Two new mouse studies pinpoint two different groups of dorsal horn inhibitory interneurons as gatekeepers of mechanical allodynia and itch. In the first study, researchers led by Reza Sharif-Naeini, McGill University, Montreal, Canada, report that parvalbumin (PV)-containing interneurons located at the border between inner laminae II and III are the gatekeepers of touch-evoked pain following nerve injury. The findings were published online October 29 in Cell Reports.
“This exciting paper is an important stone in the building of our understanding of the dorsal horn circuitry underlying mechanical allodynia,” said Daniel Voisin, University of Bordeaux, France, who was not involved in the new work. “It also brings us some hope that we can identify new targets for treating neuropathic pain,” he added.
In the second study, researchers led by Qiufu Ma, Harvard Medical School, Boston, US, and Martyn Goulding, Salk Institute for Biological Studies, La Jolla, US, implicate a different population of spinal cord inhibitory interneurons—those containing neuropeptide Y (NPY) that are found primarily in the middle lamina of the dorsal horn—in the gating of mechanical itch. The results were published October 30 in Science.
This is the first indication that there is gating of the pathway underlying mechanical itch, said Mark Hoon, National Institute of Dental and Craniofacial Research, Bethesda, US, who was not involved in either study. “It is a really interesting discovery paper that opens up a lot of new questions and will stimulate the whole field to do additional experiments,” he continued.
Together, the studies add to a growing body of evidence that identifies some of the key cellular players involved in the gate control theory of pain. First put forth by Melzack and Wall in 1965 (Melzack and Wall, 1965), this theory suggests that activation of spinal cord interneuron “gates” by innocuous sensory signals silences excitatory neurons that transmit pain information. Genetic strategies similar to those employed in the current studies have recently been used to identify glycine- and dynorphin-containing interneurons as gatekeepers of pain (see PRF related news articles here and here).
Gating mechanical allodynia
In the Cell Reports study, first author Hugues Petitjean and colleagues used the spared nerve injury (SNI) model of neuropathic pain, characterized by mechanical allodynia, to investigate the role of PV interneurons as gatekeepers of touch-pain circuitry in the dorsal horn. The existence of PV interneurons in the dorsal horn has been recognized for more than 30 years, but their contribution to sensory information processing remained largely unknown. About half of the nerve terminals of large-diameter fibers, presumably Aβ fibers (which are known to respond to light touch), receive inputs from PV neurons in the spinal cord (Hughes et al., 2012), suggesting that these interneurons may play a role in the processing of innocuous stimuli.
Some have suggested that SNI can lead to inhibitory interneuron loss (Scholz et al., 2005), so in the new study, the researchers first examined the fate of PV neurons following nerve injury. Using transgenic mice expressing a fluorescent marker in PV neurons, they found no change in the number of PV neurons at three, five, or eight weeks post-SNI, suggesting that the nerve injury did not lead to a loss of PV neurons.
To assess the function of dorsal horn PV neurons, the researchers then turned to the designer receptors exclusively activated by designer drugs (DREADD) system. Activation of PV neurons in the lumbar spinal cord of naïve mice increased baseline mechanical withdrawal thresholds but did not affect thermal withdrawal latencies, suggesting that these neurons act as modality-specific filters of somatosensory information.
“The next question was, If by activating PV neurons we can render the mice less sensitive to touch, what would be the impact of activating the cells in a neuropathic pain model in which touch is actually painful?” Sharif-Naeini said. Consistent with the researchers’ hypothesis, DREADD activation of PV neurons ipsilateral to the nerve injury two to three weeks after SNI significantly and dose-dependently attenuated mechanical allodynia. This attenuation was also observed in an inflammatory pain model known to induce both mechanical allodynia and thermal hyperalgesia. The finding that PV neuron activation did not affect thermal sensitivity in that model further supported the idea that PV neurons selectively filter mechanical sensory information.
To further explore the circuitry responsible for converting touch to pain, the researchers then investigated the specific targets of the PV neurons, suspecting excitatory interneurons containing the γ subunit of protein kinase C (PKCγ) in inner laminae II, whose activity is crucial to the development of mechanical allodynia following nerve injury (Malmberg et al., 1997; Miraucourt et al., 2007). Electron microscopy experiments revealed that PV interneurons in the dorsal horn synapse directly onto PKCγ excitatory neurons. However, after nerve injury, the PKCγ neurons received fewer inputs from the PV neurons than when the nerves were intact.
