Accepted

There has been a long-standing debate regarding the role of peripheral afferents in mediating rapid-onset anorexia among other responses elicited by peripheral inflammatory insults. Thus, the current study assessed the sufficiency of peripheral afferents expressing toll-like receptor 4 (TLR4) to the initiation of the anorexia caused by peripheral bacterial lipopolysaccharide (LPS). We generated a Tlr4 null (Tlr4) mouse in which Tlr4 expression is globally disrupted by a loxP-flanked transcription blocking cassette. This novel mouse model allowed us to restore the endogenous TLR4 expression in specific cell types. Using Zp3-Cre and Na1.8-Cre mice, we produced mice that express TLR4 in all cells (Tlr4 X Zp3-Cre) and in peripheral afferents (Tlr4 X Na1.8-Cre), respectively. We validated the Tlr4 mice, which were phenotypically identical to previously reported global TLR4 knock-out mice. Contrary to our expectations, the administration of LPS did not cause rapid-onset anorexia in mice with Na1.8-restricted TLR4. The later result prompted us to identify Tlr4-expressing vagal afferents using hybridization. , we found that Tlr4 mRNA was primarily enriched in vagal Na1.8 afferents located in the jugular ganglion that co-expressed Calcitonin gene-related peptide (CGRP). , the application of LPS to cultured Na1.8-restricted TLR4 afferents was sufficient to stimulate the release and expression of CGRP. In summary, we demonstrated using a new mouse model that vagally-expressed TLR4 is selectively involved in stimulating the release of CGRP, but not in causing anorexia. Using a new transgenic mouse model, our data establish that TLR4 is both sufficient and required for the release of CGRP from a subset of vagal afferents. This finding may be relevant to the understanding of how bacterial infections modulate nerves.

The voltage-gated sodium channel Na1.8 mediates the tetrodotoxin-resistant (TTX-R) Na current in nociceptive primary sensory neurons, which has an important role in the transmission of painful stimuli. Here, we describe the functional modulation of the human Na1.8 α-subunit in oocytes by auxiliary β subunits. We found that the β3 subunit down-regulated the maximal Na current amplitude and decelerated recovery from inactivation of hNa1.8, whereas the β1 and β2 subunits had no such effects. The specific regulation of Na1.8 by the β3 subunit constitutes a potential novel regulatory mechanism of the TTX-R Na current in primary sensory neurons with potential implications in chronic pain states. In particular, neuropathic pain states are characterised by a down-regulation of Na1.8 accompanied by increased expression of the β3 subunit. Our results suggest that these two phenomena may be correlated, and that increased levels of the β3 subunit may directly contribute to the down-regulation of Na1.8. To determine which domain of the β3 subunit is responsible for the specific regulation of hNa1.8, we created chimeras of the β1 and β3 subunits and co-expressed them with the hNa1.8 α-subunit in oocytes. The intracellular domain of the β3 subunit was shown to be responsible for the down-regulation of maximal Na1.8 current amplitudes. In contrast, the extracellular domain mediated the effect of the β3 subunit on hNa1.8 recovery kinetics.

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.