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Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10–50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP+ neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP+ cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid—reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP+ DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP+ neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα+ DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8+/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.
Citation: McCoy ES, Taylor-Blake B, Zylka MJ (2012) CGRPα-Expressing Sensory Neurons Respond to Stimuli that Evoke Sensations of Pain and Itch. PLoS ONE 7(5): e36355. https://doi.org/10.1371/journal.pone.0036355
Editor: Hiroaki Matsunami, Duke University, United States of America
Received: February 4, 2012; Accepted: April 2, 2012; Published: May 1, 2012
Copyright: © 2012 McCoy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants to MJZ from The Searle Scholars Program, The Klingenstein Foundation, The Rita Allen Foundation and NINDS (R01NS060725, R01NS067688). Confocal imaging was performed at the UNC Confocal and Multiphoton Imaging Core. The BAC Core and Imaging Core are funded by grants from NINDS (P30NS045892) and NICHD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Small-to-medium-diameter neurons in the dorsal root ganglia (DRG) have classically been divided into peptidergic and nonpeptidergic subsets [1], [2]. Many of these neurons respond to noxious thermal, mechanical and chemical stimuli, making them nociceptive, whereas others respond to innocuous stimuli, such as warming and cooling. The most widely recognized markers of peptidergic neurons are CGRP and substance P, while IB4-binding and fluoride-resistant acid phosphatase (FRAP; also known as Prostatic acid phosphatase, PAP) classically mark nonpeptidergic neurons [3], [4].
The sensory functions of these circuits were recently examined through the use of sophisticated genetic and physiological techniques. Nonpeptidergic, Mrgprd-expressing neurons are unmyelinated and contribute to mechanosensation but not thermosensation or cold sensation [5], [6]. Peptidergic CGRP+ neurons are myelinated (A-fibers) or unmyelinated (C-fibers) and, depending on fiber type, respond to nociceptive stimuli or guard hair displacement [7], [8]. TRPV1+ neurons, a subset of which are peptidergic [9], detect noxious thermal stimuli and some pruritogens [5], [10], [11], [12], [13]. However, the extent to which the broader class of peptidergic CGRP+ neurons is required for innocuous and noxious stimulus detection in mammals is currently unknown.
CGRP is not a single peptide but two separate peptides (CGRPα and CGRPβ) encoded by separate genes (Calca and Calcb). Calca is alternatively spliced, giving rise to CGRPα in neurons and calcitonin in thyroid C cells [14]. And, CGRPα and CGRPβ are nearly identical at the amino acid level. As a result, antibodies typically cannot distinguish CGRPα from CGRPβ, necessitating use of the term "CGRP-immunoreactivity" (CGRP-IR). CGRP-IR cells and fibers are present in multiple tissues, including the brain, stomach, intestine, skin and bladder [15], [16], [17], [18]. In studies where expression of each gene was resolved, both CGRPα and CGRPβ were expressed in the DRG although CGRPα was expressed at two-fold higher levels [16], [17].
When released peripherally from neurons, CGRPα causes vasodilatation, relaxes smooth muscle cells and contributes to migraine pathogenesis [19]. CGRPα is also released in the dorsal spinal cord and potentiates excitation caused by noxious stimuli and pronociceptive chemicals [20], [21]. CGRPα levels also regulate sensitivity to noxious heat [22]. Notably, CGRPα knockout mice have reduced behavioral responses to capsaicin and impaired heat hyperalgesia although acute heat responsiveness is not affected [23], [24], [25].
To directly study the projections and sensory functions of CGRPα neurons, we generated a knock-in mouse that expresses an axonal tracer and a conditional cell ablation construct from the Calca/Cgrpα locus. We used these mice to prospectively identify peptidergic DRG neurons in culture and show that they respond to agonists that evoke sensations of pain and itch.
CGRPα-GFP+ neurons respond to agonists that evoke sensations of pain and itch
At the time we began this study, there were no ways to prospectively identify CGRP+ sensory neurons for physiological studies. To permit direct visualization of CGRP+ sensory neurons and axons, we knocked-in a floxed (LoxP flanked) membrane-tethered axonal tracer (farnesylated enhanced GFP) to the Calca locus (Fig. 1A) [26]. This floxed GFP also conditionally blocks expression of downstream DTR (Cre recombinase-dependent expression of DTR will be described in a subsequent study). Heterozygous (CGRPα-GFP+/−) mice, which contain one functional Calca allele and one GFP allele, were used throughout this study. The mice were viable and showed no obvious phenotypic or behavioral abnormalities.
(A) Farnesylated GFP-DTR knocked-in to the start codon of CGRPα. Expression of DTR is conditionally blocked by LoxP flanked GFP and three polyadenylation signals (not shown). LoxP sites are oriented so that the first ATG encountered is in GFP. (B) Representative images of cultured DRG neurons from a CGRPα-GFP+/− mouse after loading with Fura2-AM. Arrows point to CGRPα-GFP+ neurons. (C–J) Responses of CGRPα-GFP+ neurons from heterozygous mice to (C) capsaicin (1 µM), (D) mustard oil (100 µM), (E) menthol (200 µM), (F) icilin (4 µM), (G) histamine (100 µM), (H) chloroquine (1 mM), (I) ATP (100 µM) and (J) acidic pH, followed by stimulation with 100 mM KCl to identify healthy neurons. Scale bar in (B) right panel is 50 µm.
We next loaded cultured DRG neurons from CGRPα-GFP+/− mice with the calcium indicator Fura2-AM. CGRPα-GFP+ neurons were readily identifiable based on intrinsic GFP fluorescence and accounted for 39.9% of all Fura2-loaded neurons (Fig. 1B, arrowheads; n = 1292 Fura2+ neurons analyzed). No CGRPα-GFP+ cells were present from wild-type littermate controls. A majority (54.8%) of these CGRPα-GFP+ neurons were 17–30 µm in diameter, with the remainder being either smaller or larger.
We then monitored calcium responses in all CGRPα-GFP+ and CGRPα-GFP− neurons to capsaicin, mustard oil, menthol, icilin, histamine, chloroquine, ATP and acidic pH (Fig. 1C–J, Table 1, Table S1, Table S2). Capsaicin is a ligand for the noxious heat- (>43°C) and acid-sensitive TRPV1 receptor [27] and activated approximately 50% of all CGRPα-GFP+ neurons. Mustard oil, an agonist of the irritant and noxious cold receptor TRPA1, activated 36.8±2.0% of all CGRPα-GFP+ neurons. Menthol, a nonselective modulator of TRPA1 and TRPM8 [28], [29], activated 14.3±5.0% of all CGRPα-GFP+ neurons (Table 1). A low concentration of icilin, which selectively activates TRPM8 [30], activated 2.2±1.3% of all CGRP+ neurons. Histamine and chloroquine evoke the sensation of itch [31] and activated ∼11% of all CGRPα-GFP+ neurons. The nonselective P2X and P2Y purinergic receptor agonist ATP activated 12.8±2.2% of all CGRPα-GFP+ neurons. Lastly, lowering the pH to between 5.0 and 6.0 activated 27.0±2.2% of all CGRPα-GFP+ neurons. For all ligands studied, the majority of all CGRPα-GFP+ responsive cells were small- to medium-diameter (Table S1). Taken together, these data indicate that peptidergic CGRPα+ neurons can detect diverse ligands that evoke sensations of pain and itch.
CGRPα-GFP genetically marks a circuit that is largely distinct from nonpeptidergic and TRPM8+ sensory circuits
