Animal Studies

Presynaptic Ca2.2 channels control calcium entry that triggers neurotransmitter release at both central and peripheral synapses. The Cacna1b gene encodes the α1-pore forming subunit of Ca2.2 channels. Distinct subsets of splice variants of Ca2.2 derived from cell-specific alternative splicing of the Cacna1b pre-mRNA are expressed in specific subpopulations of neurons. Four cell-specific sites of alternative splicing in Cacna1b that alter Ca2.2 channel function have been described in detail: three cassette exons (e18a, e24a, and e31a) and a pair of mutually exclusive exons (e37a/e37b). Cacna1b mRNAs containing e37a are highly enriched in a subpopulation of nociceptors where they influence nociception and morphine analgesia. E37a-Cacna1b mRNAs are also expressed in brain, but their cell-specific expression in this part of the nervous system, their functional consequences in central synapses and their role on complex behavior have not been studied. In this report, we show that e37a-Cacna1b mRNAs are expressed in excitatory projection neurons where Ca2.2 channels are known to influence transmitter release at excitatory inputs from entorhinal cortex (EC) to dentate gyrus (DG). By comparing behaviors of WT mice to those that only express e37b-Ca2.2 channels, we found evidence that e37a-Ca2.2 enhances behavioral responses to aversive stimuli. Our results suggest that alternative splicing of Cacna1b e37a influences excitatory transmitter release and couples to complex behaviors.

Although dysfunction of the mesolimbic dopaminergic system has been implicated in chronic pain, the underlying mechanisms remain to be elucidated. We hypothesized that increased inhibitory inputs to the neuronal pathway from the dorsolateral bed nucleus of the stria terminalis (dlBNST) to the ventral tegmental area (VTA) during chronic pain may induce tonic suppression of the mesolimbic dopaminergic system. To test this hypothesis, male Sprague-Dawley rats were subjected to spinal nerve ligation to induce neuropathic pain and then spontaneous inhibitory postsynaptic currents (sIPSCs) were measured in this neuronal pathway. Whole-cell patch-clamp electrophysiology of brain slices containing the dlBNST revealed that the frequency of sIPSCs significantly increased in VTA-projecting dlBNST neurons 4 weeks after surgery. Next, the role of corticotropin-releasing factor (CRF) signaling within the dlBNST in the increased sIPSCs was examined. CRF increased the frequency of sIPSCs in VTA-projecting dlBNST neurons in sham-operated controls, but not in chronic pain rats. By contrast, NBI27914, a CRF type 1 receptor antagonist, decreased the frequency of sIPSCs in VTA-projecting dlBNST neurons in the chronic pain rats but not in the control animals. In addition, histological analyses revealed the increased expression of CRF mRNA in the dlBNST. Finally, bilateral injections of NBI27914 into the dlBNST of chronic pain rats activated mesolimbic dopaminergic neurons and induced conditioned place preference. Taken together, these results suggest that the mesolimbic dopaminergic system is tonically suppressed during chronic pain by enhanced CRF signaling within the dlBNST via increased inhibitory inputs to VTA-projecting dlBNST neurons.The comorbidity of chronic pain and depression has long been recognized. Although dysfunction of the mesolimbic dopaminergic system has been implicated in both chronic pain and depression, the underlying mechanisms remain to be elucidated. Here we show that the inhibitory inputs to the neuronal pathway from the dorsolateral bed nucleus of the stria terminalis (dlBNST) to the ventral tegmental area increase during chronic pain. This neuroplastic change is mediated by enhanced corticotropin-releasing factor signaling within the dlBNST that leads to tonic suppression of the mesolimbic dopaminergic system, which may be involved in the depressive mood and anhedonia under the chronic pain condition.

