRG neuron. (D) Dose-response curves to different concentrations of capsaicin. The capsaicin EC50 is indicated. Numbers indicate numbers of single neurons tested with the indicated capsaicin doses at the respective data points. Electrophysiology data was generated by single neuron patch clamping. Data are presented as mean +/− sem. ** p<0.01, *** p<0.001 (Mann-Whitney u-test).

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