Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.

A distinct subset of sensory neurons are thought to directly sense changes in thermal energy through their termini in the skin. Very little is known about the molecules that mediate thermoreception by these neurons. Vanilloid Receptor 1 (VR1), a member of the TRP family of channels, is activated by noxious heat. Here we describe the cloning and characterization of TRPM8, a distant relative of VR1. TRPM8 is specifically expressed in a subset of pain- and temperature-sensing neurons. Cells overexpressing the TRPM8 channel can be activated by cold temperatures and by a cooling agent, menthol. Our identification of a cold-sensing TRP channel in a distinct subpopulation of sensory neurons implicates an expanded role for this family of ion channels in somatic sensory detection.

Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

Transient receptor potential A1 (TRPA1) is expressed in a subset of nociceptive sensory neurons where it acts as a sensor for environmental irritants, including acrolein, and some pungent plant ingredients such as allyl isothiocyanate and cinnamaldehyde. These exogenous compounds activate TRPA1 by covalent modification of cysteine residues. We have used electrophysiological methods and mea- Ϫ/Ϫ mice. These data demonstrate that multiple agents produced during episodes of oxidative stress can activate TRPA1 expressed in sensory neurons.

Osteoarthritis (OA) is a major healthcare burden, with increasing incidence. Pain is the predominant clinical feature, yet therapy is ineffective for many patients. While there are considerable insights into the mechanisms underlying tissue remodelling, there is poor understanding of the link between disease pathology and pain. This is in part owing to the lack of animal models that combine both osteoarthritic tissue remodelling and pain. Here, we provide an analysis of pain related behaviours in two models of OA in the rat: partial medial meniscectomy and iodoacetate injection. Histological studies demonstrated that in both models, progressive osteoarthritic joint pathology developed over the course of the next 28 days. In the ipsilateral hind limb in both models, changes in the percentage bodyweight borne were small, whereas marked mechanical hyperalgesia and tactile allodynia were seen. The responses in the iodoacetate treated animals were generally more robust, and these animals were tested for pharmacological reversal of pain related behaviour. Morphine was able to attenuate hyperalgesia 3, 14 and 28 days after OA induction, and reversed allodynia at days 14 and 28, providing evidence that this behaviour was pain related. Diclofenac and paracetamol were effective 3 days after arthritic induction only, coinciding with a measurable swelling of the knee. Gabapentin varied in its ability to reverse both hyperalgesia and allodynia. The iodoacetate model provides a basis for studies on the mechanisms of pain in OA, and for development of novel therapeutic analgesics.

A recent major conceptual advance has been the recognition of the importance of immune system-neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible CatS inhibitor, morpholinurea-leucinehomophenylalanine-vinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant CatS (rrCatS) induced hyperalgesia and allodynia in naïve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for CatS cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN-and CatSinduced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators.chemokines ͉ microglia ͉ proteases ͉ allodynia ͉ fractalkine

1. In current-clamp recordings, 1 /,M prostaglandin E2 (PGE2) increased the excitability of neonatal rat dorsal root ganglion neurones. The current threshold for firing was reduced, and the response to a constant suprathreshold stimulation was modified such that a single evoked action potential was converted to a train of action potentials. The excitatory action of PGE2 was still apparent when action potentials were evoked in the presence of 500 nm tetrodotoxin.2. In voltage-clamp experiments 1 /,M PGE2 frequently increased the magnitude of the peak currents recorded, and caused a hyperpolarizing shift (of approximately 6 mV) in the activation curve for the tetrodotoxin-resistant sodium current (TTX-R 'Na). In some cells, the hyperpolarizing shift in the activation curve was accompanied by a decrease in peak conductance. PGE2 also caused a hyperpolarizing shift in the steady-state inactivation curve for the sodium current. 3. Extracellular application of the cAMP analogue dibutyryl cAMP (dbcAMP) at a concentration of 1 mm produced effects on both the current-voltage relationship and the steady-state inactivation curve for the TTX-R 'Na which were indistinguishable from those observed with PGE2. Prior exposure of the neurones to dbcAMP occluded the effect of a subsequent treatment with PGE2. 4. Forskolin (10 uM), a direct activator of adenylate cyclase, mimicked the effects of PGE2 and dbcAMP on TTX-R INa, The inactive congener of forskolin, 1,9-dideoxyforskolin (10 #M), reduced the amplitude of ITX-R INa, but did not evoke a hyperpolarizing shift in the activation curve. 5. Intracellular perfusion of the neurones with an inhibitor of protein kinase A inhibited the effect of PGE2 on TTX-R 'Na* 6. PGE2 also reduced the amplitude of voltage-gated potassium currents (IK), which will contribute to the excitatory action. The mechanisms underlying the changes in IK have yet to be elucidated. 7. We propose that the PGE2-mediated increase in excitability in sensory neurones may be due, at least in part, to the cAMP-protein kinase A-dependent modulation of the tetrodotoxinresistant sodium channel.It has been recognized for many years that prostaglandins are able to sensitize primary afferent neurones to noxious stimuli (Ferreira, 1972; for recent review see Dray, 1995). In vitro, prostaglandins, particularly PGE2, can enhance the responsiveness of nociceptive neurones to excitatory substances such as bradykinin and capsaicin (Yanagisawa,

1 Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin.2 Capsazepine (10 JAM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 JM y-aminobutyric acid (GABA) and S JM adenosine 5'-triphosphate (ATP) were unaffected. 3 The effects of capsazepine were examined quantitatively with radioactive ion flux experiments.Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2" uptake in cultures of rat DRG neurones with an IC50 of 420 ± 46 nM (mean ± s.e.mean, n = 6). The 45Ca2" uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 JAM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 ± 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4 Capsazepine also inhibited the capsaicin-and RTX-evoked efflux of 86Rb+ from cultured DRG capsazepine the inhibition by Ruthenium Red (10-500nM in DRG and 0.5-10AM in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex, non-competitive inhibition.