Animal Studies

Peripheral nerve injury is associated with spinal microgliosis which plays a pivotal role in the development of neuropathic pain behavior. Several agents of primary afferent origin causing the microglial reaction have been identified, but the type(s) of primary afferents that release these mediators are still unclear. In this study, specific labeling of C-fiber spinal afferents by lectin histochemistry and selective chemodenervation by capsaicin were applied to identify the type(s) of primary afferents involved in the microglial response. Comparative quantitative morphometric evaluation of the microglial reaction in central projection territories of intact and injured peripheral nerves in the superficial (laminae I and II) and deep (laminae III and IV) spinal dorsal horn revealed a significant, about three-fold increase in microglial density after transection of the sciatic or the saphenous nerve. Prior perineural treatment of these nerves with capsaicin, resulting in a selective defunctionalization of C-fiber afferent fibers failed to affect spinal microgliosis. Similarly, peripheral nerve injury-induced increase in microglial density was unaffected in rats treated neonatally with capsaicin known to result in a near-total loss of C-fiber dorsal root fibers. Perineural treatment with capsaicin per se did not evoke a significant increase in microglial density. These observations indicate that injury-induced spinal microgliosis may be attributed to phenotypic changes in injured myelinated primary afferent neurons, whereas the contribution of C-fiber primary sensory neurons to this neuroimmune response is negligible. Spinal myelinated primary afferents may play a hitherto unrecognized role in regulation of neuroimmune and perisynaptic microenvironments of the spinal dorsal horn.

Painful or threatening experiences trigger escape responses that are guided by nociceptive neuronal circuitry. Although some components of this circuitry are known and conserved across animals, how this circuitry is regulated at the genetic and developmental levels is mostly unknown. To escape noxious stimuli, such as parasitoid wasp attacks, larvae generate a curling and rolling response. Rover and sitter allelic variants of the () gene differ in parasitoid wasp susceptibility, suggesting a link between and nociception. By optogenetically activating cells associated with each of 's promoters (pr1-pr4), we show that pr1 cells regulate larval escape behavior. In accordance with rover and sitter differences in parasitoid wasp susceptibility, we found that rovers have higher pr1 expression and increased sensitivity to nociception relative to sitters. The null mutants display impaired responses to thermal nociception, which are rescued by restoring expression in pr1 cells. Conversely, knockdown of in pr1 cells phenocopies the null mutant. To gain insight into the circuitry underlying this response, we used an intersectional approach and activity-dependent GFP reconstitution across synaptic partners (GRASP) to show that pr1 cells in the ventral nerve cord (VNC) are required for the nociceptive response, and that multidendritic sensory nociceptive neurons synapse onto pr1 neurons in the VNC. Finally, we show that activation of the pr1 circuit during development suppresses the escape response. Our data demonstrate a role of in larval nociceptive behavior. This function is specific to pr1 neurons in the VNC, guiding a developmentally plastic escape response circuit.

Gap junctions play a pivotal role in contributing to the formation of astroglial networks and in chronic pain. However, the mechanisms underlying the dysfunction of astroglial gap junctions in chronic pain have not been fully elucidated.

Activation of nociceptor sensory neurons by noxious stimuli both triggers pain and increases capillary permeability and blood flow to produce neurogenic inflammation, but whether nociceptors also interact with the immune system remains poorly understood. Here we report a neurotechnology for selective epineural optogenetic neuromodulation of nociceptors and demonstrate that nociceptor activation drives both protective pain behavior and inflammation. The wireless optoelectronic system consists of sub-millimeter-scale light-emitting diodes embedded in a soft, circumneural sciatic nerve implant, powered and driven by a miniaturized head-mounted control unit. Photostimulation of axons in freely moving mice that express channelrhodopsin only in nociceptors resulted in behaviors characteristic of pain, reflecting orthodromic input to the spinal cord. It also led to immune reactions in the skin in the absence of inflammation and potentiation of established inflammation, a consequence of the antidromic activation of nociceptor peripheral terminals. These results reveal a link between nociceptors and immune cells, which might have implications for the treatment of inflammation.

As one of the thalamic midline nuclei, the thalamic paraventricular nucleus (PVT) is considered to be an important signal integration site for many descending and ascending pathways that modulate a variety of behaviors, including feeding, emotions and drug-seeking. A recent study has demonstrated that the PVT is implicated in the acute visceral pain response, but it is unclear whether the PVT plays a critical role in the central processing of chronic pain. Here, we report that the neurons in the posterior portion of the PVT (pPVT) and their downstream pathway are involved in descending nociceptive facilitation regarding the development of neuropathic pain conditions in male rats. Lesions or inhibition of pPVT neurons alleviated mechanical allodynia induced by spared nerve injury (SNI). The excitability of pPVT-central amygdala (CeA) projection neurons was significantly increased in SNI rats. Importantly, selective optogenetic activation of the pPVT-CeA pathway induced obvious mechanical hypersensitivity in naïve rats. In addition, we used rabies virus-based and cell-type-specific retrograde transsynaptic tracing techniques to define a novel neuronal circuit in which glutamatergic neurons in the vlPAG were the target of the pPVT-CeA descending facilitation pathway. Our data suggest that this pPVT-CeA-vlPAG circuit mediates central mechanisms of descending pain facilitation underlying persistent pain conditions.Studies have shown that the interactions between the posterior portion of the thalamic paraventricular nucleus (pPVT) and central amygdala (CeA) play a critical role in pain-related emotional regulation. However, most reports have associated this circuit with fear and anxiety behaviors. Here, an integrative approach of behavioral tests, electrophysiology, and immunohistochemistry was used to advance the novel concept that the pPVT-CeA pathway activation facilitates neuropathic pain processing. Using rabies virus-based and cell-type-specific retrograde transsynaptic tracing techniques, we found that glutamatergic neurons in the vlPAG were the target of the pPVT-CeA pathway. Thus, this study indicates the involvement of a pPVT-CeA-vlPAG pathway in a descending facilitatory mechanism underlying neuropathic pain.

