SUMMARY The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side-effect of this widely used anti-malarial drug. Here we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and co-express Gastrin-Releasing Peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.

Itch and pain are two distinct sensations. Although our previous study suggested that gastrinreleasing peptide receptor (GRPR) is an itch-specific gene in the spinal cord, a long-standing question of whether there are separate neuronal pathways for itch and pain remains unsettled. Here we selectively ablated lamina I neurons expressing GRPR in the spinal cord of mice. These mice showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli tested, irrespective of their histamine-dependence. In contrast, pain behaviors were unaffected. Our data also suggest that GRPR + neurons are different from the spinothalamic tract (STT) neurons which have been the focus of the debate. Together, the present study suggests that GRPR + neurons constitute a long-sought labeled line for itch sensation in the spinal cord.Itch has long been considered to be a sub-modality or sub-quality of pain (1-4), because both sensations share many similarities (5). Whether itch and pain, two distinct sensations, are mediated by distinct neural circuits has been the subject of controversy (6-8). In the spinal cord, arguments for the "labeled line" came from electrophysiological recordings in cat showing the presence of a small subset of histamine-responsive, mechanically, thermally and mustard oil insensitive lamina I STT neurons (9). Recent studies in primates, however, found that histamine-sensitive STT neurons were all responsive to noxious mechanical and chemical stimuli, notably capsaicin, arguing against the "labeled line" for itch (10,11). Although our previous data suggested that GRPR is an itch-specific gene in the spinal cord (12), they could not be extrapolated to imply that GRPR + neurons are itch-specific, simply because neurons expressing one sensory modality-specific gene may also express other sensory modalityspecific genes as often seen in sensory neurons (13). One way to address this issue is to selectively ablate a subset of itch-signaling neurons and assess whether pain behaviors are altered in the absence of these neurons. We selectively ablated GRPR + neurons in the spinal cord of mice by intrathecal administration of bombesin-saporin (bombesin-sap), a toxincoupled to bombesin that binds with high affinity to GRPR and results in GRPR internalization and cell death ( fig.S1) (14,15).We first determined the optimal dose and time course of bombesin-sap treatment. Ablation of GRPR + neurons reduced pruritogen-induced scratching behaviors in a dose-dependent manner ( fig. S2). Most of GRPR + neurons (>75%) were lost two weeks after single intrathecal injection of bombesin-sap (400 ng, Fig. 1. A to C). To determine the specificity of bombesin-sap treatment, we analyzed several subpopulations of neurons in the spinal cord by using laminaspecific molecular markers. Expression of neuromedin U receptor 2 (NMUR2) and prodynorphin was not affected in lamina I of mice treated with bombesin-sap ( Fig. 1. D We next examined scratching behaviors of mice treated with bombesin-sap in response to intradermal i...

To understand the role of twist during mammalian development, we generated twist-null mice. twist-null embryos died at embryonic day 11.5. Their most prominent phenotype was a failure of the cranial neural folds to fuse. Mutant embryos also had defects in head mesenchyme, somites, and limb buds. Chimera analysis suggested that head mesenchyme was required for cranial neural tube closure and that twist acted in a cell-autonomous manner in this tissue. In addition, in the head mesenchyme region of chimeras, twist-null cells were segregated from wild-type cells, and in the forebrain they lacked mesenchymal characteristics. These results suggest that twist regulates the cellular phenotype and behavior of head mesenchyme cells that are essential for the subsequent formation of the cranial neural tube.

f/f/p mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b f/f/p mice rapidly became hypothermic when exposed to an ambient temperature of 4°C, decreasing core temperature to 30°C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO 2 chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO 2 . We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.