To determine if CGRPα-GFP was expressed in peptidergic sensory neurons, we next immunostained sections of lumbar DRG with antibodies to GFP and various neuronal markers. We found that the vast majority (88.9±0.5%) of all CGRP-IR neurons were CGRPα-GFP+ (Fig. 2A–C, Table 2). Conversely, 67.8±0.8% of all CGRPα-GFP+ neurons were CGRP-IR. This lack of complete overlap was likely due to the greater sensitivity of GFP immunostaining—GFP filled cells in their entirety and was easier to detect than CGRP-IR, especially in cells with low levels of CGRP-IR. Interestingly, ∼10% of the CGRP-IR neurons did not colocalize with CGRPα-GFP. Because the CGRP antibody we used recognizes CGRPα and CGRPβ, these CGRP-IR-only cells could represent DRG neurons that express CGRPβ alone [16], [17]. In addition, approximately 50% of the CGRPα-GFP+ neurons expressed TRPV1 (Fig. 2D–F, Table 2), consistent with our functional studies above.
Sections of L4-L6 DRG from CGRPα-GFP+/− mice were stained with antibodies to GFP (A,D,G,J,M,P) and the indicated markers. Images were acquired by confocal microscopy. Scale bar in (R) is 50 µm.
In contrast, there was little (<1%) overlap between CGRPα-GFP and TRPM8 (Fig. 2G–I), a receptor that is activated by icilin, menthol and cool temperatures [32], [33]. There was also limited overlap between CGRPα-GFP and nonpeptidergic markers (IB4 and PAP; Fig. 2J–O, Table 2), which was consistent with previous studies [3], [34]. Some (18.1±0.5%) CGRPα-GFP+ neurons colocalized with neurofilament-200 (NF200) (Fig. 2P–R), a marker of large-diameter neurons with myelinated axons. Importantly, DTR was not expressed in any DRG neurons (Table 2), indicating that the floxed GFP insert effectively blocks downstream transcription.
In the spinal cord, CGRPα-GFP (Fig. 3A) and CGRP-IR (Fig. 3B) were colocalized in lamina I and lamina II outer, with fibers extending into lamina V and towards lamina X. There was little overlap between CGRPα-GFP+ and IB4-binding terminals in lamina II (Fig. 3C, D), revealing segregation between peptidergic and nonpeptidergic spinal circuitry. When taken together, our data indicate that CGRPα-GFP genetically marks a distinct subset of small-to-medium- and large-diameter DRG neurons in adult mice and constitutes a circuit that is largely distinct from nonpeptidergic circuits and TRPM8+/cool temperature-sensing circuits.
Sections of lumbar spinal cord from CGRPα-GFP+/− mice were stained with antibodies to (A) GFP and (B) CGRP. (C) IB4-binding. (D) Merged image. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (D) is 100 µm.
CGRPα-GFP is expressed in motor neurons and a small population of neurons intrinsic to the dorsal spinal cord
CGRP-IR in the dorsal horn is typically attributed to primary afferent axons and their terminals; however CGRP-IR is also present in a subset of dorsal horn neurons in rats and mice [35], [36]. To detect these cells immunohistochemically, these groups performed dorsal rhizotomies or treated animals with colchicine (colchicine arrests axonal transport, allowing CGRP to accumulate). Scattered Cgrpα/Calca-expressing cells were also detected in the dorsal horn by in situ hybridization, in Allan Brain Atlas adult spinal cord images [37]. The high sensitivity of the membrane-tethered GFP axonal tracer allowed us to detect these intrinsic CGRPα+ neurons without manipulating mice surgically or chemically. When examined at higher magnification, these spinal neurons were located between axon terminals of CGRPα-GFP+ and IB4+ sensory neurons, with CGRPα-GFP+ neurons being predominantly located in lamina II inner and lamina III (Fig. 4A–F, arrowheads). Very few of these intrinsic CGRPα-GFP+ dorsal horn neurons contained PKCγ (Fig. 4C,F), a marker of some lamina II and III neurons [38], [39]. In the ventral horn, CGRPα-GFP labeled many CGRP-IR motor neurons (Fig. 4G–I) along with their axons, which terminate at motor endplates in skeletal muscle (Fig. 5A–C). There were also a number of CGRP-IR motor neurons that lacked CGRPα-GFP, likely reflecting a subset of motor neurons that only express CGRPβ [40].
Sections of lumbar spinal cord from CGRPα-GFP+/− mice were stained with (A–F) antibodies to GFP (green) and PKCγ (red). IB4-binding (blue). Arrowheads point to GFP+ cells. (D–F) Single confocal scan image from box in (A) reveals a CGRPα-GFP+ neuron located in lamina II. (G–I) Motor neurons stained with antibodies to GFP (green) and CGRP (red). Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (C and F) is 25 µm and in (I) is 100 µm.
Sections from the glabrous skin of the hindpaw from CGRPα-GFP+/? mice were stained with antibodies to (A) GFP and (B) PGP9.5. (C) Merged images were stained with the nuclear marker DRAQ5. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (C) is 50 μm.
CGRPα-GFP+ neurons innervate cutaneous and visceral tissues
CGRP-IR fibers are present in cutaneous and visceral tissues [16], [18], [26], [41], [42], [43]. Whether these fibers originate from CGRPα- and/or CGRPβ-expressing sensory neurons is unknown. To address this question, we stained a number of peripheral tissues from CGRPα-GFP+/− mice with antibodies to GFP and the pan-nerve fiber marker PGP9.5. We observed CGRPα-GFP+ free nerve endings in the epidermis of glabrous skin (Fig. 5D–I, Fig. 6). Most of these CGRPα-GFP+ endings had a straight and stubby morphology that was distinct from the meandering "zig-zag" shape of PGP9.5+/CGRPα-GFP− (presumably nonpeptidergic) fibers. We previously observed this same morphological distinction between peptidergic and nonpeptidergic fibers when targeting farnesylated GFP to Mrgprd+/nonpeptidergic neurons [26]. Interestingly, we also noticed that some of the epidermal CGRPα-GFP+ fibers had small spheres at their tips (see arrowheads, Fig. 5G-inset). These spheres may simply result from membrane budding or intriguingly might constitute a novel transduction unit at the tips of some peptidergic afferents. CGRPα-GFP+ afferents were also present within sweat glands of glabrous skin (Fig. 5G–I). These afferents, which were also PGP9.5+, are likely sensory in origin because CGRPα is not expressed in sympathetic ganglia of mice [17]. In hairy skin, CGRPα-GFP+ fibers progressed through the dermis and terminated in the epidermis and on guard hair follicles (Fig. 5J–L).
Sections of hindpaw from CGRPα-GFP+/? mice were stained with antibodies to (A,D,G,J) GFP and (B,E,H,K) the pan-nerve fiber marker PGP9.5. (C,F,I,L,G-inset) Merged images were stained with the nuclear marker DRAQ5 to visualize skin cells. (AC) Nerve bundle in the subdermis and motor end plates (ep). (DF) Epidermis (epi) and upper dermis (d) from glabrous skin, (GI) sweat gland (sg) in glabrous skin and (JL) guard hair follicle (hf) in hairy skin. Arrowheads point to putative transduction spheres. Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bar in (L) is 100 μm.
In addition, CGRPα-GFP+ fibers were present in the submucosal/smooth muscle layers of the small intestine (Fig. 7A–C), consistent with previous studies [44], [45]. There were also numerous green fluorescent cells in intestinal villi; however, these cells were not CGRPα-GFP+ because: a) they did not co-stain for CGRP-IR (Fig. 7B) and more importantly, b) they were detectable in wild-type mice (i.e., mice lacking GFP; Fig. 7C-inset). These cells are likely a population of autofluorescent stromal cells [46]. There were also a large number of CGRP-IR cells in the intestinal villi that were not CGRPα-GFP+ (Fig. 7B). These CGRP-IR-only cells likely express CGRPβ, particularly given that CGRPβ/Calcb is the primary CGRP gene expressed in the gut [16], [17]. Lastly, we observed CGRPα-GFP+ afferents in the bladder (Fig. 7D–I), a visceral tissue that is innervated by sensory afferents. When taken together, our data indicate that CGRPα-GFP+ neurons innervate diverse cutaneous and visceral structures.
