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Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.
Citation: Barabas ME, Kossyreva EA, Stucky CL (2012) TRPA1 Is Functionally Expressed Primarily by IB4-Binding, Non-Peptidergic Mouse and Rat Sensory Neurons. PLoS ONE 7(10): e47988. https://doi.org/10.1371/journal.pone.0047988
Editor: Bradley Taylor, University of Kentucky Medical Center, United States of America
Received: August 17, 2012; Accepted: September 19, 2012; Published: October 25, 2012
Copyright: © 2012 Barabas 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: Supported by National Institutes of Health grants NS40538 and NS070711 to CLS. 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.
Sensory nerve terminals detect peripheral stimuli in order to discern touch, temperature and pain. These neurons make up a considerably heterogeneous population that can be classified into subgroups based on peripheral targets, functional properties and central projections[1]–[3]. Larger-diameter neurons (≥27 um) tend to have myelinated axons, as with Aβ and Aδ fibers in vivo, whereas smaller-diameter neurons (<27 µm) tend to have unmyelinated, C fiber axons in vivo [4]. Calcitonin Gene-Related Peptide (CGRP) expression and isolectin B4 (IB4) binding are two common histochemical markers used to define subpopulations of small-diameter neurons[1]–[4]. CGRP is expressed by peptidergic sensory neurons[3]–[6], whereas IB4 binds to α-D-galactose carbohydrate residues typically expressed on the plasma membrane of non-peptidergic neurons[4], [7]–[9]. In peripheral skin targets, IB4-binding neurons terminate superficially between keratinocytes within the epidermis, whereas peptidergic neurons terminate in deeper layers of the epidermis and dermis [10]. Centrally, peptidergic neurons terminate in the outer laminae (I and outer II) of the spinal dorsal horn and target spinal neurons that transmit nociceptive information to the thalamus or brainstem nuclei, regions that mediate the sensory discriminative aspects of pain[10]–[16]. In contrast, the non-peptidergic, IB4-binding population primarily terminates on interneurons within the inner lamina II of the spinal cord and target interneurons that express PKCγ[10], [10]–[14], [17]. Rostrally, the input from IB4-binding neurons ultimately projects to brain areas including the amygdala and hypothalamus, regions involved in affective components of pain [15]. Therefore, it is reasonable to hypothesize that specific receptors that transduce environmental or endogenous stimuli would distribute preferentially between peptidergic and non-peptidergic subpopulations in order to provide selective input to these diverse neural pain pathways.
The Transient Receptor Potential Ankyrin 1 (TRPA1) channel has been the focus of intense interest for its role in inflammatory nociception[18]–[21] and its potential function in transduction of mechanical and cold signals[20], [22]–[25]. Previous investigations of the distribution of TRPA1 among peptidergic and non-peptidergic (IB4-positive) neurons have largely employed in situ hybridization and immunohistochemical techniques, which have produced disparate results[18], [21], [26]–[28]. Some studies indicate greater TRPA1 expression in peptidergic, IB4-negative neurons [18], [21], [26], [27], while others found more TRPA1 in non-peptidergic, IB4-binding neurons[27]–[29]. For example, Story and colleagues [26] found TRPA1 mRNA in 3.6% of dorsal root ganglia (DRG) neurons from adult rat, and a majority of these TRPA1-positive cells expressed CGRP. On the other hand, Caspani and colleagues [27] reported that 28% of lumbar 3–6 DRG neurons from adult mouse contain TRPA1 mRNA but only 2–3% of these neurons were CGRP-positive. Discrepancies between studies may have resulted from the inherent limitations of in situ hybridization and immunohistochemistry techniques. For in situ hybridization, the presence of mRNA does not always accurately predict that the respective protein will be expressed, as RNA can have a high turnover rate and become degraded prior to translation. Immunohistochemistry can sometimes lead to false-positives due to non-specific binding of antibodies or false-negatives due to lower sensitivity of antibodies. Further, receptors might be retained in internal organelles and not functionally expressed at the cell membrane, as has been shown for TRPA1 [30]. The sensitivity of both mRNA and antibody staining approaches depends on the thresholds established for positive versus negative cells.
