Endogenous opioids gate the locus coeruleus pain generator.

The locus coeruleus (LC) plays a paradoxical role in chronic pain. Although largely known as a potent source of endogenous analgesia, increasing evidence suggests injury can transform the LC into a chronic pain generator. We sought to clarify the role of this system in pain. Here, we show optogenetic inhibition of LC activity is acutely antinociceptive. Following long-term spared nerve injury, the same LC inhibition is analgesic - further supporting its pain generator function. To identify inhibitory substrates that may naturally serve this function, we turned to endogenous LC mu opioid receptors (LC-MOR). These receptors provide powerful LC inhibition and exogenous activation of LC-MOR is antinociceptive. We therefore hypothesized that endogenous LC-MOR-mediated inhibition is critical to how the LC modulates pain. Using cell type-selective conditional knockout and rescue of LC-MOR receptor signaling, we show these receptors bidirectionally regulate thermal and mechanical hyperalgesia - providing a functional gate on the LC pain generator.

An electrochemical approach for rapid, sensitive, and selective detection of dynorphin.

The endogenous opioid peptide systems are critical for analgesia, reward processing, and affect, but research on their release dynamics and function has been challenging. Here, we have developed microimmunoelectrodes (MIEs) for the electrochemical detection of opioid peptides using square-wave voltammetry. Briefly, a voltage is applied to the electrode to cause oxidation of the tyrosine residue on the opioid peptide of interest, which is detected as current. To provide selectivity to these voltammetric measurements, the carbon fiber surface of the MIE is coated with an antiserum selective to the opioid peptide of interest. To test the sensitivity of the MIEs, electrodes are immersed in solutions containing different concentrations of opioid peptides, and peak oxidative current is measured. We show that dynorphin antiserum-coated electrodes are sensitive to increasing concentrations of dynorphin in the attomolar range. To confirm selectivity, we also measured the oxidative current from exposure to tyrosine and other opioid peptides in solution. Our data show that dynorphin antiserum-coated MIEs are sensitive and selective for dynorphin with little to no oxidative current observed in met-enkephalin and tyrosine solutions. Additionally, we demonstrate the utility of these MIEs in an in vitro brain slice preparation using bath application of dynorphin as well as optogenetic activation of dynorphin release. Future work aims to use MIEs in vivo for real-time, rapid detection of endogenous opioid peptide release in awake, behaving animals.

Macrophage depletion lowers blood pressure and restores sympathetic nerve α2-adrenergic receptor function in mesenteric arteries of DOCA-salt hypertensive rats.

We tested the hypothesis that vascular macrophage infiltration and O2 (-) release impairs sympathetic nerve α2-adrenergic autoreceptor (α2AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3-5, 10-13, and 18-21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22(phox) were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O2 (-) was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18-21. O2 (-) and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22(phox) were localized near sympathetic nerves. Impaired α2AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O2 (-) while restoring α2AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O2 (-), which disrupts α2AR function, causing enhanced NE release from sympathetic nerves.