Accepted

Sensory neurons respond to noxious stimuli by relaying information from the periphery to the central nervous system via action potentials driven by voltage-gated sodium channels, specifically Nav1.7 and Nav1.8. These channels play a key role in the manifestation of inflammatory pain. The ability to screen compounds that modulate voltage-gated sodium channels using cell-based assays assumes that key channels present in vivo is maintained in vitro. Prior electrophysiological work in vitro utilized acutely dissociated tissues, however, maintaining this preparation for long periods is difficult. A potential alternative involves multi-electrode arrays which permit long-term measurements of neural spike activity and are well suited for assessing persistent sensitization consistent with chronic pain. Here, we demonstrate that the addition of two inflammatory mediators associated with chronic inflammatory pain, nerve growth factor (NGF) and interleukin-6 (IL-6), to adult DRG neurons increases their firing rates on multi-electrode arrays in vitro. Nav1.7 and Nav1.8 proteins are readily detected in cultured neurons and contribute to evoked activity. The blockade of both Nav1.7 and Nav1.8, has a profound impact on thermally evoked firing after treatment with IL-6 and NGF. This work underscores the utility of multi-electrode arrays for pharmacological studies of sensory neurons and may facilitate the discovery and mechanistic analyses of anti-nociceptive compounds.

Substantial evidence from preclinical models of pain suggests that basal and noxious nociceptive sensitivity, as well as antinociceptive responses to drugs, show significant heritability. Individual differences to these responses have been observed across species from rodents to humans. The use of closely related C57BL/6 inbred mouse substrains can facilitate gene mapping of acute nociceptive behaviors in preclinical pain models. In this study, we investigated behavioral differences between C57BL/6 J (B6 J) and C57BL/6 N (B6 N) substrains in the formalin test, a widely used tonic inflammatory pain model, using a battery of pain-related phenotypes, including reflexive tests, nesting, voluntary wheel running, sucrose preference and anxiety-like behavior in the light/dark test at two different time points (1-h and 24-h). Our results show that these substrains did not differ in reflexive thermal and mechanical responses at the 1-h time point. However, B6 N substrain mice showed increased sensitivity to spontaneous pain-like behaviors. In addition, B6 N substrain continued to show higher levels of mechanical hypersensitivity compared to controls at 24-h. indicating that mechanical hypersensitivity is a more persistent pain-related phenotype induced by formalin. Finally, no sex differences were observed in our outcome measures. Our results provide a comprehensive behavioral testing paradigm in response to an inflammatory agent for future mouse genetic studies in pain.

The purpose of this study was to explore the role and mechanism of the PI3K/AKT/mTOR signaling pathway in painful diabetic neuropathy (PDN). The diabetes mellitus (DM) model was established by intraperitoneal injection of streptozocin into SD rats. After 3 weeks of modeling, the DM + LY group was treated with PI3K inhibitor, the DM + vehicle group was treated with DMSO, and the DM group was untreated. The paw mechanical withdrawal thresholds (MWT) was measured by Von Frey filaments, and the expression of PI3K/AKT/mTOR pathway-related proteins and autophagy marker proteins were analyzed by Western blotting. We found that 3 weeks after modeling, the MWT values of diabetic rats were significantly reduced, p-PI3K, p-AKT and p-mTOR proteins expression in the spinal cord was increased, and Beclin1 and LC3-II expressions were reduced (P < 0.05). After administration of PI3K inhibitor, the MWT values in DM + LY group were improved, and the expressions of p-PI3K, p-AKT and p-mTOR proteins in the spinal cord were decreased significantly, and the expressions of Beclin1 and LC3-II were increased (P < 0.05). However, there were no significant changes in the DM + vehicle group compared with the DM group (P > 0.05). Therefore, we conclude that activation of the PI3K/AKT/mTOR pathway and impaired autophagy may be key factors that cause PDN. Inhibition of the PI3K/AKT/mTOR pathway could promote autophagy activity and alleviate PDN.

Chronic neuropathic pain resulting from damage to the central or peripheral nervous system is a prevalent and debilitating condition affecting 7-18% of the population. Symptoms include spontaneous pain, dysesthesia, paresthesia, allodynia and hyperalgesia. The reported sensory symptoms are comorbid with behavioral disabilities such as insomnia and depression. Neonatal anoxia, a worldwide clinical problem in both neonatal and pediatric care, causes long-term deficits similar to those mentioned. The effect of neonatal anoxia on the maturation of nociceptive pathways has been sparsely explored. To address this question and to determine whether the effects differ depending on sex, a neonatal anoxia model was used in which Wistar rat pups approximately 30 hours old and of both sexes were placed in a chamber with 100% nitrogen flow at 3.5 L/min for 25 min at 36 °C ± 1 °C. After recovery, the animals (n = 16 in each group (anoxia and control; males and females)) were returned to their mothers. The control animals were subjected to the same conditions, but no gas exchange was performed. At postnatal day (PND) 18 and PND43, the animals were subjected to pain testing by stimulation of the hind paws with von Frey monofilaments. The results revealed a significant reduction (approximately 50%) in the pain threshold in the animals exposed to anoxia in comparison with their respective controls. The pain threshold increased between PND18 and PND43. A sex-based difference was observed in the male control group at PND18. Histological analysis revealed decreased cell numbers in the ventral posterolateral thalamic nucleus (VPL), with sex differences. These results demonstrate the long-lasting negative impact of neonatal anoxia and indicate the relevance of performing suitable approaches taking in consideration the possible sex differences.

Voltage-gated calcium channels (VGCCs) are multi-subunit complexes that play a crucial role in neuronal signaling. Auxiliary α2δ subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming α1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of α2δ subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism and epilepsy. However, the contribution of distinct α2δ subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathological conditions remains unclear. Here, we show that α2δ1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of α2δ3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that α2δ1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired upon α2δ1 knock-down. Overexpression of α2δ3 increases the excitatory synaptic density as well, but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonal outgrowth in immature interneurons. Together, our findings demonstrate that α2δ1 and α2δ3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in α2δ surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that in early development auxiliary α2δ3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development α2δ1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of α2δ1 and α2δ3 subunits of calcium channels.