Calcium imaging combined with live cell markers is a useful approach to determine functional expression of receptors. However, inconsistent results have also been reported for the TRPA1 agonists, allyl isothiocyanate (AITC) and cinnamaldehyde (CINN). Investigators report rates as low as 3–7% [19], [31] to as high as 30% [32] for response to 100 µM CINN. Reports for AITC vary in kind, with 18 to 45% of neurons responding to 50 µM AITC [27], [31]. The discrepant results may be due to the wide range of agonists concentrations and duration of application used in these studies [19], [21], [21,32], the use of different species (mouse versus rat), or the duration neurons are maintained in culture.
Therefore, we set out to resolve the discrepancies in functional TRPA1 expression in identified populations of mouse and rat DRG neurons by using ratiometric calcium imaging and internally consistent parameters. We used lumbar 1–6 DRG ganglia because somata within these DRGs project to skin areas typically probed by behavior assays, such as the plantar hind paw. For both mouse and rat, we used similar isolation protocols, culture duration and media. We did not routinely add exogenous growth factors to the media because adult DRG neurons do not require growth factors for survival [33], and growth factors, such as nerve growth factor (NGF), have been shown to increase the functional expression of TRP channels, including TRPA1 [34], [35]. We identified populations of C fiber-type, small-diameter neurons in live cultures by using IB4-FITC as well as a novel CGRP-GFP mouse where GFP is expressed in CGRPα-expressing neurons. Under these conditions, functional TRPA1 is predominantly found in small-diameter neurons that are IB4-positive and CGRP-negative.
Materials and Methods
Cinnamaldehyde (CINN) and allyl isothiocyanate (AITC) were purchased from Sigma. The same lot was used throughout all experiments. Stock solutions of 100 mM AITC and CINN were made in ethanol and working solutions were prepared in extracellular buffer every 4–5 hours during experiments. Cells were superfused with CINN for 3 min or with AITC for 1 min.
Unless otherwise specified, experiments were conducted on male C57BL/6J mice, ages 2–4 months (purchased from Jackson Laboratories). One experiment utilized TRPA1−/− (KO) mice of both sexes (2–12 months old) in which the exons essential for the Trpa1 gene function were deleted [23]. These TRPA1 KO mice were back-crossed to the C57BL/6J background for over 10 generations. Other experiments utilized CGRP-GFP+/− (CGRP-GFP) mice, a gift from Mark Zylka [36] (males, 3–4 months old), and these mice were also created on a C57BL/6J background. Male Sprague-Dawley rats were purchased from Jackson Laboratories, ages 3–6 months old. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
DRG Culture
Mice were briefly anesthetized with isoflurane (Midwest Veterinary Supply) via inhalation and euthanized by decapitation. Lumbar (L) dorsal root ganglia (DRG) 1–6 were isolated bilaterally, unless otherwise specified, and placed into 1 ml Hank's Balanced Salt Solution (Gibco). After DRG extraction, 1 ml HBSS was replaced with Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F-12 (DMEM/Hams-F12; Gibco). The ganglia were incubated at 37°C and 5% CO2 with 1 mg/ml collagenase Type IV (Sigma) for 40 min, followed by incubation with 0.05% trypsin (Sigma) for 45 min. We used the same protocol for culturing rat DRG neurons except that 2 mg/ml collagenase Type IV (Sigma) was used. Ganglia were washed and resuspended in complete cell medium (see below), then dissociated into single somata via trituration through a P200 pipette tip. The neurons were plated onto laminin-coated glass coverslips and incubated for 2 hours at 37°C and 5% CO2 to allow adherence. Coverslips were then flooded with complete cell medium consisting of DMEM/Hams-F12, 10% heat-inactivated horse serum, 2 mM L-glutamine, 0.8% D-glucose, 100 units penicillin and 100 µg/ml streptomycin. Unless noted, no exogenous growth factors were added. For experiments that indicate the inclusion of nerve growth factor (NGF) in the growth media, 100 ng/ml NGF was added to the complete media and incubated with the cells overnight. Most calcium imaging experiments were performed 18–24 hr after cells were plated, with the exception of the experiments specifically conducted at earlier time points (4.5–8.5 hrs and 10.5–14.5 hrs) as noted.