Fewer inhibitory synaptic contacts between the two populations should result in disinhibition of the PKCγ neurons and subsequent mechanical allodynia, explained Sharif-Naeini. If so, then blocking the activity of the PKCγ neurons after nerve injury should eliminate mechanical hypersensitivity, he said. Consistent with the researchers’ hypothesis, intrathecal administration of a PKCγ inhibitor significantly attenuated mechanical allodynia in nerve-injured but not control mice. In addition, after nerve injury, light brushing of the paw produced activation of neurons in the superficial dorsal horn (measured by Fos expression), suggesting that nociceptive circuits had been stimulated. However, fewer of these neurons were activated by light brushing after PKCγ inhibition, again suggesting that the activation of the nociceptive circuits depends in large part on the activation of PKCγ neurons, Sharif-Naeini added.
In a final set of experiments, the researchers selectively killed PV neurons on one side of the spinal cord in naïve animals using viral injection of the ribosome inactivating protein saporin. Two weeks later, these animals displayed reduced mechanical thresholds without any effect on sensitivity to thermal stimulation, demonstrating that PV neuron activity is sufficient to inhibit mechanical allodynia. These mice also displayed a significant loss of PV neuron appositions onto PKCγ neuron cell bodies, suggesting a disinhibition of the excitatory cells, while blockade of PKCγ neuron activity restored normal mechanical sensitivity.
“The new study identifies a novel pathway through which inhibitory neurons can block touch inputs from activating nociceptive circuits,” said Sharif-Naeini. “If we could activate PV neurons in neuropathic pain patients, perhaps there would be some analgesia provided.”
Gating mechanical itch
In the Science study, co-first authors Steeve Bourane, Salk Institute, La Jolla, US, and Bo Duan, Harvard Medical School, Boston, US, focused on a different population of spinal inhibitory interneurons, those expressing neuropeptide Y (NPY), that were found primarily in laminae III and IV and to a lesser extent in laminae I and II. The Ma and Goulding labs have been working together for a number of years to silence different molecularly defined neuron populations in the dorsal horn. “Then we let the animals’ behavior tell us what the function of the neurons is,” explained Ma. They have previously used this approach to identify somatostatin and dynorphin neurons as gatekeepers of mechanical pain (see PRF related news).
In the new study, the researchers selectively ablated NPY neurons in the dorsal horn by expressing diphtheria toxin receptor in the cells and then injecting diphtheria toxin into mice. Two weeks following injection, the animals displayed spontaneous scratching and developed skin lesions throughout the body. “[From this phenotype] we knew that NPY neurons must somehow gate or suppress some kind of itch pathway. Without these neurons, this itch pathway can be spontaneously and excessively activated,” Ma said.
Itch research has primarily focused on the sensation of chemical itch, such as that experienced following a mosquito bite, which is elicited by chemical mediators such as histamine. The researchers found that the NPY-ablated mice had normal chemical itch sensation. However, low- but not high-threshold mechanical stimulation of the nape of the neck elicited higher levels of scratching than mice with intact NPY neurons. A similar phenotype was observed when NPY neurons were acutely silenced using DREADD technology.
Additional experiments revealed that the touch-evoked itch pathway gated by NPY neurons was independent of the circuitry known to govern chemical itch. Indeed, neither blockade nor ablation of histamine receptors or gastrin-releasing peptide receptors (which also mediate chemical itch) altered the NPY neuron-ablated animals’ heightened mechanical itch phenotype.
The new findings underscore that itch is not only caused by chemical mediators, Hoon said. “Now the big question is, What are those cells that detect this [mechanical itch] stimulus?” he added. People experience both chemical and mechanical itch as the same sensation, so further studies are also needed to determine how these two separate signals become integrated into a single perception, Hoon said.
Both of the new studies shed light on the inhibitory interneurons that serve as gatekeepers of sensory information. “Together, the two papers demonstrate that different sensory modalities have their own microcircuits, and each one uses a molecularly distinct inhibitory interneuron population to gate that sensory information,” Ma told PRF.
Allison Marin, PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.
Image credit: Petitjean et al., 2015