Jianguo G. Gu

TRPM8 Mechanism of Cold Allodynia after Chronic Nerve Injury

The cold- and menthol-sensitive receptor TRPM8 (transient receptor potential melastatin 8) has been suggested to play a role in cold allodynia, an intractable pain seen clinically. We studied how TRPM8 is involved in cold allodynia using rats with chronic constrictive nerve injury (CCI), a neuropathic pain model manifesting cold allodynia in hindlimbs. We found that cold allodynic response in the CCI animals was significantly attenuated by capsazepine, a blocker for both TRPM8 and TRPV1 (transient receptor potential vanilloid 1) receptors, but not by the selective TRPV1 antagonist I-RTX (5-iodoresiniferatoxin). In L5 dorsal root ganglion (DRG) sections of the CCI rats, immunostaining showed an increase in the percentage of TRPM8-immunoreactive neurons when compared with the sham group. Using the Ca2+-imaging technique and neurons acutely dissociated from the L5 DRGs, we found that CCI resulted in a significant increase in the percentage of menthol- and cold-sensitive neurons and also a substantial enhancement in the responsiveness of these neurons to both menthol and innocuous cold. These changes occurred in capsaicin-sensitive neurons, a subpopulation of nociceptive-like neurons. Using patch-clamp recordings, we found that membrane currents evoked by both menthol and innocuous cold were significantly enhanced in the CCI group compared with the sham group. By retrograde labeling afferent neurons that target hindlimb skin, we showed that the skin neurons expressed TRPM8 receptors, that the percentage of menthol-sensitive/cold-sensitive/capsaicin-sensitive neurons increased, and that the menthol- and cold-evoked responses were significantly enhanced in capsaicin-sensitive neurons after CCI. Together, the gain of TRPM8-mediated cold sensitivity on nociceptive afferent neurons provides a mechanism of cold allodynia.

Distribution of P2X1, P2X2, and P2X3 receptor subunits in rat primary afferents: relation to population markers and specific cell types.

We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.

Menthol-Induced Ca2+ Release from Presynaptic Ca2+ Stores Potentiates Sensory Synaptic Transmission

Menthol and many of its derivatives produce profound sensory and mental effects. The receptor for menthol has been cloned and named cold- and menthol-sensitive receptor-1 (CMR1) or transient receptor potential channel M8 (TRPM8) receptor. Using a dorsal root ganglion (DRG) and dorsal horn (DH) coculture system as a model for the first sensory synapse in the CNS, we studied menthol effects on sensory synaptic transmission and the underlying mechanisms. We found that menthol increased the frequency of miniature EPSCs (mEPSCs). The effects persisted under an extracellular Ca2+-free condition but were abolished by intracellular BAPTA and pretreatment with thapsigargin. Menthol-induced increases of mEPSC frequency were blocked by 2-aminoethoxydiphenylborane (2-APB) but not affected by the phospholipase C inhibitor U73122 or by the cADP receptor inhibitor 8-bromo-cADPR (8Br-cADPR). Double-patch recordings from DRG-DH pairs showed that menthol could potentiate evoked EPSCs (eEPSCs) and change the paired-pulse ratio of eEPSCs. A Ca2+ imaging study on DRG neurons demonstrated that menthol could directly release Ca2+ from intracellular Ca2+ stores. Menthol-induced Ca2+ release was abolished by 2-APB but not affected by U73122 or 8Br-cADPR. Taken together, our results indicate that menthol can act directly on presynaptic Ca2+ stores of sensory neurons to release Ca2+, resulting in a facilitation of glutamate release and a modulation of neuronal transmission at sensory synapses. Expression of TRPM8 receptor on presynaptic Ca2+ stores, a novel localization for this ligand-gated ion channel, is also strongly suggested.

