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...

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.

Background and purpose: Although CB 1 receptor activation evokes neuroprotection in response to cannabinoids, some cannabinoids have been reported to be peroxisome proliferator activated receptor (PPAR) ligands, offering an alternative protective mechanism. We have, therefore, investigated the ability of a range of cannabinoids to activate PPARa and for N-oleoylethanolamine (OEA), an endogenous cannabinoid-like compound (ECL), to evoke neuroprotection. Experimental approach: Assays of PPARa occupancy and gene transactivation potential were conducted in cell-free and transfected HeLa cell preparations, respectively. In vivo estimates of PPARa activation through fat mobilization and gene transcription were conducted in mice. Neuroprotection in vivo was investigated in wild-type and PPARa gene-disrupted mice. Key results: The ECLs OEA, anandamide, noladin ether and virodhamine were found to bind to the purified PPARa ligand binding domain and to increase PPARa-driven transcriptional activity. The high affinity synthetic CB 1/2 cannabinoid agonist WIN 55212-2 bound to PPARa equipotently with the PPARa agonist fenofibrate, and stimulated PPARa-mediated gene transcription. The phytocannabinoid D 9 tetrahydrocannabinol was without effect. OEA and WIN 55212-2 induced lipolysis in vivo, while OEA pre-treatment reduced infarct volume from middle cerebral artery occlusion in wild-type, but not in PPARa-null mice. OEA treatment also led to increased expression of the NFkB-inhibitory protein, IkB, in mouse cerebral cortex, while expression of the NFkB-regulated protein COX-2 was inhibited. Conclusions and implications: These data demonstrate the potential for a range of cannabinoid compounds, of diverse structures, to activate PPARa and suggest that at least some of the neuroprotective properties of these agents could be mediated by nuclear receptor activation.

Fusion between the membrane of HIV and the membrane of a host cell is a crucial step in HIV infection and is catalyzed by the binding of the fusion peptide domain (HFP) of the HIV gp41 protein to the host cell membrane. The HFP by itself induces vesicle fusion and is a useful model system to understand the fusion peptide/host cell membrane interaction. This article reports an experimental correlation between the membrane locations of different HFP constructs and their fusogenicities. The constructs were the HFP monomer with Val-2 to Glu-2 mutation (HFPmn_mut), wild type HFP monomer (HFPmn), and wild type HFP trimer (HFPtr). All constructs have predominant ␤ sheet structure in membranes with physiologically relevant cholesterol content. HFPmn_mut does not fuse vesicles, HFPmn has moderate fusion rate, and HFPtr has the putative oligomerization state of HIV gp41 and a very rapid fusion rate. The HFP membrane locations were probed with solid-state NMR measurements of distances between labeled carbonyl ( 13 CO) nuclei in the HFP backbone and lipid nuclei in the surface or interior regions of the membrane bilayer. HFPmn_mut is located at the membrane surface, HFPmn is inserted into a single membrane leaflet, and HFPtr is the most deeply inserted construct with contact with the center of the membrane. These results show a clear positive correlation between the insertion depths and the fusion activities of the HFP constructs. Other disease-causing enveloped viruses contain fusion peptides and this correlation may be a general structure-function model for these peptides.L ike many viruses that cause human disease, HIV is enveloped by a membrane obtained during virus budding from a previously infected host cell and infection of a new cell requires fusion between the viral membrane and the cell membrane. Fusion is catalyzed by the HIV fusion protein gp41, which has Ϸ170 ectodomain residues outside the virus including a Ϸ20-residue N-terminal fusion peptide (HFP) that binds to target cell membranes (1). Studies of HIV with a truncated or mutated HFP showed that the HFP is crucial in the fusion process (2, 3). Functionally critical fusion peptides are also found in fusion proteins of other enveloped viruses such as influenza and Ebola (1). Chemically synthesized peptides with HFP sequences have been developed as fusion model systems and provide information about HFP perturbation of target membranes. Free HFPs induce vesicle fusion and there are strong correlations between the mutation/activity relationships of HFP-induced fusion and HIV/host cell fusion (3).There have been some number of HFP structural studies, but in our view, there have not yet been clear correlations between HFP structure and fusogenic function. For example, membraneassociated HFP can adopt either helical or ␤ strand conformation and there has been effort to determine a correlation between conformation and fusogenicity. However, this work has resulted in conflicting models such as: (i) the helical conformation is fusogenic and the ␤ strand conformation i...

No abstract