Sections of (A–C) small intestine, (D–I), bladder and (J–L) thyroid from CGRPα-GFP+/− mice were stained with antibodies to GFP (A, D, G, J) and the indicated markers. Nuclei in (A–C) were labeled with DRAQ5. (C-inset) Section of wild-type mouse small intestine stained for GFP (green) and DRAQ5 (blue). Images were acquired by confocal microscopy and are representative of n = 3 mice. Scale bars in (C) and (C-inset) inset are 100 µm and apply to (A–C). Scale bar in (L) is 50 µm and applies to (D–L).
CGRPα-GFP labels other cell types that express Calca, including thyroid cells and neurons in the brain
Since GFP was targeted to exon 2 of Calca, an exon that is common to CGRPα and calcitonin [14], CGRPα-GFP should be present in all tissues where Calca is expressed. Indeed, we found that CGRPα-GFP was co-localized with CGRP-IR in parafollicular cells of the thyroid (Fig. 7J–L).
We next thoroughly mapped CGRPα-GFP expression in the brain. To do this, we immunostained adult mouse brain sections and noted all locations where CGRPα-GFP+ cell bodies were found (Table 3). With the exception of the abducens nucleus, Purkinje cells, cuneiform nucleus and the dorsomedial thalamic nucleus, we detected CGRPα-GFP+ cell bodies in all regions previously known to express CGRPα [47], [48], [49], [50]. Representative regions where cellular and/or fiber staining were observed include the spinal trigeminal nucleus caudalis (Fig. 8A), the parabrachial nucleus (Fig. 8B), the peripeduncular and posterior intralaminar thalamic nuclei (Fig. 8C), the subparafascicular nucleus of the thalamus (Fig. 8D), the nucleus accumbens (Fig. 8E), the subiculum (Fig. 8F) and weakly in the visual cortex (Fig. 8F, inset). Calca-GFP BAC transgenic mice produced by the GENSAT project show a similar distribution of cellular and axonal labeling in the brain [51]. Taken together, our data indicate that CGRPα-GFP knock-in mice reproducibly mark all cells and tissues that are known to express Calca.
(A–F) Brain sections from CGRPα-GFP+/− mice were stained with antibodies to GFP. (A) Trigeminal spinal nucleus caudalis. Arrowheads point to labeled cells in lamina III, similar to those shown in Figure 4A–F. (B) Parabrachial nucleus. (C) Peripeduncular and posterior intralaminar thalamic nuclei. (D) Subparafascicular nucleus of the thalamus. (E) Nucleus accumbens. (F) Subiculum. Abbreviations: LPB = lateral parabrachial nucleus; MPB = medial parabrachial nucleus; scp = superior cerebral peduncle; MG = medial geniculate; SN = substantia nigra; fr = fasciculus retroflexus; mt = mammillothalamic tract; Sep = septum; aca = anterior commissure, anterior; S = subiculum; VC = visual cortex. Inset in each panel shows a lower magnification view. Scale bar in (A) inset is 1 mm and applies to all insets; (B) is 200 µm and applies to (B); (F) is 100 µm and applies to (A,C–F).
We generated the first knock-in reporter mouse to directly visualize and functionally study CGRPα-containing sensory neurons. While characterizing these mice, we found that CGRPα-GFP faithfully marked the peptidergic subset of DRG neurons, as well as other cell types throughout the body that express Calca. In contrast, cells that express Calcb/CGRPβ, including intramural neurons of the intestine [17], were devoid of CGRPα-GFP immunoreactivity. Our reporter mice can thus be used to discriminate Calca-expressing cells from cells that express Calcb. The membrane-tethered GFP reporter allowed us to prospectively identify live CGRPα-expressing neurons in culture for functional studies. Remarkably, half (∼50%) of all CGRPα-GFP+ DRG neurons expressed TRPV1 and half of all CGRPα-GFP+ DRG neurons responded to the TRPV1 agonist capsaicin, suggesting that CGRPα+ neurons may play a significant role in capsaicin and noxious thermal sensitivity in vivo. In addition, >50% of all histamine- and chloroquine-responsive neurons were CGRPα-GFP+, suggesting a major role of CGRPα-expressing neurons in histamine-dependent and histamine-independent itch. Likewise, there is a large degree (∼90%) of overlap between TRPV1/capsaicin-responsive neurons and histamine-responsive neurons [31], [52], suggesting thermal pain and histamine-dependent itch are encoded by the same class of sensory neurons.
We found little (<1%) overlap between CGRPα-GFP+ cells and TRPM8. And, very few (2%) CGRPα-GFP+ cells were activated by the cooling agent icilin (at a concentration that preferentially activates TRPM8 [30]). Likewise, others found no overlap between CGRP-IR and Trpm8-expression in DRG [32], [53], and no CGRP-IR neurons responded to cooling in electrophysiological studies [7]. These results collectively suggest segregation between CGRP and cool temperature-sensing/TRPM8+ circuits.
In contrast, Takashima et al. found that TRPM8-GFP and CGRP-IR overlap by ∼20% when using a BAC transgene to mark Trpm8-expressing neurons [54]. BAC reporters often drive higher levels of gene expression when compared to knock-in reporters, but can suffer from position effects that compromise expression specificity [51]. Thus, higher detection sensitivity and/or position effects could explain why there was a greater degree of overlap between CGRP-IR and BAC reporter driven Trpm8 expression than we and others observed when examining endogenous Trpm8 expression.
We also found that 14.3±5.0% of all CGRPα-GFP+ cells were menthol-responsive. Contrary to what is commonly stated in the literature, menthol is not a TRPM8 specific agonist. Menthol activates TRPA1 at sub- to low-micromolar concentrations and inhibits TRPA1 at higher concentrations [28], [29]. This bimodal modulation provides one of many explanations for why a smaller percentage of CGRPα-GFP+ neurons responded to menthol in culture than to the TRPA1 agonist mustard oil (Table 1).
With regard to position effects, it will be interesting to determine if the Calca-GFP BAC transgenic mouse line made by the GENSAT project reproduces CGRPα expression in DRG, brain and peripheral tissues to the same extent as our knock-in mouse [51]. In addition, it will be interesting to determine if this BAC transgenic line distinguishes Calca-expressing cells from Calcb-expressing cells. Calca and Calcb are located ∼80 kb apart in the mouse genome. This genomic proximity could contribute to their similar but not identical expression patterns. Baillie and colleagues recently used Calca-GFP BAC transgenic mice and optical imaging techniques to visualize an axon reflex in an individual CGRPα+ sensory afferent [55].
In what is perhaps the most comprehensive physiological study of CGRP+ sensory neurons to date, Lawson and colleagues found that CGRP-IR neurons can be classified as C-fiber and Aδ-fiber nociceptive units (responsive to noxious thermal and high threshold mechanical stimuli), unresponsive C-fibers or Aα/β guard hair afferents. None of the CGRP-IR neurons had C-cooling/cold or C-low threshold mechanoreceptive (C-LTMR) properties. These findings, combined with TRPV1 cell inactivation studies (described above) and our current work, consistently point to a role for CGRP+ neurons in sensing noxious heat.
CGRPα-GFP might also mark the CGRP-IR+ Aα/β guard hair units that were identified by Lawson and colleagues [7], particularly since CGRPα-GFP+ fibers terminated on guard hairs in hairy skin and ∼25% of all CGRPα-GFP+ neurons expressed NF200, a marker of myelinated afferents. Guard hairs add sheen to the coat of furry mammals, are often water repellent, and drive activity in sensory afferents when deflected [7], [56], [57]. Whether activation of guard hair afferents has sensory and/or non-sensory functions in mammals is currently unknown.