Overexpression of Ca3.2 T-type Ca channels in L4 dorsal root ganglion (DRG) participates in neuropathic pain after L5 spinal nerve cutting (L5SNC) in rats. The L5SNC-induced neuropathic pain also involves high mobility group box 1 (HMGB1), a damage-associated molecular pattern protein, and its target, the receptor for advanced glycation end-products (RAGE). We thus studied the molecular mechanisms for the L5SNC-induced Ca3.2 overexpression as well as neuropathic pain in rats by focusing on; 1) possible involvement of early growth response 1 (Egr-1), known to regulate transcriptional expression of Ca3.2, and ubiquitin-specific protease 5 (USP5) that protects Ca3.2 from proteasomal degradation, and 2) possible role of HMGB1/RAGE as an upstream signal. Protein levels of Ca3.2 as well as Egr-1 in L4 DRG significantly increased in the early (day 6) and persistent (day 14) phases of neuropathy after L5SNC, while USP5 protein in L4 DRG did not increase on day 6, but day 14. An anti-HMGB1-neutralizing antibody or a low molecular weight heparin, a RAGE antagonist, prevented the development of neuropathic pain and upregulation of Egr-1 and Ca3.2 in L4 DRG after L5SNC. L5SNC increased macrophages accumulating in the sciatic nerves, and the cytoplasm/nuclear ratio of immunoreactive HMGB1 in those macrophages. Our findings suggest that L5SNC-induced Ca3.2 overexpression in L4 DRG and neuropathic pain involves Egr-1 upregulation downstream of the macrophage-derived HMGB1/RAGE pathway, and that the delayed upregulation of USP5 might contribute to the persistent Ca3.2 overexpression and neuropathy.

A major portion of individuals affected by traumatic spinal cord injury (SCI) experience one or more types of chronic neuropathic pain (NP), which is often intractable to currently available treatments. The availability of reliable behavioral assays in pre-clinical models of SCI-induced NP is therefore critical to assess the efficacy of new potential therapies. Commonly used assays to evaluate NP-related behavior in rodents, such as Hargreaves thermal and von Frey mechanical testing, rely on the withdrawal response to an evoked stimulus. However, other assays that test spontaneous/non-evoked NP-related behavior or supraspinal aspects of NP would be highly useful for a more comprehensive assessment of NP following SCI. The Mouse Grimace Scale (MGS) is a tool to assess spontaneous, supraspinal pain-like behaviors in mice; however, the assay has not been characterized in a mouse model of SCI-induced chronic NP, despite the critical importance of mouse genetics as an experimental tool. We found that beginning 2 weeks after cervical contusion, SCI mice exhibited increased facial grimace features compared to laminectomy-only control mice, and this grimace phenotype persisted to the chronic time point of 5 weeks post-injury. We also found a significant relationship between facial grimace score and the evoked forepaw withdrawal response in both the Hargreaves and von Frey tests at 5 weeks post-injury when both laminectomy-only and SCI mice were included in the analysis. However, within only the SCI group, there was no correlation between grimace score and Hargreaves or von Frey responses. These results indicate both that facial grimace analysis can be used as an assay of spontaneous NP-related behavior in the mouse model of SCI and that the information provided by the MGS may be different than that provided by evoked tests of sensory function.

In the central nervous system, hyperpolarization-activated, cyclic nucleotide-gated (HCN1-4) channels have been implicated in neuronal excitability and synaptic transmission. It has been reported that HCN channels are expressed in the spinal cord, but knowledge about their physiological roles, as well as their distribution profiles, appear to be limited. We generated a transgenic mouse in which the expression of HCN4 can be reversibly knocked down using a genetic tetracycline-dependent switch and conducted genetically validated immunohistochemistry for HCN4. We found that the somata of HCN4-immunoreactive (IR) cells were largely restricted to the ventral part of the inner lamina II and lamina III. Many of these cells were either parvalbumin- or protein kinase Cγ (PKCγ)-IR. By using two different mouse strains in which reporters are expressed only in inhibitory neurons, we determined that the vast majority of HCN4-IR cells were excitatory neurons. Mechanical and thermal noxious stimulation did not induce c-Fos expression in HCN4-IR cells. PKCγ-neurons in this area are known to play a pivotal role in the polysynaptic pathway between tactile afferents and nociceptive projection cells that contributes to tactile allodynia. Therefore, pharmacological and/or genetic manipulations of HCN4-expressing neurons may provide a novel therapeutic strategy for the pain relief of tactile allodynia.