Retrograde Labeling of Cutaneous Neurons
The medial center aspect of the plantar hind paw of C57BL/6J male mice was injected with 20 µL 1% WGA-Alexafluor594 in sterile saline (Invitrogen). One week after injection, the ipsilateral lumbar 3–5 DRG neurons were cultured as described above. Our prior studies show that in C57BL/6J mice, virtually all labeling from medial plantar hind paw injections occurs in L3-5 DRG ganglia [37]. Cells that fluoresced clearly above background fluorescence levels were targeted for calcium imaging experiments.
Calcium Imaging and Analysis
Calcium imaging was performed using dual-wavelength fluorescent calcium indicator FURA-2AM (Invitrogen). Isolated DRG neurons were loaded with 2.5 µL/ml FURA-2AM in extracellular buffer containing 2% BSA for 45 min at room temperature, followed by a 30 min wash in extracellular buffer. The extracellular buffer was composed of (in mM): 150 NaCl, 10 HEPES, 8 glucose, 5.6 KCl, 2 CaCl2 and 1 MgCl2 (pH 7.4, 320±3 mOsm). Coverslips with loaded cells were mounted onto a perfusion chamber and superfused with buffer at a constant rate of 6 ml/min using an AutoMate pressurized perfusion system. A 4-way manifold system with a 2 cm bath inlet provided immediate release of buffer and chemical agents into the superfusion chamber. This superfusion system allowed no dead space and minimized air bubbles in the lines. Identical tubing was used for each input line. Experiments were conducted at room temperature (23°±1°C). Fluorescence images were captured with a cooled CCD camera (CoolSNAP FX; Photometrics, Tucson, AZ). Metafluor imaging software was utilized in order to detect and analyze intracellular calcium changes throughout the experiment (Molecular Devices, Sunnyvale, CA). A ≥20% increase in intracellular calcium from baseline was considered a response. All neurons were tested with only one concentration of one agonist. At the end of each protocol, 50 mM KCl solution was applied to depolarize neurons, thereby allowing for identification of viable neurons from non-neuronal cells or non-functioning neurons. Neuronal viability of non-responsive cells was confirmed by response to 50 mM KCl. For mouse, neurons were considered "small" if the average somata diameter was less than 27 µm. Rat neurons were considered "small" if the average somata diameter was less than 30 µm.
IB4 Staining
Upon completion of imaging protocol, cells were incubated with 10 µg/ml isolectin B4 conjugated to fluorescein isothiocyanate (IB4-FITC; Sigma) for 10 min and then washed with extracellular buffer and visualized using FITC filters. Neurons were considered to be IB4-positive neurons if they had a complete ring of FITC stain around the soma perimeter.
Determination of CGRP-GFP-positive Cells
Small diameter cells from the CGRP-GFP−/+ mice were separated into CGRP-positive and –negative subgroups. Cells were deemed CGRP-positive if their GFP fluorescence was ≥1 standard deviation above background fluorescence. Background fluorescence was determined by fluorescence levels of wild-type neurons from the CGRP-GFP mouse line that did not express GFP (fluorescence due to autofluorescence only).
TRPA1 Agonists AITC and CINN Elicit Calcium Responses with Different Latencies in Adult Mouse DRG Neurons
Allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) are commonly-used exogenous agonists of TRPA1 [19], [21], [21], [26,32]. Figure 1A shows responses of mouse lumbar 1–6 dorsal root ganglia (DRG) neurons to AITC or CINN (both 100 µM). AITC induced an increase in intracellular calcium, reaching a peak within 58±2 sec from the onset of perfusion onto cells (Fig. 1B). Alternatively, CINN-evoked excitation was preceded by a longer delay with calcium peak occurring after 175±2 sec (Fig. 1B). This difference in response latency was not due to imperfections in our superfusion system since 50 mM potassium induced an immediate response for every application (Fig. 1A). The superfusion rate for all chemicals was consistently 6 ml/min, and switching stimuli to buffer only did not elicit a change in intracellular calcium, indicating that the stimulus-induced calcium increases were not due to mechanical stimulation. Responses to 50 mM potassium also served as a positive control for neuronal viability. Due to the differences in latency to response for CINN and AITC, we superfused cells with CINN for 3 min and with AITC for 1 min (both 100 µM) to ensure we included all responders in our analysis.
