Animal Studies

Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN and T790A which underlies increased pain behaviors in the transgenic mouse strain. We show that in male rat dorsal root ganglion (DRG) neurons these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents and the T790A mutation increased them four-fold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel β4 subunits fails to significantly alter T790A current density, but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer EADs and narrower action potentials after β4 knockdown. Together our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN. This work demonstrates that two disease mutations in the voltage-gated sodium channel Na1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open channel block is a major factor in Nav1.8 associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.

A major challenge in biology is to link cellular and molecular variations with behavioral phenotypes. Here, we studied somatosensory neurons from a panel of bird species from the family Anatidae, known for their tactile-based foraging behavior. We found that tactile specialists exhibit a proportional expansion of neuronal mechanoreceptors in trigeminal ganglia. The expansion of mechanoreceptors occurs via neurons with intermediately and slowly inactivating mechanocurrent. Such neurons contain the mechanically gated Piezo2 ion channel whose expression positively correlates with the expression of factors responsible for the development and function of mechanoreceptors. Conversely, Piezo2 expression negatively correlates with expression of molecules mediating the detection of temperature and pain, suggesting that the expansion of Piezo2-containing mechanoreceptors with prolonged mechanocurrent occurs at the expense of thermoreceptors and nociceptors. Our study suggests that the trade-off between neuronal subtypes is a general mechanism of tactile specialization at the level of somatosensory system.

Pain is a subjective experience derived from complex interactions among biological, environmental, and psychosocial pathways. Sex differences in pain sensitivity and chronic pain prevalence are well established. However, the molecular basis underlying these sex dimorphisms are poorly understood particularly with regard to the role of the peripheral nervous system. Here we sought to identify shared and distinct gene networks functioning in the peripheral nervous systems that may contribute to sex differences of pain in rats after nerve injury.

Peripheral nerve injury causes neuropathic pain and microglia activation. P2Y12 receptors on microglia are thought to be a key player in the surveillance of the local environment, but whether or how these receptors are engaged in the cross-talk between microglia and neurons of the dorsal horn remain ambiguous. Using a rodent model of nerve injury-induced pain, we investigated the roles of P2Y12 in microglia activation, excitatory synaptic transmission, and nociceptive allodynia. We found that spinal nerve ligation (SNL) significantly increased the level of P2Y12 receptors specifically in the microglia of the ipsilateral dorsal horn. Injections of P2Y12 antagonists (MRS2395 or clopidogrel) attenuated microglia activation and increased the paw withdrawal latency in response to thermal stimuli on the ipsilateral side without affecting the basal threshold on the contralateral side. These effects on pain behaviors were replicated in P2Y12 knockout mice. Patch-clamp recordings further revealed that partial sciatic nerve ligation (PSNL)-induced excessive miniature excitatory postsynaptic currents (mEPSCs) were significantly attenuated in P2Y12 knockout mice. Moreover, we found that SNL activates the GTP-RhoA/ROCK2 signaling pathway and elevates the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), which was inhibited by the P2Y12 antagonist. The phosphorylation of p38 MAPK was inhibited by a ROCK inhibitor, but not vice versa, suggesting that p38 MAPK is downstream of ROCK activation. Our findings suggest that nerve injury engages the P2Y12 receptor-dependent GTP-RhoA/ROCK2 signaling pathway to upregulate excitatory synaptic transmission in the dorsal horn. This cross-talk ultimately participates in the manifestation of nociceptive allodynia, implicating P2Y12 receptor as a potential target for alleviating neuropathic pain.

Chronic pain is a major clinical problem with limited treatment options. Previous studies have demonstrated that activation of adenosine monophosphate-activated protein kinase (AMPK) can attenuate neuropathic pain. Inflammation/immune response at the site of complete Freund's adjuvant (CFA) injection is known to be a critical trigger of the pathological changes that produce inflammatory pain. However, whether activation of AMPK produces an analgesic effect through inhibiting the proinflammatory cytokines, including interleukin-1β (IL-1β), in inflammatory pain remains unknown.

Studies showed that upregulation of Nav1.6 increased the neuronal excitability and participated in neuropathic pain in the dorsal root ganglion (DRG). However, the molecular mechanisms underlying Nav1.6 upregulation were not reported yet.