Ultimately, it should be possible to directly evaluate the in vivo functions of CGRPα+ sensory neurons by taking advantage of the LoxP-stopped DTR that we knocked-in immediately behind GFP (Fig. 1A). DTR, when combined with injections of diphtheria toxin, can be used to conditionally ablate cells and neurons in adult mice [5], [58]. Importantly, DTR expression was completely blocked in DRG (Table 2). We engineered DTR so that its ATG start codon will precisely substitute for the start codon of GFP upon CRE recombinase-mediated excision. DTR should thus be expressed in all cell types that jointly express CGRPα and CRE recombinase (including cells that expressed CRE at any time during development). When crossed with sensory neuron selective lines, such as Nav1.8-Cre or Advillin-Cre [59], [60], [61], [62], this could permit selective expression of DTR in DRG neurons while maintaining GFP expression in all other Calca-expressing cell types. Given that Calca is expressed in many other cell types, this strategy could be broadly employed to genetically label, ablate and study the function of diverse peptidergic CGRPα-containing cell types throughout the brain and body.
Materials and Methods
All procedures and behavioral techniques involving vertebrate animals were approved by the Animal Care and Use Committee at the University of North Carolina at Chapel Hill.
Molecular Biology
Recombineering was used to generate Calca targeting arms from a C57BL/6-derived bacterial artificial chromosome (BAC; RP24-136021). The start codon located in exon 2 is common to CGRPα and calcitonin and was replaced with an AscI site to facilitate cloning of an axonal tracer and a conditional cell ablation construct: AscI-LoxP-EGFPf-3x pA-LoxP-DTR-pA-Frt-PGK-NeoR-Frt-AscI. EGFPf = farnesylated enhanced GFP [26]. DTR = human diphtheria toxin receptor [58]. NeoR = neomycin resistance. The LoxP sites were oriented so that the first ATG encountered was in GFP or, after Cre recombinase-mediated excision, DTR. Correct targeting was confirmed in 5.8% of all embryonic stem cell clones by Southern blotting using flanking 5′ and 3′ probes and a NeoR internal probe. High percentage chimeras were crossed to C57BL/6 females to establish germline transmission and then crossed to ACTFLPe mice (B6.Cg-Tg(ACTFLPe)9205Dym/J, Jackson Laboratory) to remove the Frt-flanked selection cassette (removal confirmed by PCR). Next, mice were backcrossed to C57BL/6 to remove the ACTFLPe allele (removal confirmed by PCR) and then backcrossed to C57BL/6 mice for 8 generations to establish the CGRPα-GFP knock-in line. As a technical note, we were only able to detect GFP expression in DRG neurons after removal of the PGK-NeoR selection cassette.
Calcium Imaging
Adult (4–6 week old) male CGRPα-GFP+/− mice were decapitated, DRG were dissected then neurons were dissociated using collagenase (1 mg/mL; Worthington, CLS1) and dispase (5 mg/mL; Gibco, 17105-041) in DH10 media (1∶1 Ham's DMEM/F12, 10% FBS and 1% penicillin/streptomycin) [63], [64]. Medium was supplemented with 25 ng/mL of glial-derived neurotrophic factor (GDNF; Upstate, GF030). The neurons were plated onto coverslips coated with 0.1 mg/mL poly-D-lysine and 5 µg/mL laminin. After 24 h, neurons were washed 2× with Hank's balanced salt solution (HBSS) and incubated for 1 h with 2 µM Fura2-AM in the dark at room temperature. Next, the cells were washed 3× with HBSS and maintained at room temperature for 30 min prior to imaging. After a 60 s baseline, agonists (1 µM capsaicin, 100 µM mustard oil, 200 µM menthol, 4 µM icilin, 100 µM ATP, 100 µM histamine, 1 mM chloroquine or acidic pH 5–6 HBSS) were perfused onto the neurons. Following activation, cells were perfused with HBSS to remove the agonist, which was followed by addition of 100 mM KCl to determine the total number of neurons present. Images were acquired on a Nikon Eclipse Ti microscope (Nikon, Melville, NY). GFP+ neurons were identified by eye, and then a 500 ms exposure was used to image the cells. CGRPα-GFP+ neurons of all sizes were included in the analysis. Following addition of agonist, only neurons with responses greater than 15% of baseline were scored as responders.
Mice were sacrificed by overdosing with pentobarbital. The thyroid, brain, bladder, hindpaw skin, lumbar DRG, lumbar spinal cord and small intestine were dissected and immersion-fixed in 4% paraformaldehyde (5 h, 24 h, 5 h, 3 h, 4 h, 8 h and 2 h, respectively) and were cryopreserved in 30% sucrose at 4°C. Tissue was embedded in TissueTek and cryosectioned (20 µm for small intestine and DRG; 40 µm for thyroid, bladder and spinal cord; 50 µm for brain and skin). Sections were either immunostained free-floating or thaw mounted onto SuperFrost Plus slides and stored at −20°C until needed.
Tissue was rehydrated in PBS, rinsed with TBST (0.05 M Tris, 2.7% NaCl, 0.3% Triton-X 100, pH 7.6), then blocked with 10% neat donkey serum (NDS) in TBST for 1 h at room temperature. Sections were incubated overnight at 4°C with primary antibodies. The following reagents were used: Isolectin Griffonia simplicifolia IB4, Alexa 568 conjugate (1∶100, Invitrogen, I21412), chicken anti-GFP (1∶600; Aves Labs, GFP-1020), rabbit anti-GFP (1∶600; Invitrogen, A11122), rabbit anti-CGRP (1∶750; Peninsula, T-4032), sheep anti-CGRP (1∶250; Enzo Life Sciences, CA1137), mouse anti-NeuN (1∶200; Millipore, MAB377), chicken anti-PAP (1∶4,000; Aves Labs), rat anti-TRPM8 (1∶100; a generous gift from Masatoshi Takeichi) [65], rabbit anti-PGP9.5 (1∶500; Ultraclone), rabbit anti-NF200 (1∶500; Sigma, N4142) and goat anti-HB-EGF (1∶1,000; R&D Systems, AF-259-NA), which labels DTR. Tissue was rinsed and then blocked for 1 h in 10% NDS in TBST. Sections were incubated for 2 h in 10% NDS in TBST with Alexa fluor-conjugated secondary antibodies (Invitrogen). DRAQ5 (1∶10; Cell Signaling, 4084) was used to label nuclei. PAP, TRPV1 and DTR immunostaining was performed using amplification, as described previously [66]. All fluorescent images were obtained using a Zeiss LSM 510 confocal microscope (Zeiss, Thornwood, NY).
For diaminobenzidine (DAB) staining, brain sections were processed as described above with chicken anti-GFP (1∶5,000). On day 2, the sections were washed in TBST and then blocked for 30 min. Sections were incubated for 2 h in biotinylated donkey anti-chicken IgG (1∶500), which was followed by washes in TBST. Sections were incubated with the Vectastain ABC complex in TBST for 2 h and washed. Sections were treated with a DAB solution (0.02% DAB, 0.01% H2O2 and 0.005% nickel ammonium sulfate in TBST) for 15 min. Following TBST washes and a PBS rinse, the sections were immersed in 0.2% gelatin in water, mounted onto Superfrost Plus slides and then air-dried for 4 days. Lastly, the sections were dehydrated with graded ethanols, cleared with xylene and coverslipped with DPX.
Supporting Information
Table S1.
Percentage of CGRPα-GFP+/− DRG neurons of a given size class (small, medium, large diameter) that respond to the indicated agonists.
Table S2.
Percentage of GFP-negative DRG neurons of a given size class (small, medium, large diameter) that respond to the indicated agonists.
We thank JrGang Cheng at the University of North Carolina (UNC) BAC Core for generating the CGRPα targeting arms, the UNC Animal Models Core for embryonic stem cell work and producing chimeras, Sarah Shoemaker for mouse colony management and small intestine immunohistochemistry, Brendan Fitzpatrick for technical support, Kenji Kohno for providing DTR (pTRECK1) plasmid and Masatoshi Takeichi for providing TRPM8 antibody.
Author Contributions
Performed the experiments: ESM BTB MJZ. Analyzed the data: ESM BTB MJZ. Wrote the paper: ESM BTB MJZ.
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After a good breakfast at a very nice hotel, we set off for the Fabled Atlanterhavsvegen - reportedly one of the great drives in the world, up the Atlantic coast. Frankly, we were underwhelmed...found a nice bridge that had been much photographed (called ...
Goodness Me!
... is only going to be for one-hour. It is time to unleash the camera and enjoy this part of Norway. Not surprisingly, the hour visit passes quickly. Sandnessjoen is a place that is worth a return visti and must be a true delight during the summer months. ...