A. Representative traces of individual, dissociated lumbar 1–6 dorsal root ganglia (DRG) neurons from C57BL/6J wild-type mice during FURA-2 calcium imaging. Separate neurons were tested with 100 µM allyl isothiocyanate (AITC; 1 min; top trace) or cinnamaldehyde (CINN; 3 min; bottom trace) at 6 ml/min. Note that the latency to peak amplitude for CINN responses was longer than that for AITC, whereas responses to 50 mM K+ (30 s) occurred almost immediately after the start of superfusion. B. Average latency from the start of superfusion to maximum amplitude of response for AITC and CINN (3 culture preparations from 5–6 animals for each group). The latency between onset of superfusion and peak response for CINN was significantly longer than that for AITC (p<0.0001; t-test). C–D. Concentration-response curves for AITC (C) and CINN (D) for percentage of responders (left two panels) and peak amplitude of calcium responses (right panel). Neurons that exhibited a ≥20% increase in FURA ratio from baseline during agonist superfusion were considered "responsive" (3 cultures from 5–6 animals for each group; AITC: 446 total neurons; CINN: 281 total neurons). All neurons were tested with only one concentration of one agonist. For percentage of responders, the EC50 for AITC was 38.5 µM and that for CINN was 60.2 µM (both calculated from 10–1000 µM data). For peak response amplitude, the EC50 for AITC was 115.3 µM (calculated from 10–300 µM data) and that for CINN was 97.5 µM (calculated from 10–1000 µM data).
We performed concentration-response curves for each agonist. A ≥20% increase in FURA 340/380 ratio over baseline during agonist superfusion was considered a response, and neurons were tested with one concentration of only one agonist as depicted in Figure 1A. For percentage of neurons activated, the EC50 for AITC was 38.5 µM whereas that for CINN was 60.2 µM. (Fig. 1C, D). Both compounds activated a maximum percentage of neurons at approximately 300 µM. We defined the magnitude of response as the amplitude of the increase in Fura-2 ratio from baseline evoked by each stimulus. For magnitude of response, the EC50 for AITC was 115.3 µM, whereas that for CINN was 97.5 µM.
Next we determined the size distribution of the somata of lumbar DRG neurons that respond to AITC and CINN. While a number of studies indicate that large neurons do not express TRPA1 immunoreactivity[24], [28], [38]–[40], prior data from our laboratory has shown that the terminals of some cutaneous Aβ fiber low-threshold neurons express TRPA1 protein, and mechanical responsiveness of several types of cutaneous Aβ and Aδ fibers are altered in global TRPA1-null mice [41]. However, here we found that very few neurons greater than 27 µm responded to either AITC (6.3%; 2/32) or CINN (3.6%; 5/140; Figs. 2A, B) and therefore, we focused the rest of the experiments on small-diameter (<27 µm) neurons.
Distribution of somata diameters (1 µm bins) of lumbar DRG neurons in culture preparations from mouse or rat. Number of neurons responding to AITC or CINN (both 100 µM) are shown in grey bar portion, non-responsive neurons are shown in white bar portion, and total cells in each size bin are reflected by the top of the bar (sum of the grey and white bars). A. Mouse lumbar 1–6 DRG neurons tested with 100 µM AITC (3 cultures from 6 animals; 271 total neurons; 160 responders; 59% responders). B. Mouse lumbar 1–6 DRG neurons tested with 100 µM CINN (7 cultures from 7 animals; 493 total neurons; 160 responders; 32% responders). C. Mouse neurons labeled via retrograde tracer injected into the medial plantar hind paw. All neurons were taken from lumbar 3–5 DRGs ipsilateral to injection. Only labeled neurons were used for recordings. Thus all neurons in the graph were labeled from the hind paw plantar skin. Neurons were tested with 100 µM CINN (5 cultures from 10 animals; 131 total neurons; 43 responders; 33% responders). D. Rat lumbar 1–6 DRG neurons tested with 100 µM CINN (3 cultures from 3 animals; 312 total neurons; 80 responders; 26% responders).
