The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a pro-nociceptive role after peripheral nerve injury upon its release from primary afferent neurons in preclinical models of neuropathic pain. We previously demonstrated a critical role for spinal cord microglial CD40 in the development of spinal nerve L5 transection (L5Tx)-induced mechanical hypersensitivity. Herein, we investigated whether CGRP is involved in the CD40-mediated behavioral hypersensitivity. First, L5Tx was found to significantly induce CGRP expression in wild-type (WT) mice up to 14 days post-L5Tx. This increase in CGRP expression was reduced in CD40 knockout (KO) mice at day 14 post-L5Tx. Intrathecal injection of the CGRP antagonist CGRP8–37 significantly blocked L5Tx-induced mechanical hypersensitivity. In vitro, CGRP induced glial IL-6 and CCL2 production, and CD40 stimulation added to the effects of CGRP in neonatal glia. Further, there was decreased CCL2 production in CD40 KO mice compared to WT mice 21 days post-L5Tx. However, CGRP8–37 did not significantly affect spinal cord CCL2 production following L5Tx in WT mice. Altogether, these data suggest that CD40 contributes to the maintenance of behavioral hypersensitivity following peripheral nerve injury in part through two distinct pathways, the enhancement of CGRP expression and spinal cord CCL2 production.

Male-male courtship is infrequent among mature adult Drosophila melanogaster. After pairs of mature adult males expressing a temperature-sensitive allele of the ecdysone receptor (EcR) gene were treated at a restrictive temperature, however, they engaged in elevated levels of male-male courtship. EcR-deficient males courted wildtype males and females, but were not courted by wildtype males. These results suggest that the ecdysone steroid hormone system may have a role in courtship initiation by adult male fruit flies.

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd1 adult Drosophila melanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed towards females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.

BackgroundNeuropathic pain is one of the most devastating kinds of chronic pain. Neuroinflammation has been shown to contribute to the development of neuropathic pain. We have previously demonstrated that lumbar spinal cord-infiltrating CD4+ T lymphocytes contribute to the maintenance of mechanical hypersensitivity in spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we further examined the phenotype of the CD4+ T lymphocytes involved in the maintenance of neuropathic pain-like behavior via intracellular flow cytometric analysis and explored potential interactions between infiltrating CD4+ T lymphocytes and spinal cord glial cells.ResultsWe consistently observed significantly higher numbers of T-Bet+, IFN-γ+, TNF-α+, and GM-CSF+, but not GATA3+ or IL-4+, lumbar spinal cord-infiltrating CD4+ T lymphocytes in the L5Tx group compared to the sham group at day 7 post-L5Tx. This suggests that the infiltrating CD4+ T lymphocytes expressed a pro-inflammatory type 1 phenotype (Th1). Despite the observation of CD4+ CD40 ligand (CD154)+ T lymphocytes in the lumbar spinal cord post-L5Tx, CD154 knockout (KO) mice did not display significant changes in L5Tx-induced mechanical hypersensitivity, indicating that T lymphocyte-microglial interaction through the CD154-CD40 pathway is not necessary for L5Tx-induced hypersensitivity. In addition, spinal cord astrocytic activation, represented by glial fibillary acidic protein (GFAP) expression, was significantly lower in CD4 KO mice compared to wild type (WT) mice at day 14 post-L5Tx, suggesting the involvement of astrocytes in the pronociceptive effects mediated by infiltrating CD4+ T lymphocytes.ConclusionsIn all, these data indicate that the maintenance of L5Tx-induced neuropathic pain is mostly mediated by Th1 cells in a CD154-independent manner via a mechanism that could involve multiple Th1 cytokines and astrocytic activation.

We previously demonstrated that CD4+ T cells contribute to the maintenance of L5 spinal nerve transection (L5Tx)-induced neuropathic pain in BALB/c mice, and T-Bet+ Th1 cells were found to be the dominant subtype of CD4+ T cells within the lumbar spinal cord following L5Tx. To investigate the specific downstream responses mediated by these infiltrating Th1 cells, we examined the cytokine expression of the spinal cord-infiltrating CD4+ T cells via flow cytometry. At day 7 post-L5Tx, the peak time for detecting the lumbar infiltrating CD4+ T cells, there were trends indicating increases in the numbers of IFNγ+ and TNFα+ lumbar spinal cord-infiltrating CD4+ T cells in the L5Tx group compared to the sham group. Further, we examined the expression of glial fibrillary acidic protein (GFAP), an indication of the level of astrocytic activation, in the L5 spinal cord via immunohistochemistry. As expected, in wild type BALB/c mice, L5Tx induced an increase in GFAP expression in the ipsilateral side of the spinal cord compared to sham-operated mice 7 days post-surgery. This increase appeared to be reduced in CD4 knockout mice post-L5Tx. Altogether our data suggest the involvement of the Th1 cytokines IFNγ and TNFα in the maintenance of L5Tx-induced neuropathic pain, which may have potential role in regulating spinal cord astrocyte activation.