The perception of visceral pain is a complex process involving the spinal cord and higher order brain structures. Increasing evidence implicates the gut microbiota as a key regulator of brain and behavior, yet it remains to be determined if gut bacteria play a role in visceral sensitivity. We used germ-free mice (GF) to assess visceral sensitivity, spinal cord gene expression and pain-related brain structures. GF mice displayed visceral hypersensitivity accompanied by increases in Toll-like receptor and cytokine gene expression in the spinal cord, which were normalized by postnatal colonization with microbiota from conventionally colonized (CC). In GF mice, the volumes of the anterior cingulate cortex (ACC) and periaqueductal grey, areas involved in pain processing, were decreased and enlarged, respectively, and dendritic changes in the ACC were evident. These findings indicate that the gut microbiota is required for the normal visceral pain sensation.

Functional gastrointestinal disorders, which have visceral hypersensitivity as a core symptom, are frequently comorbid with stress-related psychiatric disorders. Increasing evidence points to a key role for toll-like receptor 4 (TLR4) in chronic pain states of somatic origin. However, the central contribution of TLR4 in visceral pain sensation remains elusive. With pharmacological and genetic approaches, we investigated the involvement of TLR4 in the modulation of visceral pain. The TLR4-deficient and wild-type mice were exposed to chronic stress. Visceral pain was evaluated with colorectal distension. Protein expression levels for TLR4, Cd11b, and glial fibrillary acidic protein (glial cells markers) were quantified in the lumbar region of the spinal cord, prefrontal cortex (PFC), and hippocampus. To evaluate the effect of blocking TLR4 on visceral nociception, TAK-242, a selective TLR4 antagonist, was administered peripherally (intravenous) and centrally (intracerebroventricular and intra-PFC) (n = 10-12/experimental group). The TLR4 deficiency reduced visceral pain and prevented the development of chronic psychosocial stress-induced visceral hypersensitivity. Increased expression of TLR4 coupled with enhanced glia activation in the PFC and increased levels of proinflammatory cytokines were observed after chronic stress in wild-type mice. Administration of a TLR4 specific antagonist, TAK-242, attenuated visceral pain sensation in animals with functional TLR4 when administrated centrally and peripherally. Moreover, intra-PFC TAK-242 administration also counteracted chronic stress-induced visceral hypersensitivity. Our results reveal a novel role for TLR4 within the PFC in the modulation of visceral nociception and point to TLR4 as a potential therapeutic target for the development of drugs to treat visceral hypersensitivity.

Transforming growth factors-β (TGF-βs) signal through type I and type II serine–threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-β family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS.BAMBIis expressed in regions of the CNS involved in pain transmission and modulation. The lack ofBAMBIin mutant mice resulted in increased levels of TGF-β signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited byBAMBI^−/−mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses ofBAMBI^−/−mice also appeared attenuated through a mechanism involving δ-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptidesproopiomelanocortin(POMC) andproenkephalin(PENK) appeared increased in the spinal cords ofBAMBI^−/−. Transcript levels of TGF-βs and their intracellular effectors correlated directly with genes encoding opioid peptides, whereasBAMBIcorrelated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increasedPOMCand/orPENKmRNA levels. Our findings identify TGF-β family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids.