New research has uncovered a spinal cord circuit, centered on excitatory interneurons in the deep dorsal horn, that plays a key role in persistent mechanical pain. The cells transiently express the vesicular glutamate transporter 3 (VGLUT3) and, surprisingly, are found in lamina III, an area known to play a role in the sensation of touch but which has been largely overlooked in the study of pain. Researchers led by Rebecca Seal, University of Pittsburgh, US, also identified additional components of the circuit, including lamina II neurons that express the calcium-binding protein calretinin. The findings were published August 19 in Neuron.
“The present study represents a veritable tour de force in the elegant use of conditional gene deletion and systematic behavioral phenotyping coupled with modern methods of cell silencing as well as circuit mapping,” wrote Vijayan Gangadharan and Rohini Kuner, Heidelberg University, Germany, in an accompanying commentary.
“I think it’s a very important piece of work, and certainly provides yet another group of cells that are thought to be important in mechanical allodynia,” said Andrew Todd, University of Glasgow, UK, who was not involved in the current research.
Dissecting mechanical pain circuitry
Persistent mechanical pain resulting from injury or disease is one of the most intractable forms of pain, and the underlying spinal cord and brain circuitry responsible remain largely unknown. Seal and her team previously reported that VGLUT3 is required specifically for mechanical pain in mice (Seal et al., 2009). In the new study, co-first authors Cedric Peirs, Sean-Paul Williams, and colleagues generated several lines of VGLUT3 conditional knockout mice using the Cre/loxP system in order to identify the specific population of VGLUT3-expressing neurons required for mechanical pain and other components of the mechanical pain circuit.
The researchers first examined the effects of germline deletion of VGLUT3, noting reduced acute mechanical pain, measured with the Randall-Selitto paw pressure assay, as well as lower mechanical hypersensitivity in both the carrageenan model of inflammatory pain and the spared nerve injury model of neuropathic pain. This phenotype was similar to what the researchers had observed in global VGLUT3 knockout mice in their earlier study.
To determine where the VGLUT3 neurons responsible for mechanical allodynia were located, the researchers then crossed mice with two floxed alleles of VGLUT3 with several different strains of Cre mice, assessing the role of the transporter in various peripheral and spinal cord cell populations. They found that only one strain—in which Cre was expressed only in spinal dorsal horn neurons in order to delete VGLUT3 in those cells, and not in the brain, DRG, or Merkel cells (where VGLUT3 is also present)—displayed elevated withdrawal thresholds in both the acute and chronic mechanical pain assays compared to control animals. Further experiments revealed that it was specifically excitatory VGLUT3 interneurons in the deep (lamina III) dorsal horn that contributed to these behavioral alterations.
Interestingly, the group found that only short-lived VGLUT3 expression was required to produce the deficits in mechanical pain behaviors. “The fact that VGLUT3 is only transiently expressed during early development in these lamina III neurons makes it rather surprising that simply blocking VGLUT3 in these cells should produce such a dramatic pain phenotype in the adult,” said Todd. Future studies are needed to determine the extent to which these findings are the result of altered development and compensatory changes stemming from VGLUT3 knockout, he added.
An in vitro spinal cord slice preparation (Torsney and MacDermott, 2006) used for studying mechanical allodynia produced results consistent with the behavioral studies and indicated that VGLUT3 knockouts have impaired neuronal transmission within the dorsal horn. Under complete pharmacological disinhibition, low-threshold primary afferent stimulation did not cause polysynaptic activity in neurokinin 1 receptor-expressing lamina I cells in VGLUT3 knockout mice, as would normally be observed, indicating a signaling defect in the mechanical allodynia pathway in VGLUT3 knockout mice.
Subsequent experiments revealed that the deletion of VGLUT3 did not appear to cause cell death, or alterations in the architecture or afferent innervation of the dorsal horn, a concern given the observation of gross anatomical defects in mice with spinal cord gene deletions (Ross et al., 2010). Thus, the mechanical pain phenotype observed in the animals could not be attributed to those factors.
The researchers also showed that a high proportion of the lamina III VGLUT3 cells received input directly from low-threshold, myelinated Aβ fibers. That result, along with the study’s behavioral findings, suggested that the transient VGLUT3 population is an entry point into the dorsally directed mechanical allodynia circuit.
A DREADD approach
To further demonstrate the role of VGLUT3-containing neurons in mechanical pain, the researchers then turned to an excitatory Designer Receptor Exclusively Activated by Designer Drug (DREADD) approach. “DREADDs allow one to examine behavior while reversibly increasing or decreasing the synaptic output of a neuron population with a time course of hours,” wrote Seal in an email to PRF. DREADD delivery was accomplished by direct injection of an adeno-associated virus into the dorsal horn, allowing selective activation of spinal cord neurons but not DRG or brain cells.
DREADD-induced activation of lamina III VGLUT3-containing neurons induced mechanical hypersensitivity and allodynia (but did not affect heat hypersensitivity), an effect that was absent in mice lacking VGLUT3. “This is a key finding because it indicates that activating [VGLUT3] neurons is sufficient to drive activity of the downstream circuit that culminates into mechanical allodynia,” wrote Gangadharan and Kuner.
To find additional neurons in the mechanical allodynia circuit, the researchers examined the expression of c-Fos, a marker of neuronal activity, after the animals engaged in walking behavior (to elicit low-threshold stimulation) following DREADD activation of the lamina III VGLUT3-containing neurons. They observed an increase in c-Fos expression in lamina I-III primarily in the medial dorsal horn. Additional staining showed that these cells could be divided into at least four populations of excitatory interneurons: those containing calretinin, protein kinase Cγ (PKCγ), paired box 2 (Pax2), and an unidentified group.
Focusing on the calretinin neurons, the researchers found that DREADD activation of this lamina II population induced mechanical hypersensitivity without affecting thermal sensitivity, similar to the effect of activating lamina III VGLUT3 neurons. This finding contrasts with a recent study suggesting that calretinin neurons are important only for light mechanical pain, hinting at the existence of two different calretinin neuron populations in lamina II (Duan et al., 2014).
In a final set of experiments, Seal and colleagues found that distinct spinal microcircuits played a role in mechanical allodynia depending on the type of injury, though both required VGLUT3-containing neurons. In the carrageenan model of inflammatory pain, mechanical stimulation increased c-Fos expression in calretinin and Pax2 cells, but not in those expressing PKCγ. In contrast, in the spared nerve injury model of neuropathic pain, mechanical stimulation led to c-Fos expression in some PKCγ and a few calretinin neurons.
The results from the current work reveal a complicated picture of mechanical allodynia, in which signals from VGLUT3 neurons in lamina III are relayed through the different laminae of the dorsal horn to a number of different populations of neurons expressing various markers and, ultimately, to lamina I nociceptive projection neurons.
Overall, the new study “provides insight into the neuronal populations that participate in the dorsal horn circuit for mechanical hypersensitivity, which provides the anatomical foundation for understanding the mechanisms that convert touch into pain in the setting of injury,” wrote Seal.
Future studies will focus on identifying additional populations of neurons within the dorsally directed mechanical hypersensitivity circuit and delineating the connectivity among these populations, as well as identifying the specific subtypes of primary sensory neurons that activate them, Seal explained. “Understanding how descending inputs modulate the activity of this dorsal horn circuit will also be key,” she wrote. Finally, more work is required to fully understand the mechanisms by which different types of injury trigger disinhibition within the dorsal horn and to understand which excitatory and inhibitory interneurons are involved, she added.
Allison Marin (Curley), PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, Pennsylvania, US.