subset: commoncrawl


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The Vampire in the Attic
One dark night Amy and Amanda were having a sleepover. They were watching movies downstairs when the tv went off. They screamed and hurried off upstairs to bed, but they could hear strange noises coming from the attic. Suddenly the attic door opened, and out came a vampire. The girls screamed in terror. The vampire disappeared through the window right before Amy's mum hurried into the room and asked why they were screaming, Amy said
'There was a vampire in the attic!.'
'Dont be silly' said Amy's mum laughing. And Amy's mum turned the lights off and left the room. Straight after she had left the room the vampire came back through the window. Following him were many more vampires. The girls were terrified, and screamed even louder than last time, Amy's mum entered the room once again and asked... 'What is it now girls?'
'The vampire came back through the window with many more vampires and they have gone into the attic, please go and get rid of them!' said Amanda.
Amy's mum told them that there are no such things as vampires, but the girls knew otherwise so they did not believe her. Amy's mum told them to get some sleep because it was getting late, and she left the room.
They decided that she was right so Amy and Amanda went to sleep. In the morning Amy's mother came to wake them up and she began to now believe that Amy and Amanda were right about the vampires. On that day they put a lock on the attic door and made sure that the windows were closed every night, the same day the girls talked about how weird that sleepover was.


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Mark: Oh. My. God.
"Tallie?" I said. Was it her? Could she possibly have survived the Suicidals? i looked at her shoulder-length dark hair, her vibrant green eyes, her pale ivory face. She was beautiful. She didn't blink as she stared at me through the long lashes that framed her perfect eyes. I closed the distance between us in three long strides. I pulled her towards me, and kissed her passionatly.
"Yeah." She breathed when we were finished.


text:
Home > Video Gallery > Video Gallery - Workshops
Overview and Operation Manual
Build, Tune, Fly!
Assembly courtesy of 2 Brothers Fly
Part 1 - Product Overview
Part 2 - Installation
Part 3 - Data Modules
If you own a Hitec Optima receiver and haven't taken advantage of the SPC port, what are you waiting for?
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Main Site - :: Why Won't God Heal Amputees? ::
0 Members and 2 Guests are viewing this topic.
Religions need books because they don't have gods.
^^Where did you get this?<snip>
Quote from: mrbiscoop on July 06, 2013, 08:41:26 AM^^Where did you get this?<snip>There's this nifty search engine I like to call "Google".
Gee, I was wrong you really are just a dick.


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What percent of the U.S. population are farmers?
About 1 percent of people living in the United States claim farming as an occupation, according to the United States Environmental Protection Agency. About 2 percent of Americans actually live on f...
Read More »
What percent of the population experiences heartburn?
According to the International Foundation for Functional Gastrointestinal Disorders (IFFGD), occasional heartburn impacts about 60 million Americans at least once a month. About 25 percent of those...
Read More »
What percent of the population is left-handed?
Ten percent of the population is left-handed, according to an MSN Health & Fitness article by Robert Shmerling. Famous left-handed people include Barack Obama, Jimi Hendrix and Leonardo da Vinci. S...
Read More »
What Percent of the US population currently in the military?
What percent of US population has completed an ultra-marathon?
Certainly less than one tenth of one percent of the US population has attempted this physical feat successfully. A 100 kilometer run completed within 24 hours by a Battalion sized unit in full pack...
Read More »


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Geen omschrijving in het Nederlands voorhanden
Romantic Oud West Canal Houseboat offers accommodation in Amsterdam, 700 metres from Leidseplein and 1 km from Vondelpark Open Air Theatre. The unit is 1.1 km from Anne Frank House. Free WiFi is offered .
An oven, a microwave and a toaster can be found in the kitchen. Towels and bed linen are available in this boat.
Vondelpark is 1.1 km from Romantic Oud West Canal Houseboat, while Van Gogh Museum is 1.1 km from the property. The nearest airport is Schiphol Airport, 10 km from the property....
|Da Costakade 352


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Home › Forums › Campfire › Pre-Trip Planning › Suggestions for late June backpacking in CO
May 20, 2014 at 5:16 pm #1317039
Leigh BakerMember
@leighbLocale: Northeast Texas Pineywoods
Was planning on the Pecos, but after talking with the ranger it looks like the area is a tinderbox waiting to go off again this year. So, looking for suggestions in CO where there's been adequate snow fall to possibly alleviate that concern. Seems to rule out the Weminuche and South San Juans. Will possibly be driving to Cortez to see friends on to way, so southern UT is also an option, but figure it would be too late for that area. I have been intrigued by the Lost Creek Wilderness and I'm wondering what it would be like in the last week of June? Coming from Texas I like that the elevation isn't that great. Don't want to deal with a bear canister if possible. I'm open to suggestions.
As always thanks in advance.May 20, 2014 at 6:45 pm #2104540
Brendan SwihartBPL Member
@brendansLocale: Fruita CO
I don't think moisture will be a problem in the Wemenuche or SSJs. Snowpack isn't as high as other parts of the state but I just heard toady that snowpack is still at 120+% at Molas and Red Mtn. We did part of the CDT in the Wemenuche in a similar year a few years ago over fourth of July wknd and there was plenty of sloppy postholing.May 20, 2014 at 6:50 pm #2104542
HkNewmanBPL Member
@hknewmanLocale: Western US
Leigh it's dry in the Pecos but not like last May – just returned. Even some postholing Not sure what it will look like late June though unless there's moisture. I was planning the weminuche or big blue myself , … not sure about more postholing tho unless I get an age transplant.May 20, 2014 at 7:38 pm #2104553
Gordon GrayBPL Member
@gordongLocale: Front Range, CO
No bear canister needed. No fire danger – may have patches of snow at the top.
Try James Peak Wilderness starting from the East Portal.
The pics below are from Forrest Lake. My then 8 and 10 year old daughters did it.
Yes, it's this amazing.May 20, 2014 at 8:01 pm #2104560
Luke SchmidtSpectator
@cameronLocale: Idaho Falls
Leigh I love Lost Creek but I've mostly gone early, might be a bit warmer in June. The most popular loop starts at Goose Creek but I really like the western loop over McCurdy Mountain its higher and more scenic. Of course if you have time you can combine the two loops into one big one.
Feel free to PM if you'd likeMay 20, 2014 at 9:13 pm #2104573
Randy MartinBPL Member
@randalmartinLocale: Colorado
I wouldn't rule out South San Juan Wilderness. It may be a lower snow year for that part of the state but actually that may make June workable whereas in a normal or higher snow year it wouldn't be. Here is a link to a well documented trip around the Blue Lake area. On my list to do so I don't have any personal knowledge to share.May 21, 2014 at 4:27 am #2104614
Leigh BakerMember
@leighbLocale: Northeast Texas Pineywoods
Thanks everyone, I was hoping I might be wrong about southern CO as it's still fairly close. I've just been so busy with end of school year stuff and being out of town on the weekends I haven't had the time to do a lot of research yet and it's coming up pretty soon. I'll check out those links.
You must be logged in to reply to this topic.


