Animal Studies

Stress can trigger symptoms in patients with irritable bowel syndrome (IBS). Previously we demonstrated that chronic psychological stress induced microglial remodeling in the central nucleus of amygdala (CeA), and contributed to the development of visceral hypersensitivity via synaptic engulfment. However, the specific signaling mechanisms that microglia depend upon to recognize target neurons to facilitate visceral pain remain unknown. Here we test the hypothesis that the microglia in the CeA contribute to chronic stress-induced visceral hypersensitivity via complement C1q/C3-CR3 signaling-mediated synaptic remodeling. In male and female Fischer-344 rats, micropellets of corticosterone (CORT) or cholesterol (control) were stereotaxically implanted bilaterally onto the CeA. After 7 days, microglial C1q, complement receptor 3 (CR3) expression and microglia-mediated synaptic engulfment were assessed via RNAscope, quantitative PCR and immunofluorescence. The microglial inhibitor minocycline, CR3 antagonist neutrophil inhibitory factor (NIF) or vehicle were daily infused into the CeA following CORT implantations. Visceral sensitivity was assessed via a visceromotor response (VMR) to graded pressures of isobaric colorectal distension (CRD). Our results suggest that chronic exposure to elevated CORT in the CeA induced visceral hypersensitivity and amygdala microglial morphological remodeling. CORT increased microglial C1q and CR3 expression, and increased microglia-mediated synaptic engulfment. Both groups of animals with minocycline or NIF infusions reversed microglia-mediated synaptic remodeling, and attenuated CORT-induced visceral hypersensitivity. Our findings demonstrate that C1q/C3-CR3 signaling is critical for microglia-mediated synaptic remodeling in the CeA and contributes to CORT-induced visceral hypersensitivity.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Contrary to dogma that defensive venoms are simple in composition, produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

Elimination or blocking of astrocytes could ameliorate neuropathic pain in animal models. MiR-125a-5p, expressed in astrocyte derived extracellular vesicles, could mediate astrocyte function to regulate neuron communication. However, the role of miR-125a-5p in DPN (diabetic peripheral neuropathy) remains elusive.

Transient receptor potential ankyrin 1 (TRPA1) channel is expressed in a subset of nociceptive neurons. This channel integrates several nociceptive signals. Particularly, it is modulated by intracellular pH (pH). Na/H exchanger 1 (NHE1) contributes to the maintenance of pH in nociceptors. However, it is currently unknown whether the interaction between TRPA1 and NHE1 contributes to the nociceptive processing. Thus, the purpose of this study was to assess the functional interaction between NHE1 and TRPA1 in small dorsal root ganglion (DRG) neurons from primary culture obtained from adult rats. Moreover, we also evaluated their possible interaction in acute and inflammatory pain. Zoniporide (selective NHE1 inhibitor) reduced pH and increased intracellular calcium in a concentration-dependent fashion in DRG neurons. Zoniporide and allyl isothiocyanate (AITC, TRPA1 agonist) increased calcium transients in the same DRG neuron, whereas that A-967079 (TRPA1 antagonist) prevented the effect of zoniporide in DRG neurons. Repeated AITC induced TRPA1 desensitization and this effect was prevented by zoniporide. Both NHE1 and TRPA1 were localized at the membrane surface of DRG neurons in culture. Local peripheral zoniporide enhanced AITC-induced pronociception and this effect was prevented by A-967079. Likewise, zoniporide potentiated Complete Freund́s Adjuvant (CFA)-induced hypersensitivity, effect which was prevented by A-967079 in vivo. CFA paw injection increased TRPA1 and decresed NHE1 protein expression in DRG. These results suggest a functional interaction between NHE1 and TRPA1 in DRG neurons in vitro. Moreover, data suggest that this interaction participates in acute and inflamatory pain conditions in vivo.

The motivation to eat is not only shaped by nutrition but also competed by external stimuli including pain. How the mouse hypothalamus, the feeding regulation center, integrates nociceptive inputs to modulate feeding is unclear. Within the key nociception relay center parabrachial nucleus (PBN), we demonstrated that neurons projecting to the lateral hypothalamus (PBN) are nociceptive yet distinct from danger-encoding central amygdala-projecting (PBN) neurons. Activation of PBN strongly suppressed feeding by limiting eating frequency and also reduced motivation to work for food reward. Refined approach-avoidance paradigm revealed that suppression of PBN, but not PBN, sustained motivation to obtain food. The effect of PBN neurons on feeding was reversed by suppressing downstream LH neurons. Thus, distinct from a circuit for fear and escape responses, PBN neurons channel nociceptive signals to LH neurons to suppress motivational drive for feeding. Our study provides a new perspective in understanding feeding regulation by external competing stimuli.