A new study identifies a very small population of about 30 specialized parvocellular oxytocin (OT) neurons that project simultaneously to the supraoptic nucleus (SON) of the hypothalamus and to the spinal cord, and control inflammatory but not neuropathic pain. In addition, the study, led by Alexandre Charlet, University of Strasbourg, France, and Valery Grinevich, University of Heidelberg, Germany, reports that the new OT population controls nociception in rats via both peripheral and central mechanisms.
“The two fascinating parts of this story are the small number of cells that can affect behavior with miniscule increases in the circulating concentration of oxytocin, and the ability [of this population] to affect only one type of pain,” said James Eisenach, Wake Forest University, Winston-Salem, US. “The results suggest that a systemically active analogue of oxytocin might be analgesic, and this is worth exploring,” he added.
The new study was published online March 3 in Neuron.
The hormone OT stimulates uterine contractions and is widely used in medicine to induce labor. It is also critical for the milk let-down reflex of lactation. In addition, researchers have realized that it may play a role in analgesia (see PRF related news story). Rodent models have demonstrated that OT can relieve a variety of different types of pain—including mechanical, thermal, inflammatory, and neuropathic pain—and a handful of clinical trials have shown that OT can improve pain from back injury, migraine, and irritable bowel syndrome (Goodin et al., 2015).
OT, a neuropeptide, is produced mainly in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. OT is released by neurons classified as either magnocellular or parvocellular, two populations that differ in their morphology, location, and function. Magnocellular OT neurons are responsible for the release of OT into the bloodstream via the posterior pituitary gland and project mainly to the forebrain. Parvocellular OT neurons, on the other hand, project to nuclei in the brainstem as well as to the spinal cord and are thought to be the population that contributes to nociception (Condés-Lara et al., 2003). However, the exact mechanism by which parvocellular OT axons can release OT and affect nociception remained unknown.
A new population
In the new study, Charlet, Grinevich, and colleagues were not initially looking at the role of OT neurons in nociception, Charlet told PRF. “While tracing oxytocinergic projections from the PVN to the SON in an effort to understand the generation of the bursting activity of oxytocinergic neurons that is involved in lactation, we found this unique neuronal population,” he wrote in an email.
To identify PVN neurons projecting to the SON, co-first authors Marina Eliava, Meggane Melchior, H. Sophie Knobloch-Bollmann, and Jérôme Wahis injected canine adenovirus 2 (CAV2), which is retrogradely transported and monosynaptically transmitted, into the SON; CAV2 carried a green fluorescent protein marker to label OT-positive cells. Sectioning the entire PVN and counting all of the labeled neurons revealed a very small population of approximately 30 OT neurons. Subsequent electrophysiological and morphological experiments revealed that these neurons were parvocellular neurons. In addition, light and electron microscopy and electrophysiological studies demonstrated that these parvocellular OT neurons not only synapsed onto magnocellular OT neurons in the SON, but also controlled the activity of these cells, resulting in the release of OT into the bloodstream.
“[Up until now], the dogma [in the field] was that there are no connections between oxytocin neurons. We found that there is a specific group of cells that connect parvocellular and magnocellular oxytocin neurons, and that these cells are oxytocinergic as well,” explained Grinevich.
The researchers next followed the projections of this parvocellular OT neuron population into the spinal cord, finding axons primarily in the deep layers of the spinal cord and located close to neurons containing the neurokinin 1 receptor (NK1R) and oxytocin receptor (OTR), a population which prior studies have identified as sensory-wide dynamic range (WDR) neurons. These NKR1/OTR neurons were activated by noxious stimuli, as injection of capsaicin in the hind paws induced c-Fos expression, a marker of neuronal activity, in the cells.
“[Next], we decided to identify the functional circuit and find out what these cells that project to both the hypothalamus and spinal cord are doing,” Grinevich said. To do that, the researchers turned to optogenetics to investigate the effect of the parvocellular OT neurons, engineered to express the light-sensitive channelrhodopsin-2 (ChR2) protein, on the NKR1/OTR cells. As expected, WDR C-fiber-evoked spikes in response to hind paw stimulation increased when the researchers added the NK1R specific agonist SarMet-SP. But the number of spikes decreased when blue light was used to selectively stimulate the ChR2-expressing parvocellular OT neurons. Taken together, the results indicated that OT release from the parvocellular population can inhibit the activation of WDR sensory neurons and therefore prevent the transmission of a stream of sensory, especially pain, information, said Grinevich. Additional experiments demonstrated that this inhibitory action resulted from the binding of a G protein to the OTR.
Finally, a series of electrophysiological experiments revealed that the newly identified parvocellular OT population could modulate nociception in two ways: quickly, through the direct release of OT onto WDR neurons in the spinal cord, inhibiting their activity, and more slowly and indirectly, by stimulating release of OT from SON neurons into the blood, which then acts on peripheral targets.
Grinevich wasn’t too surprised by the small size of the new oxytocin population, because the nervous system has such large amplification abilities. In many areas of the brain a single neuron terminates on hundreds or thousands of downstream cells. “You need many axon terminals, but not many cells [to create a large effect],” he explained. In addition, OTRs are so sensitive to oxytocin, so a very small number of molecules can trigger a large effect in the spinal cord, Grinevich said.
Parvocellular OT neurons promote pain relief
In a final set of experiments, the researchers analyzed the effects of activating or inhibiting the small parvocellular OT neuron population identified in the study on analgesia, using inflammatory and neuropathic pain models. Painful peripheral inflammatory sensitization was achieved via a single intraplantar injection of complete Freund’s adjuvant (CFA). Neuropathic pain was modeled by cuffing the sciatic nerve.
The application of blue light to the PVN in rats expressing ChR2 specifically in the parvocellular OT neuron population partially alleviated CFA-mediated hyperalgesia in response to mechanical and thermal stimulation, but had no effect on mechanical hyperalgesia resulting from the cuffed sciatic nerve. As further evidence that this analgesic effect was mediated by activation of OT neurons, pain returned following intraperitoneal injection of a blood-brain permeable OTR antagonist.
Using the designer receptors exclusively activated by designer drugs (DREADD) system to inhibit the parvocellular OT population yielded opposite results. Here, rats expressing the DREADD hM4Di specifically in the parvocellular OT population that were then subsequently exposed to the ligand clozapine-N-oxide (CNO) showed an increase in CFA-mediated mechanical and thermal hyperalgesia, but there were no effects on mechanical hyperalgesia induced by sciatic nerve cuffing.
Importantly, neither gain nor loss of parvocellular OT function affected mechanical or thermal sensitivity in non-sensitized animals, indicating that this cell population did not affect normal sensory processing.
Overall, the new findings are quite incredible, though in need of replication, said Eisenach. “It’s a pretty amazing story. A system in the brain with a really, really small number of neurons can simultaneously release OT onto magnocellular neurons in the SON, which then release enough OT into the blood for a peripheral effect, and onto a few cells deep in the dorsal horn to affect the behavior of animals and analgesia, selectively only in inflammatory states,” he said.
Allison Marin, PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
Image credit: Eliava et al., 2016, with permission from Elsevier