To determine whether AITC and CINN at a near maximal concentration (both 100 µM) are specific agonists for TRPA1, we tested responses in small-diameter lumbar DRG neurons from global TRPA1-null mice [23]. AITC evoked calcium increases in only 4.3% (7/162) of small neurons, whereas CINN elicited responses in just 2% (4/193) of the neurons (Fisher's exact, p = 0.2386, n.s.). These data indicate that both AITC and CINN at 100 µM selectively target the TRPA1 channel protein on isolated DRG somata. Since CINN elicited slightly fewer responses in TRPA1-null neurons, we used CINN for subsequent experiments where only one TRPA1 agonist was utilized.
TRPA1 is Functionally Expressed Predominantly by Non-peptidergic, IB4-binding Neurons
To determine which populations of small-diameter neurons typically respond to TRPA1 agonists, we incubated lumbar DRG neurons from adult mouse with IB4 conjugated to FITC (IB4-FITC) after exposure to AITC or CINN. Examples of neurons stained with IB4-FITC are shown in Figure 3A. IB4-positive neurons were identified by a complete halo of FITC fluorescence around the perimeter of the soma. Approximately 50% of all small neurons from mouse lumbar ganglia stained positively for IB4 (56%; 339/601). Among all small neurons tested, 66% (158/241) responded to 100 µM AITC and 42% (155/366) responded to 100 µM CINN. Next, we sorted the small-diameter neurons into IB4-positive and IB4-negative. Among all IB4-positive neurons, 79% (125/159) responded to AITC and 72% (129/180) responded to CINN (Fig. 3B). Conversely, among all IB4 negative neurons, only 40% (33/82) responded to AITC and 14% (25/180) responded to CINN (Fig. 3B). Overall, approximately 80% of neurons responding to either AITC (79%; 125/158) or CINN (84%; 129/154) were IB4 positive. In addition, the IB4-positive responsive neurons exhibited a greater peak magnitude to both AITC and CINN than did the IB4-negative responsive neurons (Fig. 3C). These findings were consistent across ten independent experiments (ten separate animals and culture preparations). Thus, TRPA1 appears to be functionally expressed mainly by IB4-positive small-diameter neurons in adult mice.
A. Representative brightfield (left) and FITC (right) images of lumbar 1–6 DRG neurons from C57BL/6J wild-type mouse stained with IB4-FITC (60x objective). IB4-positive neurons were defined by a halo of FITC-labeling around the entire perimeter of the somata of small-diameter (<27 µm) neurons. The red arrow indicates an IB4-positive neuron, whereas the white arrow indicates an IB4-negative neuron. B. Percentage of small-diameter neurons responding to TRPA1 agonists (all 100 µM) defined by IB4-binding or CGRP expression. A greater percentage of IB4-positive neurons responded to TRPA1 agonists than the percentage of IB4-negative neurons (overall effect: Chi square p<0.0001; ***p<0.0001). Likewise, a greater percentage of CGRP-negative neurons responded to CINN than CGRP-positive neurons (*p<0.0350 Fisher's exact). CGRP data were generated from 3 animals in 2 cultures. C. Amplitude of responses to TRPA1 agonists (all 100 µM) of small-diameter DRG neurons defined by IB4-binding or CGRP expression. IB4-positive neurons responded with a greater peak average amplitude than IB4-negative neurons (overall effect: ANOVA p<0.0001; *p<0.05, Tukey). There was no difference between CGRP-positive and CGRP-negative peak amplitudes.
