Primary sensory neurons in dorsal root ganglia (DRG) are highly diverse. Activation of small- to medium-diameter DRG neurons can generate different types of somatosensation, including pain, itch, and tactile sensation. The behavioral responses to these sensations are specific and distinct. For instance, pain causes a withdrawal response (to avoid tissue injury) and itch causes a scratching response (to remove irritants and suppress itch). However, the detailed cellular and molecular mechanisms underlying each of these sensations are not completely understood, especially for itch and tactile sensation. The major difficulties in tackling the problem are the lack of molecular markers that label specific subpopulations of small- to medium-diameter neurons in DRG and the lack of cell surface receptors directly activated by specific sensory stimuli.
My goal is to understand how these three sensations are initiated by DRG neurons. To achieve this, we isolated many mouse genes specifically expressed in small- to medium-diameter neurons but not in other types of sensory neurons. In a multidisciplinary approach, we use mouse genetics, behavioral assays, electrophysiology, biochemistry, and molecular biology to study the functions of the genes in somatosensation. Since some of these genes are exclusively expressed in DRG neurons, we have used them to study the axon projection patterns of neurons expressing the genes. Based on these functional and circuitry analyses, we believe that we now have the molecular tools essential for studying all three somatosensations initiated by these neurons.
Pain Signaling and Modulation
Transient receptor potential vanilloid 1 (TRPV1) is a molecular sensor of noxious heat and capsaicin. Its channel activity can be modulated by several mechanisms, including interaction with phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 constitutes less than 1 percent of membrane phospholipids and does not diffuse freely in the membrane. It has been speculated that PIP2-sensitive channels interact with other PIP2-binding proteins. We identified a novel membrane protein, Pirt, as a regulator of TRPV1. Pirt is highly expressed in most pain-sensing neurons in the DRG, including TRPV1-positive cells, but not in the central nervous system.
Pirt-null mice show impaired responsiveness to noxious heat and capsaicin. Noxious heat- and capsaicin-sensitive currents in Pirt-deficient DRG neurons are significantly attenuated. These mutant phenotypes resemblethose observed in TRPV1–/–mice, although they are less severe. Heterologous expression of Pirt strongly enhances TRPV1-mediated currents. The C-terminus of Pirt binds to TRPV1 and several phosphoinositides, including PIP2, and can potentiate TRPV1. The PIP2 binding is dependent on the cluster of basic residues in the Pirt C-terminus and is crucial for Pirt regulation of TRPV1. The enhancement of TRPV1 by PIP2 requires Pirt. Therefore, Pirt is a key component of the TRPV1 complex and positively regulates TRPV1 activity. We also found that Pirt can potentially regulate another PIP2-sensitive TRP channel, TRPM8, a molecular sensor for cold temperature and menthol. Numerous studies have shown that PIP2 regulates various ion channels, including TRPs, voltage-gated potassium, calcium, and sodium channels. Thus, our research is not only relevant to the TRP field but also has implications for the general understanding of ion channel regulation.Manipulating the interaction between TRPV1 and Pirt may be a novel mechanism for analgesia.
Decoding Itch Sensation
Itch (formally known as pruritus) has been defined as an unpleasant sensation that elicits the desire or reflex to scratch. Itch-sensing neurons in DRG can be broadly divided into two classes: histamine-sensitive and histamine-insensitive neurons. Histamine-induced itch in human skin can be almost completely blocked by histamine receptor H1 antagonists. The histamine blockade is ineffective, however, in many other itch conditions, such as dermatitis, drug-induced side effects, and mechanically induced itch, suggesting the existence of histamine-independent types of itch. The major hurdles in studying histamine-independent itch are the lack of molecular markers that label the responsive neurons and the lack of membrane receptors directly activated by the histamine-unrelated itch-inducing agents.
We recently identified a membrane receptor that is specifically expressed in a subset of DRG neurons and directly mediates chloroquine (CQ)-induced itch. CQ is a drug that has been used in the treatment and prevention of malaria. CQ-induced itch, which is common among black Africans, is not considered a cause of allergic reaction, since pruritus is noted after first exposure. The CQ-induced itch side effect cannot be treated effectively by antihistamine drugs, suggesting a histamine-independent pathway is involved.
Previous studies show that subcutaneous injection of CQ in wild-type mice immediately evokes a pronounced scratching behavior. Strikingly, in CQ receptor–deficient mice that we have generated, CQ-induced itch is severely reduced. On the contrary, histamine-induced effects in the mutant animals are the same as those in wild-type mice, implying that this receptor specifically regulates CQ signaling. In addition, expression of the mouse and human genes for the CQ receptor in heterologous cells renders them sensitive to CQ. These data suggest that we have identified an endogenous receptor for CQ. More significantly, we now have a molecular tool to study the subpopulation of DRG neurons that is crucial for mediating histamine-independent itch.
Labeling Tactile Afferents
The skin detects both pleasant and unpleasant stimuli. Although nociception has been extensively studied, the mechanisms underlying pleasant sensations are less well understood. Innocuous mechanical stimuli are detected both by myelinated afferents and unmyelinated, C-fiber tactile (CT) afferents. Although it has been almost 70 years since CT afferents were discovered, their properties and functions remain elusive. In humans, CT fibers respond to gentle stroking, are present only in hairy skin, and engage cortical areas involved in emotion. CT afferents have not been identified in mice, limiting their study.
We employed molecular genetic methods to label a rare population of small-diameter sensory neurons that express the Mas-related G protein–coupled receptor B4 (MrgprB4). These neurons exclusively innervate hairy skin. The large (~1 mm2), scattered peripheral arbors of these neurons are similar to the receptive fields of human CT afferents. Furthermore, these neurons project to spinal lamina IIo, whose ascending projections selectively engage limbic areas. These data suggest that MrgprB4 may identify CT afferents in mice, a genetically tractable animal model. Recent studies conducted on a patient lacking large-diameter myelinated afferents showed that activation of CT afferents evokes strong responses in a limbic-related cortex (insular area) but not in somatosensory area S1 or S2. This suggests that CT afferents may be involved in the emotional perception of light stroking but not the cognitive aspects. Therefore, MrgprB4 may provide us a useful molecular tool to study the role of CT afferents in affiliative and social behaviors.
With these DRG-specific genes in hand, we are in a position to decode all three somatosensations. We would like to answer several questions: Are these sensations mediated by the same or distinct subsets of DRG neurons? Are neuronal circuitries for these sensations segregated in the spinal cord? How do they interact with each other in higher orders of neurons? Which membrane proteins are responsible for transducing histamine-independent itch and pleasant touch in sensory endings? Our research may have direct clinical applications in treating chronic pain and itch.