Peripheral neuropathy is dose limiting in paclitaxel cancer chemotherapy and can result in both acute pain during treatment and chronic persistent pain in cancer survivors. The hypothesis tested was that paclitaxel produces these adverse effects at least in part by sensitizing transient receptor potential vanilloid subtype 1 (TRPV1) through Toll-like receptor 4 (TLR4) signaling. The data show that paclitaxelinduced behavioral hypersensitivity is prevented and reversed by spinal administration of a TRPV1 antagonist. The number of TRPV1 ϩ neurons is increased in the dorsal root ganglia (DRG) in paclitaxel-treated rats and is colocalized with TLR4 in rat and human DRG neurons. Cotreatment of rats with lipopolysaccharide from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS), a TLR4 inhibitor, prevents the increase in numbers of TRPV1 ϩ neurons by paclitaxel treatment. Perfusion of paclitaxel or the archetypal TLR4 agonist LPS activated both rat DRG and spinal neurons directly and produced acute sensitization of TRPV1 in both groups of cells via a TLR4-mediated mechanism. Paclitaxel and LPS sensitize TRPV1 in HEK293 cells stably expressing human TLR4 and transiently expressing human TRPV1. These physiological effects also are prevented by LPS-RS. Finally, paclitaxel activates and sensitizes TRPV1 responses directly in dissociated human DRG neurons. In summary, TLR4 was activated by paclitaxel and led to sensitization of TRPV1. This mechanism could contribute to paclitaxel-induced acute pain and chronic painful neuropathy.

The role of excitatory amino acids (EAAs) in the excitation of monkey spinothalamic tract (STT) neurons following activation of cutaneous primary afferent fibers by noxious and non-noxious stimuli was investigated. The responses of STT neurons to either NMDA or non-NMDA EAA ligands were blocked by infusion of specific antagonists through a microdialysis fiber into the region surrounding the cells. Our results show that blockade of non-NMDA receptors results in a nearly complete elimination of the responses of STT neurons to all stimuli. Blockade of NMDA receptors results in an attenuation of the responses to noxious stimuli but, in addition, prevents the development of the sensitization of STT neurons that is often observed after intradermal injection of capsaicin. These observations further support a role of EAAs in the transmission of sensory information from primary afferent fibers to dorsal horn neurons and a role for NMDA receptors in the generation of hyperalgesia.

Physiological modulation of neuronal activity has been associated with elevations in cytosolic calcium concentrations induced by calcium influx through postsynaptic ligandactivated channels, voltage-dependent calcium channels and calcium release from intracellular stores (e.g. McBurney & Neering, 1987;Blaustein, 1988;Keller et al. 1992;Yuste & Tank, 1996). Voltage-dependent calcium influx occurs through a heterogeneous population of underlying calcium channels, which can be distinguished by their profile of voltage dependence, gating behaviour and subtype-specific pharmacological properties (Hess, 1990;Llinas et al. 1992). For a given influx pathway, the effective elevation in intra-cellular calcium concentrations depends on several components, including calcium uptake into intracellular stores, extrusion mechanisms and endogenous calcium buffering (McBurney & Neering, 1987;Blaustein, 1988;Baimbridge et al. 1992;Neher, 1995). Endogenous buffering can be quantified by the calcium binding ratio êS, i.e. the ratio between the number of calcium ions bound to endogenous buffers compared to the number of calcium ions that make up the free calcium concentration in the cytosol (Neher & Augustine, 1992;Zhou & Neher, 1993). It can be experimentally determined by the 'added buffer' method based on a controlled loading of cells with fluorescent

Therapeutic application of stem cell derivatives requires large quantities of cells produced in defined media that cannot be produced via conventional adherent culture. We have applied human induced pluripotent stem (hiPS) cells expressing eGFP under control of the OCT4 promoter to establish the expansion of undifferentiated human embryonic stem (hES) and hiPS cells in suspension culture. A defined culture medium has been identified that results in up to six-fold increase in cell numbers within four days. Our culture system is based on initial single cell dissociation which is critical for standardized process inoculation. HES / hiPS cells were expanded for up to 17 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers. The ability to expand HES / hiPS cells in a scalable suspension culture represents a critical step towards standardized production in stirred bioreactors.

1. Responses of spinothalamic tract (STT) neurons to mechanical and thermal stimulation of skin were recorded under urethane and pentobarbital anesthesia in 12 control rats and in 20 rats with experimental neuropathy. Activity of the STT cells in neuropathic rats was recorded 7, 14, and 28 days after inducing the neuropathy by placing four loose ligatures on the sciatic nerve. 2. All neuropathic animals showed guarding of the injured hindpaw and a shorter withdrawal latency from a radiant heat source of the neuropathic hindpaw than that of the sham-operated paw. 3. STT neurons in neuropathic animals showed the most profound changes 7 and 14 days after the nerve ligation. When compared with STT cells in unoperated animals, approximately half of the neurons had high background activity, responses to innocuous stimuli represented a larger percentage of the total evoked activity in wide dynamic range neurons, and the occurrence and magnitude of afterdischarges to mechanical and thermal stimuli were increased. 4. The mean threshold temperatures of heat-evoked responses of the STT cells in neuropathic animals were not different than those of cells from control animals. However, in neuropathic rats, cells reacting to small heat stimuli usually already had afterdischarges. 5. The increase in the background activity of STT cells is consistent with behavioral observations of spontaneous pain in this model of experimental neuropathy. Furthermore, the afterdischarges of STT cells may parallel the prolonged paw withdrawal in response to noxious stimuli that is seen in these animals and that is evidence for hyperalgesia. However, there was no indication of a lowered threshold for thermal stimuli as might be expected if the animals have thermal allodynia. Mechanical allodynia may have resulted from a relative increase in responsiveness to innocuous mechanical stimuli. However, responses to noxious mechanical stimuli were reduced compared with control, at least at 28 days after the ligation. Peripheral and central mechanisms responsible for the changes in responses of STT cells in neuropathic animals are suggested.

