Sensory nerves detect an extensive array of somatosensory stimuli, including environmental temperatures. Despite activating only a small cohort of sensory neurons, cold temperatures generate a variety of distinct sensations that range from pleasantly cool to painfully aching, prickling, and burning. Psychophysical and functional data show that cold responses are mediated by both C-and A␦-fibers with separate peripheral receptive zones, each of which likely provides one or more of these distinct cold sensations. With this diversity in the neural basis for cold, it is remarkable that the majority of cold responses in vivo are dependent on the cold and menthol receptor transient receptor potential melastatin 8 (TRPM8). TRPM8-null mice are deficient in temperature discrimination, detection of noxious cold temperatures, injury-evoked hypersensitivity to cold, and nocifensive responses to cooling compounds. To determine how TRPM8 plays such a critical yet diverse role in cold signaling, we generated mice expressing a genetically encoded axonal tracer in TRPM8 neurons. Based on tracer expression, we show that TRPM8 neurons bear the neurochemical hallmarks of both C-and A␦-fibers, and presumptive nociceptors and non-nociceptors. More strikingly, TRPM8 axons diffusely innervate the skin and oral cavity, terminating in peripheral zones that contain nerve endings mediating distinct perceptions of innocuous cool, noxious cold, and first-and second-cold pain. These results further demonstrate that the peripheral neural circuitry of cold sensing is cellularly and anatomically complex, yet suggests that cold fibers, caused by the diverse neuronal context of TRPM8 expression, use a single molecular sensor to convey a wide range of cold sensations.

TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo.

Cold temperatures robustly activate a small cohort of somatosensory nerves, yet during a prolonged cold stimulus their activity will decrease, or adapt, over time. This process allows for the discrimination of subtle changes in temperature. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel expressed on a subset of peripheral afferent fibers. We and others have reported that TRPM8 channels also adapt in a calcium-dependent manner when activated by the cooling compound menthol. Additionally, TRPM8 activity is sensitive to the phospholipid phosphoinositol 4,5-bisphosphate (PIP 2 ), a substrate for the enzyme phospholipase C (PLC). These results suggest an adaptation model whereby TRPM8-mediated Ca 2؉ influx activates PLC, thereby decreasing PIP 2 levels and resulting in reduced TRPM8 activity. Here we tested this model using pharmacological activation of PLC and by manipulating PIP 2 levels independent of both PLC and Ca 2؉ . PLC activation leads to adaptation-like reductions in cold-or menthol-evoked TRPM8 currents in both heterologous and native cells. Moreover, PLCindependent reductions in PIP 2 had a similar effect on cold-and menthol-evoked currents. Mechanistically, either form of adaptation does not alter temperature sensitivity of TRPM8 but does lead to a change in channel gating. Our results show that adaptation is a shift in voltage dependence toward more positive potentials, reversing the trend toward negative potentials caused by agonist. These data suggest that PLC activity not only mediates adaptation to thermal stimuli, but likely underlies a more general mechanism that establishes the temperature sensitivity of somatosensory neurons.The detection of temperature is a fundamental task of the nervous system. Temperature-sensing sensory afferent neurons reside in either the trigeminal (TG) 2 or dorsal root (DRG) sensory ganglia and project peripherally, terminating as free nerve endings that innervate areas of the head or trunk, respectively (1, 2). Subpopulations of these afferents respond to distinct sub-modalities of thermal stimuli, including noxious heat, innocuous cooling and warmth, and painfully cold temperatures. Each carries thermal information to the dorsal horn of the spinal cord, synapsing with neurons that project centrally (1, 3). The discovery of thermosensitive ion channels of the transient receptor potential (TRP) family demonstrated an underlying molecular mechanism for temperature detection (4). Cold temperature sensation is largely mediated by TRPM8, a nonselective cation channel expressed on a small subset of neurons (5, 6). TRPM8 is activated by cooling compounds, such as menthol, as well as cold temperatures below ϳ28°C, in vitro (7,8). Recent reports on the behavioral phenotype of TRPM8-null mice suggest that this lone channel is required for the majority of cold sensing in vivo (5, 9 -11). These and other data strongly implicate TRPM8 in not only the detection of both innocuous cool and some aspects of ...

Schedulers confronted with significant processing time uncertainty often discover that a schedule which is optimal with respect to a deterministic or stochastic scheduling model yields quite poor performance when evaluated relative to the actual processing times. In these environments, the notion of schedule robustness, i.e., determining the schedule with the best worst-case performance compared to the corresponding optimal solution over all potential realizations of job processing times, is a more appropriate guide to schedule selection. In this paper, we formalize the robust scheduling concept for scheduling situations with uncertain or variable processing times. To illustrate the development of solution approaches for a robust scheduling problem, we consider a single-machine environment where the performance criterion of interest is the total flow time over all jobs. We define two measures of schedule robustness, formulate the robust scheduling problem, establish its complexity, describe properties of the optimal schedule, and present exact and heuristic solution procedures. Extensive computational results are reported to demonstrate the efficiency and effectiveness of the proposed solution procedures.production scheduling, processing time uncertainty, worst-case analysis

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