HIV-1 infection of the nervous system causes various neurological diseases, and synaptic degeneration is likely a critical step in the neuropathogenesis. Our prior studies revealed a significant decrease of synaptic protein specifically in the spinal dorsal horn (SDH) of HIV-1 patients who developed pain, suggesting a potential contribution of synaptic degeneration to the pathogenesis of HIV-associated pain. However, the mechanism by which HIV-1 causes the spinal synaptic degeneration is unclear. Here, we identified a critical role of microglia in the synaptic degeneration. In primary cortical cultures (DIV14) and spinal cords of 3-5-month-old mice (both genders), microglial ablation inhibited gp120-induced synapse decrease. Fractalkine (FKN), a microglia-activation chemokine specifically expressed in neurons, was up-regulated by gp120, and knockout of the FKN receptor CX3CR1, which is predominantly expressed in microglia, protected synapses from gp120-induced toxicity. These results indicate the neuron-to-microglia intercellular FKN/CX3CR1 signaling plays a role in gp120-induced synaptic degeneration. To elucidate the mechanism controlling this intercellular signaling, we tested the role of the Wnt/β-catenin pathway in regulating FKN expression. Inhibition of Wnt/β-catenin signaling blocked both gp120-induced FKN up-regulation and synaptic degeneration, and gp120 stimulated Wnt/β-catenin-regulated FKN expression via NMDA receptors. Furthermore, NMDAR antagonist APV, Wnt/β-catenin signaling suppressor DKK1 or knockout of CX3CR1 alleviated gp120-induced mechanical allodynia in mice, suggesting a critical contribution of the Wnt/β-catenin/FKN/CX3R1 pathway to gp120-induced pain. These findings collectively suggest that HIV-1 gp120 induces synapse degeneration in the spinal pain neural circuit by activating microglia via Wnt3a/β-catenin-regulated FKN expression in neurons.HIV patients with chronic pain develop synaptic degeneration in the spinal cord dorsal horn, but the patients without the pain disorder do not show this neuropathology, indicating a pathogenic contribution of the synaptic degeneration to the development of HIV-associated pain. However, the mechanism underlying the synaptic degeneration is unclear. We report here that HIV-1 gp120, a neurotoxic protein that is specifically associated with the manifestation of pain in HIV patients, induces synapse loss via microglia. Further studies elucidate that gp120 activates microglia by stimulating Wnt/β-catenin-regulated fractalkine in neuron. The results demonstrate a critical role of microglia in the pathogenesis of HIV-associated synaptic degeneration in the spinal pain neural circuit.

Regulator of G protein signaling 4 (RGS4) is a potent modulator of G protein-coupled receptor (GPCR) signal transduction that is expressed throughout the pain matrix. Here, we use genetic mouse models to demonstrate a role of RGS4 in the maintenance of chronic pain states in male and in female mice. Using paradigms of peripheral inflammation and nerve injury, we show that prevention of RGS4 action leads to recovery from mechanical and cold hypersensitivity and increases motivation for wheel running. Similarly, RGS4KO eliminates the duration of nocifensive behavior in the second phase of the formalin assay. Using the Complete Freud's adjuvant (CFA) model of hind paw inflammation we also demonstrate that downregulation of RGS4 in the adult ventral posterolateral thalamic nuclei (VPL-THL) promotes recovery from mechanical and cold allodynia. RNA sequencing analysis of thalamus (THL) from RGS4WT and RGS4KO mice points to many signal transduction modulators and transcription factors that are uniquely regulated in CFA-treated RGS4WT cohorts. Ingenuity Pathway Analysis suggests that several components of glutamatergic signaling are differentially affected by CFA treatment between RGS4WT and RGS4KO groups. Notably, western blot analysis shows increased expression of metabotropic glutamate receptor 2 (mGluR2) in THL synaptosomes of RGS4KO mice at time points at which they recover from mechanical allodynia. Overall, our study provides information on a novel intracellular pathway that contributes to the maintenance of chronic pain states and points to RGS4 as a potential therapeutic target.There is an imminent need for safe and efficient chronic pain medications. RGS4 is a multifunctional signal transduction protein, widely expressed in the pain matrix. Here, we demonstrate that RGS4 plays a prominent role in the maintenance of chronic pain symptoms in male and female mice. Using genetically modified mice we show a dynamic role of RGS4 in recovery from symptoms of sensory hypersensitivity deriving from hind paw inflammation or hind limb nerve injury. We also demonstrate an important role of RGS4 actions in gene expression patterns induced by chronic pain states in the mouse thalamus. Our findings provide novel insight into mechanisms associated with the maintenance of chronic pain states and demonstrate that interventions in RGS4 activity promote recovery from sensory hypersensitivity symptoms.