Spicarova D, Palecek J. The role of the TRPV1 endogenous agonist N-oleoyldopamine in modulation of nociceptive signaling at the spinal cord level. J Neurophysiol 102: 234 -243, 2009. First published April 15, 2009 doi:10.1152/jn.00024.2009. Transient receptor potential vanilloid (TRPV1) receptors are abundant in a subpopulation of primary sensory neurons that convey nociceptive information from the periphery to the spinal cord dorsal horn. The TRPV1 receptors are expressed on both the peripheral and central branches of these dorsal root ganglion (DRG) neurons and can be activated by capsaicin, heat, low pH, and also by recently described endogenous lipids. Using patch-clamp recordings from superficial dorsal horn (DH) neurons in acute spinal cord slices, the effect of application of the endogenous TRPV1 agonist N-oleoyldopamine (OLDA) on the frequency of miniature excitatory postsynaptic currents (mEPSCs) was evaluated. A high concentration OLDA (10 M) solution was needed to increase the mEPSC frequency, whereas low concentration OLDA (0.2 M) did not evoke any change under control conditions. The increase was blocked by the TRPV1 antagonists SB366791 or BCTC. Application of a low concentration of OLDA evoked an increase in mEPSC frequency after activation of protein kinase C by phorbol ester (PMA) and bradykinin or in slices from animals with peripheral inflammation. Increasing the bath temperature from 24 to 34°C enhanced the basal mEPSC frequency, but the magnitude of changes in the mEPSC frequency induced by OLDA administration was similar at both temperatures. Our results suggest that presumed endogenous agonists of TRPV1 receptors, like OLDA, could have a considerable impact on synaptic transmission in the spinal cord, especially when TRPV1 receptors are sensitized. Spinal TRPV1 receptors could play a pivotal role in modulation of nociceptive signaling in inflammatory pain.

1. Activation of neurokinin receptors contributes to the excitation of many dorsal horn neurons by cutaneous sensory stimuli, particularly noxious stimuli. In the present study we investigate the role of neurokinin receptors in the activation of primate spinothalamic tract (STT) neurons by cutaneous mechanical stimuli and by intradermal injection of capsaicin. This was done by testing the responses of these neurons to a battery of cutaneous stimuli before and during infusion by microdialysis of antagonists selective for NK1 and NK2 receptors. 2. The NK1 receptor antagonists cis-3-(2-methoxybenzyl-amino-2-benzhydrylquinuclidine (CP96345) and D-Pro9-[Spiro-y-lactam]-Leu10,Trp11)-Physalaemin(1-11) (GR82334) did not significantly reduce the responses of STT cells to mechanical stimulation of the skin. Both NK1 antagonists did, however, produce a significant reduction in the responses of STT neurons to an intradermal injection of capsaicin. Finally, despite having no effects on responses to mechanical stimuli, both NK1 antagonists prevented the sensitization of the responses to cutaneous stimuli that is usually observed after intradermal injections of capsaicin. 3. The NK2 selective antagonists PhCO-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (GR98400) and [Tyr5,D-Trp6,8,9,Lys10]-NKA (4-10) (MEN10376) had effects very similar to those of the NK1 antagonists, but with an important difference. Neither NK2 antagonist affected the responses of STT neurons to noxious or innocuous mechanical stimulation of the skin, but they did reduce the responses to intradermal capsaicin injections. These compounds failed to prevent capsaicin-induced sensitization. In fact, cells exposed to GR98400 or MEN10376 showed unusually sustained increases in the responses to mechanical stimuli after the first capsaicin injection, suggesting that these compounds actually induced sensitization. 4. These results support the contention that both neurokinin receptors participate in the processing of nociceptive information in the dorsal horn, especially responses to strong stimuli such as intradermal injection of capsaicin. NK1 receptors are also involved in the sensitization of STT neurons after peripheral injury. A clearer understanding of the role of NK2 receptors in sensitization requires further studies with improved antagonists.

Nitric oxide (NO) has been proposed to contribute to the development of hyperalgesia by activating the NO/guanosine 3',5'-cyclic monophosphate (cGMP) signal transduction pathway in the spinal cord. We have examined the effects of NO on the responses of primate spinothalamic tract (STT) neurons to peripheral cutaneous stimuli and on the sensitization of STT cells following intradermal injection of capsaicin. The NO level within the spinal dorsal horn was increased by microdialysis of a NO donor, 3-morpholinosydnonimine (SIN-1). SIN-1 enhanced the responses of STT cells to both weak and strong mechanical stimulation of the skin. This effect was preferentially on deep wide dynamic range STT neurons. The responses of none of the neurons tested to noxious heat stimuli were significantly changed when SIN-1 was administered. Intradermal injection of capsaicin increased dramatically the content of NO metabolites, NO-2/NO-3, within the dorsal horn. This effect was attenuated by pretreatment of the spinal cord with a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Sensitization of STT cells induced by intradermal injection of capsaicin was also prevented by pretreatment of the dorsal horn with the NOS inhibitors, L-NAME or 7-nitroindazole. Blockade of NOS did not significantly affect the responses of STT cells to peripheral stimulation in the absence of capsaicin injection. The data suggest that NO contributes to the development and maintenance of central sensitization of STT cells and the resultant mechanical hyperalgesia and allodynia after peripheral tissue damage or inflammation. NO seems to play little role in signaling peripheral stimuli under physiological conditions.