Somatostatin (SOM)-positive excitatory neurons in the spinal cord are key conduits for mechanical pain, according to a report published online November 20 in Cell. In the study, Qiufu Ma of the Dana-Farber Cancer Institute and Harvard Medical School, Boston, US, in collaboration with Martyn Goulding at the Salk Institute in La Jolla, California, US, found that ablating spinal cord SOM neurons in mice abolished the animals’ responses to painful mechanical stimulation, yet preserved responses to heat and cold. SOM-ablated mice were also protected from touch allodynia after chronic inflammation or nerve injury. In addition, the researchers identified dynorphin (Dyn)-positive inhibitory neurons that, when ablated, promoted painful responses to innocuous touch.
The results suggest that SOM and Dyn neurons might eventually be harnessed therapeutically to control mechanical pain without affecting other sensations. The findings also help illuminate the thicket of spinal cord wiring for pain transmission, placing the neurons in a circuit diagram proposed by the gate control theory of pain 50 years ago.
“We identified a cellular target for selective mechanical pain treatment that might at the same time preserve other basic somatic sensations,” Ma told PRF. “But it’s a long way to go before we have something for the clinic.”
The specificity of the observed effects on mechanical pain is unexpected, given how many different kinds of sensory afferents converge in the spinal cord.
Yet the new results suggest that a specific “line” for mechanical nociception exists, particularly within lamina 2 of the spinal cord, which contains the SOM-positive excitatory interneurons.
“Our work suggests that lamina 2 is required to transmit nociceptive mechanical information,” Ma said, adding that SOM neurons might respond to heat, but that they are not necessary for expressing a heat response.
"I think it’s very provocative that they have no heat phenotype,” said Allan Basbaum of the University of California, San Francisco, who studies spinal cord circuitry. “Now the question is, How did that come about?” Basbaum was not involved in the study.
Basbaum noted that the results fit with much earlier studies pointing to anti-nociceptive powers of antibodies to SOM that were specific to mechanical pain (Kuraishi et al., 1985; Ohno et al., 1988).
Gate control
Ma teamed with Goulding to generate mice in which subsets of spinal interneurons were marked and ablated using a novel intersectional genetics technique that crosses three mouse lines (Dymecki and Kim, 2007). The researchers made diphtheria toxin receptor expression dependent on the combination of a cell type-specific promoter and a dorsal spinal cord- and hindbrain-specific promoter. In the engineered mice, introducing diphtheria toxin destroyed specific sets of spinal cord interneurons, allowing the researchers to examine their roles in pain one cell type at a time. Unlike previous attempts to study specific pools of spinal cord interneurons, this approach leaves afferent inputs intact.
The researchers reported results from six different ablations—three of excitatory and three of inhibitory interneurons. Among these, the SOM neuron-ablated mice stood out as completely different. They had no response to a painful pinch stimulus, despite being able to sense non-painful touch stimuli, heat, and cold. Inflammatory or neuropathic pain conditions did not produce sensitivity to innocuous touch, or mechanical allodynia, either. The assays included gentle brushing of the skin, which models the dynamic allodynia that is difficult to treat in humans.
Electrophysiological recordings in spinal cord slices revealed that almost all SOM neurons received inputs from small, pain-transmitting C and Aδ fibers, as well as the large-diameter Aβ fibers that relay innocuous touch stimulation. This made them akin to a pain-transmitting “T cell” in the circuit diagram proposed by gate control theory in 1965, long before the many diverse interneuron types of the spinal cord were known (Melzack and Wall, 1965; Braz et al., 2014).
The gate theory accounted for the phenomenon where stimulating sensory neurons that respond to harmless touch could bring pain relief. In the proposed circuit, innocuous sensory signals access the pain-transmitting T cell via a gate embodied as an inhibitory interneuron. Stimulating sensory fibers like Aβ fibers could activate the inhibitory interneuron, thus closing the gate and diminishing any pain signals sent by the T cell to the brain.
Consistent with this scenario, Aβ fiber stimulation could evoke inhibitory currents in SOM neurons, and blocking inhibition enabled Aβ inputs to drive SOM neurons to fire action potentials. Unlike the theory, however, SOM neurons do not project to the brain.
This leaves open the question of whether the SOM neurons are involved in generating a true pain message destined for the brain, or function in a reflex circuit restricted to the spinal cord that results in mechanical hypersensitivity, Basbaum said.
The findings also deal with only one-half of the theory. “Gate control theory postulated that, not only do the large fibers close the gate, but the small fibers open the gate by shutting off the closing system,” Basbaum said. “That’s a part of the circuit everyone’s still trying to understand.”
Itch puzzle
The researchers also found that Dyn-ablated mice developed mechanical allodynia, which could not be worsened by inflammation or nerve injury. Dyn neurons had electrophysiological properties consistent with inhibitory interneurons in the gate control circuit. Gently touching Dyn-ablated mice resulted in widespread activation of SOM neurons, suggesting that Dyn neurons normally curb SOM responses to innocuous stimuli.
Dyn-ablated mice did not develop excessive itchiness, contrary to a recent report of elevated itch in a knockout mouse line lacking inhibitory interneurons containing dynorphin (Kardon et al., 2014; see also PRF conference coverage). The two techniques—Bhlhb5 transcription factor knockout or genetically targeted ablation—affect overlapping but distinct populations of interneurons, and it remains to be determined how each gives rise to the divergent itch behaviors. “It’s really paradoxical, and it needs a more detailed explanation,” Basbaum said.
Michele Solis is a science writer and former neuroscientist who lives in Seattle, Washington, US.
Image: Adapted from Duan et al., 2014. Reprinted with permission from Elsevier
