Pharmacology/Drug Development

Opioids play a major role at descending pain modulation but the effects of neuropathic pain on the brain opioidergic system remain understudied. Since descending facilitation is enhanced during neuropathic pain, we studied the opioidergic modulation of the dorsal reticular nucleus (DRt), a medullary pain facilitatory area, in the spared nerve injury (SNI) model of neuropathic pain. We first performed a series of behavioral experiments in naïve-animals to establish the role of μ-opioid receptor (MOR) in the effects of endogenous and exogenous opioids at the DRt. Specifically, we showed that lentiviral-mediated MOR-knockdown at the DRt increased sensitivity to thermal and mechanical stimuli while the MOR agonist DAMGO induced the opposite effects. Additionally, we showed that MOR-knockdown and the pharmacological blockade of MOR by CTAP at the DRt decreased and inhibited, respectively, the analgesic effects of systemic morphine. Then, we performed microdialysis to measure enkephalin peptides in the DRt and evaluated MOR expression in the DRt at mRNA, protein and phosphorylated form levels by quantitative real-time PCR and immunohistochemistry, respectively. SNI-animals, compared to sham control, showed higher levels of enkephalin peptides, lower MOR-labeled cells without alterations in MOR mRNA levels, and higher phosphorylated MOR-labeled cells. Finally, we performed behavioral studies in SNI animals to determine the potency of systemic morphine and the effects of the pharmacologic and genetic manipulation of MOR at the DRt. We showed a reduced potency of the antiallodynic effects of systemic morphine in SNI-animals compared to the antinociceptive effects in sham animals. Increasing MOR-cells at the DRt of SNI-animals by lentiviral-mediated MOR-overexpression produced no effects on mechanical allodynia. DAMGO induced anti-allodynia only after MOR-overexpression. These results show that MOR inhibits DRt pain facilitatory actions and that this action contributes to the analgesic effects of systemic opioids. We further show that the inhibitory function of MOR is impaired during neuropathic pain. This is likely due to desensitization and degradation of MOR which are adaptations of the receptor that can be triggered by MOR phosphorylation. Skipping counter-regulatory pathways involved in MOR adaptations might restore the opioidergic inhibition at pain facilitatory areas.

Growing evidence indicates cannabinoid receptors as potential therapeutic targets for chronic pain. Consequently, there is an increasing interest in developing cannabinoid receptor agonists for treating human and veterinary pain. To better understand the actions of a drug, it is of paramount importance to know the cellular distribution of its specific receptor(s). The distribution of canonical and putative cannabinoid receptors in the peripheral and central nervous system of dogs is still in its infancy. In order to help fill this anatomical gap, the present study has been designed to identify the cellular sites of cannabinoid and cannabinoid-related receptors in canine spinal ganglia. In particular, the cellular distribution of the cannabinoid receptors type 1 and 2 (CB and CB) and putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), and transient receptor potential vanilloid type 1 (TRPV1) have been immunohistochemically investigated in the C6-C8 cervical ganglia of dogs. About 50% of the neuronal population displayed weak to moderate CB receptor and TRPV1 immunoreactivity, while all of them were CB-positive and nearly 40% also expressed GPR55 immunolabeling. Schwann cells, blood vessel smooth muscle cells, and pericyte-like cells all expressed CB receptor immunoreactivity, endothelial cell being also PPARα-positive. All the satellite glial cells (SGCs) displayed bright GPR55 receptor immunoreactivity. In half of the study dogs, SGCs were also PPARα-positive, and limited to older dogs displayed TRPV1 immunoreactivity. The present study may represent a morphological substrate to consider in order to develop therapeutic strategies against chronic pain.