Merkel Cells Transduce and Encode Tactile Stimuli to Drive Aβ-Afferent Impulses

Sensory systems for detecting tactile stimuli have evolved from touch-sensing nerves in invertebrates to complicated tactile end organs in mammals. Merkel discs are tactile end organs consisting of Merkel cells and Aβ-afferent nerve endings and are localized in fingertips, whisker hair follicles, and other touch-sensitive spots. Merkel discs transduce touch into slowly adapting impulses to enable tactile discrimination, but their transduction and encoding mechanisms remain unknown. Using rat whisker hair follicles, we show that Merkel cells rather than Aβ-afferent nerve endings are primary sites of tactile transduction and identify the Piezo2 ion channel as the Merkel cell mechanical transducer. Piezo2 transduces tactile stimuli into Ca(2+)-action potentials in Merkel cells, which drive Aβ-afferent nerve endings to fire slowly adapting impulses. We further demonstrate that Piezo2 and Ca(2+)-action potentials in Merkel cells are required for behavioral tactile responses. Our findings provide insights into how tactile end-organs function and have clinical implications for tactile dysfunctions.

Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain?

Neuropathic pain is a common clinical condition. Current treatments are often inadequate, ineffective, or produce potentially severe adverse effects. Understanding the mechanisms that underlie the development and maintenance of neuropathic pain will be helpful in identifying new therapeutic targets and developing effective strategies for the prevention and/or treatment of this disorder. The genesis of neuropathic pain is reliant, at least in part, on abnormal spontaneous activity within sensory neurons. Therefore, voltage-gated sodium channels, which are essential for the generation and conduction of action potentials, are potential targets for treating neuropathic pain. However, preclinical studies have shown unexpected results because most pain-associated voltage-gated channels in the dorsal root ganglion are down-regulated after peripheral nerve injury. The role of dorsal root ganglion voltage-gated channels in neuropathic pain is still unclear. In this report, we describe the expression and distribution of voltage-gated sodium channels in the dorsal root ganglion. We also review evidence regarding changes in their expression under neuropathic pain conditions and their roles in behavioral responses in a variety of neuropathic pain models. We finally discuss their potential involvement in neuropathic pain.

Modulation of inhibitory synaptic activity by a non-α4β2, non-α7 subtype of nicotinic receptors in the substantia gelatinosa of adult rat spinal cord

&NA; The GABA/glycine‐mediated inhibitory activity in the substantia gelatinosa (SG) of the spinal cord is critical in the control of nociceptive transmission. We examined whether and how SG inhibitory activity might be regulated by neuronal nicotinic receptors (nAChRs). Patch‐clamp recordings were performed in SG neurons of spinal slice preparations from adult rats. We provided electrophysiological evidence that inhibitory presynaptic terminals in the SG expressed nAChRs and their activation resulted in large increases in the frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in over 90% SG neurons tested. The enhancement of inhibitory activity was mediated by increases in the release of GABA/glycine, and direct Ca2+ entry through SG presynaptic nAChRs appeared to be involved. Miniature IPSC frequency could be enhanced by the nAChR agonists nicotine or cytisine. Nicotine could still elicit large increases in mIPSC frequency in the presence of the &agr;4&bgr;2 nAChR antagonist dihydro‐beta‐erythroidine (5 &mgr;M) and the &agr;7 nAChR‐selective antagonist methyllycaconitine (40 nM). However, nicotine did not produce a significant enhancement of mIPSC frequency in the presence of the broad spectrum nAChR antagonist mecamylamine (5 &mgr;M). Nicotinic agonist‐evoked whole‐cell currents from SG neurons and the antagonist profiles also indicated the presence of a subtype of nAChRs, which were different from the major central nervous system nAChR subtypes, i.e. &agr;4&bgr;2* or &agr;7 nAChRs. Together, our results suggest that a subtype of nAChR, possibly &agr;3&bgr;4* nAChR or a new nAChR type, is highly expressed at the inhibitory presynaptic terminals in SG of adult rats and play a role in the control of inhibitory activity in SG.

