Osmosensitivity of Transient Receptor Potential Vanilloid 1 Is Synergistically Enhanced by Distinct Activating Stimuli Such as Temperature and Protons

In animals, body-fluid osmolality is continuously monitored to keep it within a narrow range around a set point (∼300 mOsm/kg). Transient receptor potential vanilloid 1 (TRPV1), a cation channel, has been implicated in body-fluid homeostasis in vivo based on studies with the TRPV1-knockout mouse. However, the response of TRPV1 to hypertonic stimuli has not been demonstrated with heterologous expression systems so far, despite intense efforts by several groups. Thus, the molecular entity of the hypertonic sensor in vivo still remains controversial. Here we found that the full-length form of TRPV1 is sensitive to an osmotic increase exclusively at around body temperature using HEK293 cells stably expressing rat TRPV1. At an ambient temperature of 24°C, a slight increase in the intracellular calcium concentration ([Ca2+]i) was rarely observed in response to hypertonic stimuli. However, the magnitude of the osmosensitive response markedly increased with temperature, peaking at around 36°C. Importantly, the response at 36°C showed a robust increase over a hypertonic range, but a small decrease over a hypotonic range. A TRPV1 antagonist, capsazepine, and a nonspecific TRP channel inhibitor, ruthenium red, completely blocked the increase in [Ca2+]i. These results endorse the view that the full-length form of TRPV1 is able to function as a sensor of hypertonic stimuli in vivo. Furthermore, we found that protons and capsaicin likewise synergistically potentiated the response of TRPV1 to hypertonic stimuli. Of note, HgCl2, which blocks aquaporins and inhibits cell-volume changes, significantly reduced the osmosensitive response. Our findings thus indicate that TRPV1 integrates multiple different types of activating stimuli, and that TRPV1 is sensitive to hypertonic stimuli under physiologically relevant conditions

Hyperpolarization-Activated Current (Ih) in Ganglion-Cell Photoreceptors

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and serve as the primary retinal drivers of non-image-forming visual functions such as circadian photoentrainment, the pupillary light reflex, and suppression of melatonin production in the pineal. Past electrophysiological studies of these cells have focused on their intrinsic photosensitivity and synaptic inputs. Much less is known about their voltage-gated channels and how these might shape their output to non-image-forming visual centers. Here, we show that rat ipRGCs retrolabeled from the suprachiasmatic nucleus (SCN) express a hyperpolarization-activated inwardly-rectifying current (Ih). This current is blocked by the known Ih blockers ZD7288 and extracellular cesium. As in other systems, including other retinal ganglion cells, Ih in ipRGCs is characterized by slow kinetics and a slightly greater permeability for K+ than for Na+. Unlike in other systems, however, Ih in ipRGCs apparently does not actively contribute to resting membrane potential. We also explore non-specific effects of the common Ih blocker ZD7288 on rebound depolarization and evoked spiking and discuss possible functional roles of Ih in non-image-forming vision. This study is the first to characterize Ih in a well-defined population of retinal ganglion cells, namely SCN-projecting ipRGCs

A simplified microwave-based motion detector for home cage activity monitoring in mice

Background: Locomotor activity of rodents is an important readout to assess well-being and physical health, and is pivotal for behavioral phenotyping. Measuring homecage-activity with standard and cost-effective optical methods in mice has become difficult, as modern housing conditions (e.g. individually ventilated cages, cage enrichment) do not allow constant, unobstructed, visual access. Resolving this issue either makes greater investments necessary, especially if several experiments will be run in parallel, or is at the animals' expense. The purpose of this study is to provide an easy, yet satisfying solution for the behavioral biologist at novice makers level. Results: We show the design, construction and validation of a simplified, low-cost, radar-based motion detector for home cage activity monitoring in mice. In addition we demonstrate that mice which have been selectively bred for low levels of anxiety-related behavior (LAB) have deficits in circadian photoentrainment compared to CD1 control animals. Conclusion: In this study we have demonstrated that our proposed low-cost microwave-based motion detector is well-suited for the study of circadian rhythms in mice

Differential arousal regulation by prokineticin 2 signaling in the nocturnal mouse and the diurnal monkey

The temporal organization of activity/rest or sleep/wake rhythms for mammals is regulated by the interaction of light/dark cycle and circadian clocks. The neural and molecular mechanisms that confine the active phase to either day or night period for the diurnal and the nocturnal mammals are unclear. Here we report that prokineticin 2, previously shown as a circadian clock output molecule, is expressed in the intrinsically photosensitive retinal ganglion cells, and the expression of prokineticin 2 in the intrinsically photosensitive retinal ganglion cells is oscillatory in a clock-dependent manner. We further show that the prokineticin 2 signaling is required for the activity and arousal suppression by light in the mouse. Between the nocturnal mouse and the diurnal monkey, a signaling receptor for prokineticin 2 is differentially expressed in the retinorecipient suprachiasmatic nucleus and the superior colliculus, brain projection targets of the intrinsically photosensitive retinal ganglion cells. Blockade with a selective antagonist reveals the respectively inhibitory and stimulatory effect of prokineticin 2 signaling on the arousal levels for the nocturnal mouse and the diurnal monkey. Thus, the mammalian diurnality or nocturnality is likely determined by the differential signaling of prokineticin 2 from the intrinsically photosensitive retinal ganglion cells onto their retinorecipient brain targets