Sigma-1 (σ) receptor antagonists are promising tools for neuropathic pain treatment, but it is unknown whether σ receptor inhibition ameliorates the neuropathic signs induced by nerve transection, in which the pathophysiological mechanisms and response to drug treatment differ from other neuropathic pain models. In addition, σ antagonism ameliorates inflammatory pain through modulation of the endogenous opioid system, but it is unknown whether this occurs during neuropathic pain. We investigated the effect of σ inhibition on the painful hypersensitivity associated with the spared nerve injury (SNI) model in mice. Wild-type (WT) mice developed prominent cold (acetone test), mechanical (von Frey test), and heat hypersensitivity (Hargreaves test) after SNI. σ receptor knockout (ခσ-KO) mice did not develop cold allodynia and showed significantly less mechanical allodynia, although they developed heat hyperalgesia after SNI. The systemic acute administration of the selective σ receptor antagonist S1RA attenuated all three types of SNI-induced hypersensitivity in WT mice. These ameliorative effects of S1RA were reversed by the administration of the σ agonist PRE-084, and were absent in σ-KO mice, indicating the selectivity of S1RA-induced effects. The opioid antagonist naloxone and its peripherally restricted analog naloxone methiodide prevented S1RA-induced effects in mechanical and heat hypersensitivity, but not in cold allodynia, indicating that opioid-dependent and -independent mechanisms are involved in the effects of this σ antagonist. The repeated administration of S1RA twice a day during 10 days reduced SNI-induced cold, mechanical, and heat hypersensitivity without inducing analgesic tolerance during treatment. These effects were observed up to 12 h after the last administration, when S1RA was undetectable in plasma or brain, indicating long-lasting pharmacodynamic effects. These data suggest that σ antagonism may have therapeutic value for the treatment of neuropathic pain induced by the transection of peripheral nerves.

Optopharmacology is a very promising approach based on the use of light-deliverable drugs, which allows manipulating physiological processes with high spatiotemporal resolution. Light-dependent drugs (i.e. caged-compounds) targeting G protein-coupled receptors (GPCRs) have been developed to provide great pharmacological precision on the control of pain. Metabotropic glutamate type 5 (mGlu) receptors are widely expressed through the pain neuraxis and play a key role in pain transmission. In line with this, selective mGlu receptor negative allosteric modulators (NAMs) have consistently shown analgesic activity in experimental animal models of inflammatory pain. Accordingly, we synthesized a light-deliverable drug (i.e. caged compound) using the chemical structure of raseglurant, a mGlu receptor NAM, as a molecular scaffold. And thereafter, we evaluated the analgesic activity of the caged compound in formalin-injected (hind paw) mice upon light irradiation (405 nm). Of note, light was both delivered at the peripheral (i.e. hind paw) and central level (i.e. thalamus), by means of brain-implanted fiber-optics. The novel light-deliverable drug, JF-NP-26, showed antinociceptive activity upon violet light irradiation either of the hind paw or the thalamus, demonstrating the ability of precisely activating, in time and space, the caged compound. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin nociception test in mice using an optopharmacology approach (i.e. light-deliverable compounds).

Of the fast ionotropic synapses, glycinergic synapses are the least well understood, but are vital for the maintenance of inhibitory signaling in the brain and spinal cord. Glycinergic signaling comprises half of the inhibitory signaling in the spinal cord, and glycinergic synapses are likely to regulate local nociceptive processing as well as the transmission to the brain of peripheral nociceptive information. Here we have investigated the rapid and prolonged potentiation of glycinergic synapses in the superficial dorsal horn of young male and female mice after brief activation of NMDA receptors (NMDARs). Glycinergic inhibitory postsynaptic currents (IPSCs) evoked with lamina II-III stimulation in identified GABAergic neurons in lamina II were potentiated by bath-applied Zn2+ and were depressed by the prostaglandin PGE2, consistent with the presence of both GlyRα1- and GlyRα3-containing receptors. NMDA application rapidly potentiated synaptic glycinergic currents. Whole-cell currents evoked by exogenous glycine were also readily potentiated by NMDA, indicating that the potentiation results from altered numbers or conductance of postsynaptic glycine receptors. Repetitive depolarization alone of the postsynaptic GABAergic neuron also potentiated glycinergic synapses, and intracellular EGTA prevented both NMDA-induced and depolarization-induced potentiation of glycinergic IPSCs. Optogenetic activation of trpv1 lineage afferents also triggered NMDAR-dependent potentiation of glycinergic synapses. Our results suggest that during peripheral injury or inflammation, nociceptor firing during injury is likely to potentiate glycinergic synapses on GABAergic neurons. This disinhibition mechanism may be engaged rapidly, altering dorsal horn circuitry to promote the transmission of nociceptive information to the brain.