Role of spinal cord glutamate transporter during normal sensory transmission and pathological pain states

Glutamate is a neurotransmitter critical for spinal excitatory synaptic transmission and for generation and maintenance of spinal states of pain hypersensitivity via activation of glutamate receptors. Understanding the regulation of synaptically and non-synaptically released glutamate associated with pathological pain is important in exploring novel molecular mechanisms and developing therapeutic strategies of pathological pain. The glutamate transporter system is the primary mechanism for the inactivation of synaptically released glutamate and the maintenance of glutamate homeostasis. Recent studies demonstrated that spinal glutamate transporter inhibition relieved pathological pain, suggesting that the spinal glutamate transporter might serve as a therapeutic target for treatment of pathological pain. However, the exact function of glutamate transporter in pathological pain is not completely understood. This report will review the evidence for the role of the spinal glutamate transporter during normal sensory transmission and pathological pain conditions and discuss potential mechanisms by which spinal glutamate transporter is involved in pathological pain.

P2X purinoceptors and sensory transmission

The involvement of P2X purinoreceptors (P2X receptors) in somatosensory transmission is herein reviewed with a focus on those receptors that are expressed on sensory neurons to elucidate their roles in the initiation of sensory excitation from primary afferent neurons, in modulating synaptic transmission at the first sensory synapses formed between primary afferent central terminals and dorsal horn neurons, in directly mediating sensory synaptic transmission to the spinal cord dorsal horn, and in modulating synaptic transmission among spinal cord dorsal horn neurons. Research on P2X receptors has indicated that these receptors play a significant role in both physiological and pathological pain states. As a result, P2X receptors may serve as therapeutic targets for the treatment of pathological pain conditions associated with nerve injury, tissue inflammation, cancer, and other diseases.

Molecular pain, a new era of pain research and medicine

Molecular pain is a relatively new and rapidly expanding research field that represents an advanced step from conventional pain research. Molecular pain research addresses physiological and pathological pain at the cellular, subcellular and molecular levels. These studies integrate pain research with molecular biology, genomics, proteomics, modern electrophysiology and neurobiology. The field of molecular pain research has been rapidly expanding in the recent years, and has great promise for the identification of highly specific and effective targets for the treatment of intractable pain. Although several existing journals publish articles on classical pain research, none are specifically dedicated to molecular pain research. Therefore, a new journal focused on molecular pain research is needed. Molecular Pain, an Open Access, peer-reviewed, online journal, will provide a forum for molecular pain scientists to communicate their research findings in a targeted manner to others in this important and growing field.

Glutamate acts as a neurotransmitter for gastrin releasing peptide-sensitive and insensitive itch-related synaptic transmission in mammalian spinal cord

Itch sensation is one of the major sensory experiences of human and animals. Recent studies have proposed that gastrin releasing peptide (GRP) is a key neurotransmitter for itch in spinal cord. However, no direct evidence is available to indicate that GRP actually mediate responses between primary afferent fibers and dorsal horn neurons. Here we performed integrative neurobiological experiments to test this question. We found that a small population of rat dorsal horn neurons responded to GRP application with increases in calcium signaling. Whole-cell patch-clamp recordings revealed that a part of superficial dorsal horn neurons responded to GRP application with the increase of action potential firing in adult rats and mice, and these dorsal horn neurons received exclusively primary afferent C-fiber inputs. On the other hands, few Aδ inputs receiving cells were found to be GRP positive. Finally, we found that evoked sensory responses between primary afferent C fibers and GRP positive superficial dorsal horn neurons are mediated by glutamate but not GRP. CNQX, a blocker of AMPA and kainate (KA) receptors, completely inhibited evoked EPSCs, including in those Fos-GFP positive dorsal horn cells activated by itching. Our findings provide the direct evidence that glutamate is the principal excitatory transmitter between C fibers and GRP positive dorsal horn neurons. Our results will help to understand the neuronal mechanism of itch and aid future treatment for patients with pruritic disease.