Pharmacology/Drug Development

The amygdala plays a key role in emotional-affective aspects of pain and in pain modulation. The central nucleus (CeA) serves major amygdala output functions related to emotional-affective behaviors and pain modulation. Our previous studies implicated the corticotropin-releasing factor (CRF) system in amygdala plasticity and pain behaviors in an arthritis model. We also showed that serotonin (5-HT) receptor subtype 5-HTR in the basolateral amygdala (BLA) contributes to increased CeA output and neuropathic pain-like behaviors. Here, we tested the novel hypothesis that 5-HTR in the BLA drives CRF1 receptor activation to increase CeA neuronal activity in neuropathic pain. Extracellular single-unit recordings of CeA neurons in anesthetized adult male rats detected increased activity in neuropathic rats (spinal nerve ligation model) compared to sham controls. Increased CeA activity was blocked by local knockdown or pharmacological blockade of 5-HTR in the BLA, using stereotaxic administration of 5-HTR short hairpin RNA (shRNA) viral vector or a 5-HTR antagonist (SB242084), respectively. Stereotaxic administration of a CRF1 receptor antagonist (NBI27914) into the BLA also decreased CeA activity in neuropathic rats and blocked the facilitatory effects of a 5-HTR agonist (WAY161503) administered stereotaxically into the BLA. Conversely, local (BLA) knockdown of 5-HTR eliminated the inhibitory effect of NBI27914 and the facilitatory effect of WAY161503 in neuropathic rats. The data suggest that 5-HTR activation in the BLA contributes to neuropathic pain-related amygdala (CeA) activity by engaging CRF1 receptor signaling.

Metformin, an AMP-activated protein kinase (AMPK) activator, is an oral hypoglycemic drug widely used to treat patients with type 2 diabetes. As AMPK plays a role in the nociceptive processing, investigating the effects induced by metformin in experimental models of pain is warranted. In the present study, we further evaluated the effects induced by metformin in models of nociceptive and neuropathic pain and investigated mechanisms that could mediate such effects. Metformin was administered per os (p.o.) in mice. Nociceptive response induced by heat (hot-plate) and mechanical allodynia induced by chronic constriction injury (CCI) were used as pain models. Naltrexone (intraperitoneal) and glibenclamide (p.o.) were used to investigate mechanisms mediating metformin effects. A single administration of metformin (500 or 1000 mg/kg) inhibited the nociceptive response in the hot-plate model. Single and repeated administration of metformin (250, 500 or 1000 mg/kg) inhibited the mechanical allodynia induced by CCI. Metformin (250, 500 or 1000 mg/kg) did not affect the time mice spent in the rota-rod apparatus. The activity of metformin (1000 mg/kg) in both pain models was attenuated by naltrexone (10 mg/kg), but not by glibenclamide. Concluding, metformin exhibited activity in models of nociceptive and neuropathic pain. In the model of neuropathic pain, preventive and therapeutic effects were observed. Activation of opioidergic pathways partially mediates metformin antinociceptive activity. Altogether, the results indicate that metformin should be further investigated aiming its repositioning in the treatment of patients with different painful conditions.

The development of effective therapeutics for cancer-induced bone pain (CIBP) remains a tremendous challenge owing to its unclear mechanisms. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). Emerging studies have shown that disinhibition in the spinal cord dorsal horn may account for the development of chronic pain. However, the role of GABA in the development of CIBP remains elusive. In addition, accumulating evidence has shown that neuroglial cells in the peripheral nervous system, especially astrocytes and microglial cells, played an important role in the maintenance of CIBP. In this study, we investigated the expression of GABA and Gamma-aminobutyric acid transporter-1 (GAT-1), a transporter of GABA. Our results demonstrate that GABA was decreased in CIBP rats as expected. However, the expression of glutamic acid decarboxylase (GAD) 65 was up-regulated on day 21 after surgery, while the expression of GAD 67 remained unchanged after surgery. We also found that the expression of GAT-1 was up-regulated mainly in the astrocytes of the spinal cord. Moreover, we evaluated the analgesic effect of exogenous GABA and the GAT-1 inhibitor. Intrathecal administration of exogenous GABA and NO-711 (a GAT-1 selective inhibitor) significantly reversed CIBP-induced mechanical allodynia in a dose-dependent manner. These results firstly show that neuron-glia interactions, especially on the GABAergic pathway, contribute to the development of CIBP. In conclusion, exogenous GABA and GAT-1 inhibitor might be alternative therapeutic strategies for the treatment of CIBP.

TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.

The role of spinal α subunit-containing GABA (α-GABA) receptors in chronic pain is controversial. The purpose of this study was to investigate the participation of spinal α-GABA receptors in the reserpine-induced pain model. Reserpine administration induced tactile allodynia and muscle hyperalgesia in female and male rats. Intrathecal injection of L-655,708 and TB 21007 (7 days after the last reserpine injection) decreased tactile allodynia and, at a lesser extent, muscle hyperalgesia in female rats. The effects of these drugs produced a lower antiallodynic and antihyperalgesic effect in male than in female rats. Contrariwise, these drugs produced tactile allodynia and muscle hyperalgesia in naïve rats and these effects were lower in naïve male than female rats. Intrathecal L-838,417 prevented or reversed L-655,708-induced antiallodynia in reserpine-treated female rats. Repeated treatment with α-GABA receptor small interfering RNA (siRNA), but not scramble, siRNA reduced reserpine-induced allodynia in female rats. Accordingly, α-GABA receptor siRNA induced nociceptive hypersensitivity in naïve female rats. Reserpine enhanced α-GABA receptors expression in spinal cord and DRG, while it increased CD11b (OX-42) and glial fibrillary acidic protein (GFAP) fluorescence intensity in the lumbar spinal cord. In contrast, reserpine diminished K-Cl co-transporter 2 (KCC2) protein in the lumbar spinal cord. Data suggest that spinal α-GABA receptors play a sex-dependent proallodynic effect in reserpine-treated rats. In contrast, these receptors have a sex-dependent antiallodynic role in naïve rats.