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Hi, I'm 18 and looking to get into hunting. I was wondering, if i were to complete the 10hour course and get a permit, I would still have to get a NYS rifle license permit correct?
The rifle permit is for ages 21 and older. So if my dad were to get a license permit, am I able to use the rifle he purchased?
It states that he has to carry the permit with him while in possession of the rifle. So can he sit in the car as I drive upstate with the rifle.Then I take it out and shoot with it while I'm in the field and he is sitting in the car or something? Or does he have to always be with it no matter where it goes?
He has back problems and cannot be out in the woods with me.


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Guest reviews
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The hotel is ideally located at Renhe Zhen. The hotel is 8 km from Baiyun International Airport. Free WiFi and free parking are provided at the hotel. All air-condition rooms are equipped with electric kettle, slippers, television and independent writing desk. Each unit is fitted with complimentary bottled water and electronic card lock. The hotel come with wake-up service and elevator. Guests can have a nice trip.


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Order Subtotal
Midnight outdoor wall sconce features a square etched white opal glass shade diffuser. Finish available in black and vista silver. Available in square or round version. One 60 watt, 120 volt, A19 medium base incandescent lamp not included. General light distribution. UL listed. Rated for wet location. 5W x 7H x 6 inch depth.


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5 Things To Know
Toronto officially gets a new top cop today. Argo's reportedly sold to Bell, Larry Tanenbaum. Defective airbags represent largest auto recall ever. Ticks found in Toronto. Talks between province and teachers resume today. +**Tolls suggested for non-Torontonians to use Gardiner Expressway.** Canadians coping with record debt levels.** David Letterman's final show airs tonight.**
He's already been on the job for the last couple of weeks, but an official swearing in ceremony will be held this morning for Mark Saunders. Exactly a month ago Saunders was named chief, becoming the city's first black Police Chief. He succeeds Bill Blair, who is currently seeking the Liberal nomination in Scarborough South-West, elevating him to the force's biggest job after 32-years with Toronto Police Service. A ceremony will be held at police headquarters, and will be attended by Mayor John Tory and other dignitaries.
"As the first black Chief, it's important to make sure that I have the skills necessary and I think I do. Being black is fantastic but it doesn't give me superpowers, so if you're expecting that all of a sudden, the earth will open up and miracles are going to happen, that's not going to happen…public safety is the most important thing." – Chief Mark Saunders
Mark Saunders to be officially sworn in as Toronto police chief today http://t.co/eY1326lBEZ pic.twitter.com/N8OVjcpwpp— Global News Toronto (@globalnewsto) May 20, 2015
A source has confirmed to the Canadian Press that the Toronto Argonauts have been sold to Bell and MLSE chairman Larry Tanenbaum. The official announcement is expected to be made on Wednesday. The deal includes a lease agreement for the Argo's to play at BMO field as early as next season, pending further renovations needed to accommodate the CFL field. The CFL team will play this season at the Rogers Centre. Their lease is set to expire after the 2017 campaign, although they have the option of leaving earlier if they choose to do so. Rogers, as part of the MLSE, is absent from the deal, and was reportedly uninterested in acquiring the CFL team. Rogers prompted the Argo search for a new home following its plan to install grass into the dome.
BREAKING NEWS: @TorontoArgos announcing new ownership Wednesday with @Bell, Larry Tanenbaum. http://t.co/BhueotCU1U pic.twitter.com/Jv4oIL8WD7— SportsCentre (@SportsCentre) May 19, 2015
33.8 million defective airbags now represent the largest auto recall in history. Auto parts company Takata Corp. bowed to pressure from US safety regulators after it was revealed that the chemical which inflates the airbags can explode, blowing apart a metal inflator which could spew shrapnel in the passenger compartment. The faulty inflators are responsible for six deaths. The National Highway Traffic Safety Administration has established a website allowing owners to search for their vehicle to see if it's affected. Transport Canada has no reported complaints at this time.
Toronto Public Health says as as a precaution going into summer, they're monitoring ticks for Lyme Disease. Jennifer Veenboer says blacklegged ticks being tested were taken from Rouge Valley, Morningside Park and Algonquin Island, sent to the National Microbiology Laboratory. She says the city has found blacklegged ticks last year and this spring from those locations.
TPH is stressing that the risk of Lyme disease in the city is low, but the public can take the following precautions:
– Wear closed-toe shoes, long-sleeved shirts and pants
– Pull socks over pant legs
– Wear light-coloured clothes to spot ticks easier
– Use insect repellent containing DEET (active ingredient to keep bugs away) or Icaridin (always follow directions)
– Shower or bathe within 2 hours of being outdoors to wash away loose ticks
– Do a daily "full-body" check for ticks on yourself, children and pets
To reduce the risk of ticks living near your home, they recommend keeping your grass mowed, remove leaf litter, and keep firewood piles away from your house. For more information on the prevention of Lyme disease, click here.
It's back to the bargaining table today for striking high school teachers in Ontario. A new mediator is joining the discussions, a move the union is pleased with. Meanwhile, board officials in Durham – where the strike is into its 5th week – are looking at scrapping exams and using the 2 weeks they'd normally be held to pack in as much lost curriculum as possible.
Tolls Suggested For Non-Torontonians To Use Gardiner
If one Toronto Councillor has his way, those who don't live in the 416 would be paying a toll to use the Gardiner Expressway. A decision on what to do with the eastern portion of the highway is due to be made this summer, and James Pasternak says council should look at having non-residents pay to use it. He says people who live in the city already pay their share through property taxes and they shouldn't be asked to pay even more. Pasternak says if that doesn't get the thumbs-up, maybe it's time for the province to take over both the Gardiner and the DVP. At it's next meeting, council will debate whether the elevated portion of the Gardiner should be torn down, or go ahead with the hybrid option which will maintain a link to the Parkway.
Canadians Coping With Record Debt Levels
Debt is still an issue for Canadians but they are managing their finances carefully. A Fraser institute study shows the advance of household borrowing has slowed in the last 6 years and that the impact of servicing debt has low impact on their income. Shifting their borrowing to mortgages instead of consumer credit has helped Canadians to benefit from lower interest rates. Stats-Can reports showed Canadians owed about $1.63 in mortgage and consumer credit for every dollar of disposable income in the fourth quarter of last year.
David Letterman's Final Show Airs Tonight
It's the end of the line for David Letterman. After 33-years on late night TV, he airs his las show tonight. Executives are promising a jam packed show with all kinds of special guests.


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The Virtual Board Room: Veeam CEO Ratmir Timashev with Greg Stuart
Recently I was able to sit down with Veeam CEO and co-founder Ratmir Timashev. I was impressed with the ease of which he answered the questions I asked him and he seemed to really be at the helm of this company which is something I don't think you always get with CEOs. He's not the corporate suit type, he came across as knowing his customer base and understanding where Veeam is going and where he wants Veeam to be. I think you'll enjoy the look at Veeam from their CEO's perspective.