IntroductionThe renin-angiotensin system is a regulatory cascade that plays an essential role in the regulation of blood pressure, electrolyte, and volume homeostasis. The first and rate-limiting component of this cascade is renin, a protease synthesized and secreted predominantly by the juxtaglomerular (JG) apparatus in the nephron. Renin cleaves angiotensin I (Ang I) from liver-derived angiotensinogen, which is then converted to Ang II by the angiotensin-converting enzyme. Ang II, through binding to its receptors, exerts diverse actions that affect the electrolyte, volume, and blood pressure homeostasis (1). Inappropriate stimulation of the renin-angiotensin system has been associated with hypertension, heart attack, and stroke.The renin-producing granulated cells are mainly located in the afferent glomerular arterioles in the kidney (2). It is well established that renin secretion is regulated by renal perfusion pressure, renal sympathetic nerve activity, and tubular sodium load (1, 2). Renin secretion is stimulated by factors such as prostaglandins, NO, and adrenomedullin, and inhibited by other factors, including Ang II (feedback), endothelin, vasopressin, and adenosine (1, 2). Stimulation of renin secretion is often mediated by an increase in intracellular cAMP and is accompanied by increases in renin gene transcription (3). In the renin gene promoter, several cAMP response elements have been identified. Recently, steroid hormone receptors LXRα and RAR/RXR complex, transcriptional factors CREB/CREM and USF1/USF2, and HOX gene family members have been found to be involved in the activation of murine renin gene transcription (4-7).Vitamin D is a primary regulator of calcium homeostasis. Genetic inactivation of either the vitamin D receptor (VDR), a member of the nuclear receptor superfamily that mediates the action of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], or 25-hydroxyvitamin D 3 1α-hydroxylase, the rate-limiting enzyme for the biosynthesis of 1,25(OH) 2 D 3 , results in impaired calcium homeostasis, leading to hypocalcemia, secondary hyperparathyroidism, and rickets (8-11). However, the wide tissue distribution of VDR suggests that the vitamin D endocrine system has additional physiological functions beyond calcium homeostasis. Indeed, vitamin D and VDR have been shown to play important roles in the immune system, cardiovascular system, reproductive system, and hair growth. Inappropriate activation of the renin-angiotensin system, which plays a central role in the regulation of blood pressure, electrolyte, and volume homeostasis, may represent a major risk factor for hypertension, heart attack, and stroke. Mounting evidence from clinical studies has demonstrated an inverse relationship between circulating vitamin D levels and the blood pressure and/or plasma renin activity, but the mechanism is not understood. We show here that renin expression and plasma angiotensin II production were increased severalfold in vitamin D receptor-null (VDR-null) mice, leading to hypertension, cardiac hypertrophy, and ...

Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Here we show that wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice were indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, were reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Intra-ACC administration of forskolin rescued behavioral allodynia defective in the AC1&8 DKO mice. Our studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia.

The parathyroid glands are the only known source of circulating parathyroid hormone (PTH), which initiates an endocrine cascade that regulates serum calcium concentration. Glial cells missing2 (Gcm2), a mouse homologue of Drosophila Gcm, is the only transcription factor whose expression is restricted to the parathyroid glands. Here we show that Gcm2-deficient mice lack parathyroid glands and exhibit a biological hypoparathyroidism, identifying Gcm2 as a master regulatory gene of parathyroid gland development. Unlike PTH receptor-deficient mice, however, Gcm2-deficient mice are viable and fertile, and have only a mildly abnormal bone phenotype. Despite their lack of parathyroid glands, Gcm2-deficient mice have PTH serum levels identical to those of wild-type mice, as do parathyroidectomized wild-type animals. Expression and ablation studies identified the thymus, where Gcm1, another Gcm homologue, is expressed, as the additional, downregulatable source of PTH. Thus, Gcm2 deletion uncovers an auxiliary mechanism for the regulation of calcium homeostasis in the absence of parathyroid glands. We propose that this backup mechanism may be a general feature of endocrine regulation.

SUMMARY Why do opiates make human beings itch ? Spinal opioid-induced itch, a prevalent side effect of pain management, has been considered to occur as a result of pain inhibition. We report that morphine-induced scratching (MIS) is abolished in mice lacking either gastrin-releasing peptide receptor (GRPR) or the μ opioid receptor (MOR). Using exon-specific knockdown, we identified the MOR1D isoform as essential for MIS, whereas MOR1 is important for morphine-induced analgesia (MIA) with no cross activity present. MOR1D and GRPR form constitutive heterodimers in the spinal cord and relay itch information upon morphine activation. Morphine induces internalization of both GRPR and MOR1D, whereas GRP induces that of GRPR but not MOR1D, when co-expressed. Moreover, GRP-induced scratching (GIS) is independent of MOR activation. These results suggest a unidirectional cross-activation of GRPR signaling by MOR1D via heterodimerization, and that opioid-induced itch is an active process concomitant with but independent of opioid analgesia.