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Dog adoption saves lives. Adopt a dog and you'll have a friend for life! Contact us, or contact another local humane society, animal shelter or SPCA.
"Never doubt that a small group of thoughtful, committed citizens can change the world; indeed, it's the only thing that ever has". --Margaret Mead
All known information about an individual dog is provided in it's listing. We do our best to provide accurate information, but adopters must understand that each home is different and the dog may behave differently in a new home. Dogs are creatures of their environment and you help make the dog what it will be.
Homes considering adopting a puppy must be prepared for:
1.) Flexible schedules for potty training. Puppies can only "hold it" for one hour per month of age (ex. a 4 month old puppy can only go 4 hours without a potty break).
2.) Crate training until the puppy is at least one year old, to prevent chewing on inappropriate things when you can't supervise.
3.) Socialization: The more positive and varying experiences as a puppy the better, both in and out of your home.
4.) Puppy behavior and life stages are equivalent to a human toddler. It takes at least a full year to have a calmer, well adjusted dog. Patience is required and when your dog's behavior is a positive experience for you and those around you, your patience will be rewarded tenfold, for years to come.
Please do not consider adopting a puppy if you have not thoroughly thought through the pros and cons of having one. So many people end up returning them after 3-5 months because they didn't realize the amount of work involved in raising a puppy.
Patience, appropriate toys, socialization, and obedience training are all musts. All are time consuming, and can be expensive.
All Dogs Require:Supervision with children and obedience training.
Adopters that want to have good dogs must be prepared to put the time and effort into training a dog. Any dog requires work and effort, but a well t rained, well socialized dog is more than worth the effort put in to get them there.
Why should you adopt?
Dog adoption and cat adoption saves lives. Adopt a dog or adopt a cat and you'll have a friend for life! What is the difference between adopting a dog or puppy versus getting dogs for sale or puppies for sale from a dog breeder? When someone is breeding puppies, they are creating new dogs who need homes. Some people are interested in a very specific breed of dog or puppy and they think the only way to find that specific breed is to buy a dog for sale from a puppy breeder. Yet animal shelters are filled with dogs who must find homes. So rather than buying a dog or puppy for sale from a dog breeder, we encourage people to adopt a dog or adopt a puppy at their local animal shelter, SPCA, humane society or pet rescue group.


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Hi AT LA,
I have a question. When I first saw the Anthropologie Cirrus duvet in this Small Cool
2008 entry I had to have it. But I don't have $200 to shell out. Do you or any of your readers know where I could buy a similar duvet that costs less?
Hi Emily--We haven't seen anything similar but we totally get why you want it, and why you want it cheaper. Readers? Anyone? For reference, here's the duvet on the Anthropologie site.


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|Reviews for It Worked
| BuzzCat chapter 1 . 3/9/2011
BuzzCat likes. BuzzCat enjoys reading this story muchly. BuzzCat also likes third person writing.
| redcarrot chapter 1 . 7/14/2010
Whoa! More more more, loved it. The idea with the Truth or Dare thing is absolutely brilliant. I guess the image of Gillian in Cal's pyjamas inspired me to draw...yes, that's what I plan to do. Thanks a lot for sharing your fantasies with other close junkies. 3
| LolaRedMuse chapter 1 . 6/15/2010
very nice n kinda funny :)
| lie-2-me-fan68 chapter 1 . 5/17/2010
I loved this story i wish they would really get together and that was an awesome idea to make it happen over a truth or dare game
| Longing for Oblivion chapter 1 . 5/14/2010
Freakin' AWESOME!
| Rugbygirrl chapter 1 . 5/11/2010
I love dirt pie! Almost as much as I love good Callian stories (which this definitely was)!
| locisvu82 chapter 1 . 5/11/2010
you know I really really really love Emily!
Good Work
| binhereb4 chapter 1 . 5/10/2010
absolutly wicked idea...a perfect game for those two to play...loved it
| StepFurther chapter 1 . 5/10/2010
hahaha:) WOW!
it's one of the funniest fics.
i LOVED it!
She immediately said, "Truth?"
He squinted his eyes and said, "Kiss me."
haha:) He always know what to do:)
| Angelikpear chapter 1 . 5/9/2010
Awesome! LOL I enjoyed the most last part! Gillian in the kitchen LOL
Are going on with this story. Aren't you?
| nikkidog chapter 1 . 5/9/2010
That was fun - I must admit I was hoping she came out of the kitchen with the shirt inside-out. Thanks for the laughs.


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Andrew's first letter
Dear Santa,
My nemesis. Well done on escaping my lair of painful death last year in time for present delivering. I must say though, it made me laugh in a most evil mastermind way when i discovered you had to resort to ramraiding Woolworths in order to leave at least something under the trees: board games and jumpers. You fool.
Anyway, other than that one off bit of evil, I am sure you agree that I have been a very good boy this year. Well, okay, I did kill my smurf, but i'm sure you saw the tv footage, and he/she blatantly deserved it. Quite.
So. Here's my list:
- A Swimming Pool that is hidden in the floor
- 5-10 Bloodthirsty Sharks
- Some of that stuff you just find too hard to resist.. what is it... oh yeah, Brandy
- A camcorder
- A new scarf
- A chimney trap
And if you could deliver them before Xmas eve in the usual arrangement, forgetting the scarf, and then delivering that on your usual route... that would be most acceptable.
Hope you've had a good year recovering, big brother.
See you soon,
Andrew Claus


text: SEATTLE, Feb. 11, 2014 /PRNewswire/ -- Omeros Corporation (NASDAQ: OMER) today announced additional positive data from its Phase 1 program for OMS824, the lead compound in Omeros' phosphodiesterase 10 (PDE10) program. This clinical trial evaluated the extent to which OMS824 binds to PDE10, an enzyme expressed in the region of the brain that has been linked to a wide range of diseases that affect cognition. In the latest cohort enrolled in this trial, OMS824 achieved a high of approximately 70‑percent engagement at PDE10 without evidence of extrapyramidal symptoms (EPS). These data further support the potential of OMS824 to have a high therapeutic index in the treatment of patients with schizophrenia, Huntington's disease and other cognitive disorders. Omeros is completing a Phase 2a trial evaluating the drug in patients with schizophrenia and will start enrollment in a Phase 2 trial for Huntington's disease this quarter. The results reported today were in healthy male subjects receiving OMS824 once daily for 10 days. Positron emission tomography (PET) scans were used to measure the binding activity of OMS824 to PDE10 in the brain on the last day of dosing. An average of 66‑percent occupancy at PDE10 was seen in the striatum, the region of the brain that plays a critical role in cognition. The drug was well tolerated in all subjects with mild somnolence as the main side effect. Since the maximally tolerated dose has not been reached, Omeros is now planning to evaluate a higher dose of OMS824 in the trial to determine whether an even higher level of PDE10 occupancy can be achieved. Effective inhibition of PDE10 is considered a novel and promising mechanism to treat central nervous system disorders. Intolerable side effects, particularly EPS, are an important limitation to PDE10 inhibitors currently in development, narrowing the therapeutic index (the ratio of the toxic dose to the effective dose) and likely prohibiting the evaluation of these compounds at higher levels of target engagement. The results in the current cohort along with previously reported data from this clinical trial indicate that OMS824 may not be similarly constrained by such side effects.


text:
Explore ID real estate listings on Homestat: The website contains information on houses for sale in ID, land for sale in ID, real estate websites for agents, ID real estate.Priest River real estate: Search homes for sale in Priest River, ID by using our listings page. Navigation is made easy using our filter options that allow you to select houses for sale in Priest River, land for sale Priest River. By clicking on "Details" button you will get detailed information on zip code demographics, nearby schools and day care information.What is HomeSTAT? Homestat.org is a vibrant, real estate search web site designed to attract consumers interested in the "community experience"and value of each property, without over complicating the search process. The website differentiates from other homes for sale websites by providing listings and statistical information on homes for sale in ID and Priest River, as well as Priest River real estate. Homestat maintains large statistical databases that enhance user experience when viewing houses in ID & Priest River. We also write articles on ID real estate market. The website provides an easy-to-navigate consumer portal that makes it fun to search for listings and explore the neighborhood. Consumers can quickly search over thousands of listings, with relevant search results. View local real estate trends and compare your home to other Priest River real estate listings and houses for sale in ID. Our unique design and layout is a combination of website and software and designed to interact with the user. We invite you to enjoy our large selection of Priest River and ID houses for sale. HOME FOR SALE Large modular family resort home on approximately one & one quarter acres. Be happy to come home!. Twelve miles north of Bishop in Chalfant Valley, California. Three bedrooms, two baths, with oak cabinets, dual pane windows, wood burning stove, propane stove. Two redwood decks, front and back. Large two car garage with sixteen foot door and electric door opener. Large metal carport, new storage building with rollup doors. Two wells, lots of water. Big lawn with automatic sprinklers. BLM land next door for recreational vehicles or horses, hunting, fishing or skiing. This home is well maintained and has good neighbors. Asking $225,000. More Real Estate in Priest River, ID
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ED Magedson – Founder
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kirby of fredericksburg8509 Indian Hills Ct. Fredericksburg, Virginia United States of America
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This report was posted on Ripoff Report on 03/20/2012 07:28 PM and is a permanent record located here: http://www.ripoffreport.com/reports/kirby-of-fredericksburg/fredericksburg-virginia-22408/kirby-of-fredericksburg-false-advertising-of-kirby-vaccum-cleaners-fredericksburg-virgin-856998. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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ED Magedson – Founder
Ripoff Report
Valuemags.comInternet United States of America
Does your business have a bad reputation?
Fix it the right way.
Corporate Advocacy Program™
Valuemags.com fraudulently places magazine subscriptions in your name, as a 3rd party. I received "Taste of Home" magazine which I did NOT order. I called them, they said "an agent ordered it for you." That agent is "Valuemags.com" they said they can't cancel the subscription since an agent ordered it, and they gave me their phone number 1-800-303-0344.
I called valuemags.com but they did not answer the phone. I submitted an email through their customer service page on their website telling them that I did NOT order this magazine, cancel it, and remove my info and do NOT place any further orders for me.
I also told them that I was reporting them to The Better Business Bureau for their fraudulent activities. I am also going to report them to my local law enforcement agency.
My Better Business Bureau complaint #94321948.
How outrageous is this? That someone can just buy your info and order things in your name without your permission?? I am assuming my information was sold to them by Glamour Magazine, as that is the ONLY magazine I have ordered online in the last several years, although of course Glamour Mag. denies this.
This report was posted on Ripoff Report on 10/26/2010 05:09 PM and is a permanent record located here: http://www.ripoffreport.com/reports/valuemagscom/internet/valuemagscom-fraudulently-internet-655536. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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1400/1420 Crist Isle Drive, Cheboygan 49721
See more homes for sale in Cheboygan, MI
Unique group of Black Lake lots. Great three sided view of Black Lake and N. Black River on the end of this private peninsula. Waterfront on three sides. Nice sandy lake bottom with plenty of spots for dock and beach. Great building site or get-a-way spot. Take a row boat ride to your island pic-nic spot. Seller financing available with acceptable credit!!
Property Information
- Status
- Active
- Address
- 1400/1420 Crist Isle Drive
- City
- Cheboygan
- Zip
- 49721
- Lot Size
- 2560.00
- Features
- Nat Gas Available
- Lot Dimensions:
- 160 x 126 +/-
- Zoning
- 401 Residential
- Water
- No Well
- Subdivision
- Plat of Crist Isle
- County
- Cheboygan
- Waterfront Desc
- Yes
- Body of Water
- Black Lake
- Taxes
- $361
Listing Video for 1400/1420 Crist Isle Drive, Cheboygan MI 49721
Mortgage Calculator
Listing Broker
Listing Courtesy of Coldwell Banker Schmidt Cheboygan
Office Address 10667 N. Straits Hwy. Po Box 217
Listings last updated: . Some properties that appear for sale on this web site may subsequently have been sold and may no longer be available.
Our Michigan real estate agents can answer all of your questions about 1400/1420 Crist Isle Drive, Cheboygan MI 49721. Real Estate One, Max Broock Realtors, and J&J Realtors are part of the Real Estate One Family of Companies and dominate the Cheboygan, Michigan real estate market. To sell or buy a home in Cheboygan, Michigan, contact our real estate agents as we know the Cheboygan, Michigan real estate market better than anyone with over 100 years of experience in Cheboygan, Michigan real estate for sale.
The data relating to real estate for sale on this web site appears in part from the IDX programs of our Multiple Listing Services. Real Estate listings held by brokerage firms other than Real Estate One includes the name and address of the listing broker where available.
IDX information is provided exclusively for consumers personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.
The data relating to real estate one this web site comes in part from the Internet Data Exchange Program of the Water Wonderland MLS (WWLX). Real Estate listings held by brokerage firms other than Real Estate One are marked with the WWLX logo and the detailed information about said listing includes the listing office. Water Wonderland MLS, Inc. © All rights reserved.


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Don't Do it Yourself: Why DIY Home Security Spells Trouble
Tuesday, January 6, 2015 by Jeff Bates under Monitoring, Personal Safety, Security, Top Tips
With outlets such as Pinterest and HGTV growing in popularity, there has been an uptick in do-it-yourself (DIY) ventures. Home security is no exception.
However, when it comes to home monitoring, taking the DIY route can put you and your family at risk.
Equipment Placement Concerns
More and more security companies are beginning to offer a DIY approach to home security. Bundled DIY packages (comprised of sensors, cameras, keypads and environmental detectors) cut costs for security companies, as installation and maintenance is left to the consumer.
But this doesn't negate the need for trained installation professionals. Though you may fancy yourself a "handyman," home security installation is much more than simply hooking up the technology.
Trained professionals understand an intruder's mindset, giving them the knowledge and knowhow to survey your home for optimal sensor and camera placement. With DIY home security, this process is often a guessing game—a dangerous one at that.
Improperly placed equipment can leave gaps in your security
It can also lead to an uptick in false alarms. For example, a temperature change of glass, pets, small children and other triggers can prompt false motion detector alarms. Professional design support ensures that no detail is overlooked and systems are reliable.
Compromising True Professional Monitoring
Professional home monitoring is a keynote element of full-service security companies. However, many DIY kit resellers neglect this very core necessity in their offerings.
Without professional monitoring, you're left to notify emergency personnel yourself if a problem arises. This can be high-risk in those situations where every second counts or when you are unable to personally respond.
DIY Maintenance Becomes a Solo Effort
Proper maintenance is key to the longevity of any home security system. When choosing the DIY route, you are left to handle this task alone.
Best vendors offer a maintenance package along with your service contract. A professional will come to your home to assess the state of your system. While there, they will:
- Check to see that batteries are functioning properly.
- Ensure that transmitted signals are being received by the monitoring center.
- Perform controlled tests on panic buttons.
- Check to see that sensors have not come loose.
- Repair any damaged or malfunctioning parts of the system.
With a DIY system, there's nobody to tell you if your equipment needs upgraded or maintained.
When it's time to make the choice, how will you decide? Share your thoughts in the comments below.
Image Source: David Goehring via Flickr


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Use our website's state-of-the-art search to pinpoint over 60s members who match your needs. Or just flip through listings and pinpoint people that you find attractive.
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You Have to Watch This Guy Ride His Dirtbike on the Ocean Like It's a Jet Ski
Robbie Maddison is your new hero.
There are certain things you aren't supposed to be able to do. Things like riding a motorcycle on the water for more than half a second. Robbie Maddison doesn't seem to think that the laws of physics should apply to him though, and he rode his dirbike on the freaking ocean.
Yes. On the ocean. And in waves.
How? Well, first, it has small boards on the side to give it some buoyancy and stability. Secondly, it's all about momentum. You have to stay on the power and let the special rear tire that looks like a paddle board do its job or else you'll be sinking.
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Maddison has said that this is one of the craziest things he's over done. He's right. This is insane. Get ready to be slack jawed.
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