To confirm our findings using an alternate label, we used CGRP as a marker for the peptidergic subgroup in neurons cultured from a novel CGRP-GFP mouse line in which GFP expression is driven by the CGRPα promoter [36] (Fig. 4A). In this mouse strain, the majority (89%) of all neurons that were immunoreactive for a CGRP antibody (which recognizes both α and β CGRP isoforms) were CGRPα-GFP-positive [36]. Likewise, the majority (68%) of CGRPα-GFP-positive neurons bound the CGRP antibody [36]. The finding that not all CGRPα-GFP-positive neurons expressed CGRP immunoreactivity was likely due to greater sensitivity and detection of the GFP marker than the CGRP antibody [36]. First we found that there was no difference between the overall percentage of small neurons that responded to 100 µM CINN or mean amplitude of responses between neurons from CGRP-GFP mice (44%; 109/248; mean response amplitude 173±14%) compared to those from wild-type (C57BL/6J) mice (42%; 155/366; mean response amplitude 155±9%), indicating that the mouse strains, which are both on a C57BL/6J background, express similar levels of TRPA1 at the lumbar DRG level. Significantly more CGRP-negative neurons (48%; 85/176) responded to CINN compared to CGRP-positive neurons (33%; 24/72; Fig. 3B). Among small neurons that responded to CINN, 78% (85/109) were CGRP-negative and only 22% (24/109) were CGRP-positive. However, there was no difference in the average amplitude of response to CINN in CGRP-negative and CGRP-positive responders (Fig. 3C). The CGRP-positive responders exhibited a trend for larger responses, but this was not significantly different.
A. Representative brightfield (left) and FITC (right) images of lumbar 1–6 DRG neurons from the CGRP-GFP+/− mouse strain (20x objective). CGRP-positive neurons were identified by GFP fluorescence levels that were three standard deviations above the average autofluorescence levels of DRG neurons from CGRP-GFP+/+ wild-type mice. The green arrows indicate CGRP-positive neurons. B. Distribution of both CGRP expression and IB4 binding among all small-diameter lumbar 1–6 DRG neurons (248 total neurons). A total of 29% of small neurons were CGRP positive. Note that 50% of these (14.5%) were also IB4-positive. C. Percentage of small diameter neurons responding to 100 µM CINN defined by IB4 and CGRP labeling. A greater percentage of IB4-positive/CGRP-negative neurons responded to CINN than the percentage of responders with other staining combinations (overall effect: Chi square p<0.0001; ***p<0.0001, **p = 0.0019, Fisher's exact for 2-group comparisons). The majority of responders (66%; 72/109) were IB4-positive and CGRP-negative. D. Amplitude of responses to 100 µM CINN grouped by CGRP and IB4 labeling. The responses of IB4-positive/CGRP-positive were significantly greater than those for IB4-negative/CGRP-negative neurons (overall effect: ANOVA p = 0.0250; *p<0.05 Tukey post hoc test).
Next, to determine the overlap in IB4 binding and CGRP expression in small neurons, we used IB4 staining with a red fluorophore (IB4-AlexaFluor594) together with the CGRP-GFP mouse neurons. Among all small-diameter lumbar DRG neurons, a total of 29% (72/248) were CGRP-positive. Surprisingly, among the CGRP-positive neurons, 50% (36/72) were co-labeled for IB4 (Fig. 4B). There was a significantly greater percentage of IB4-positive/CGRP-negative neurons responding to CINN than neurons with other staining combinations (Fig. 4C). In fact, the majority of neurons that responded to CINN were IB4-positive and CGRP-negative (66%; 72/109). IB4-negative/CGRP-negative neurons exhibited significantly smaller response amplitudes to CINN than IB4-positive/CGRP-positive neurons (Fig. 3C). Togther, these data indicate that TRPA1 is functionally expressed primarily by IB4-positive/CGRP-negative neurons in mouse. These data also suggest that IB4 staining is a better predictor of neurons that are likely to respond to TRPA1 agonists than is CGRP expression.
Plantar Skin-projecting Neurons Express TRPA1 Primarily in IB4-positive Neurons
Lumbar DRG neurons are considerably heterogeneous with respect to their peripheral target projections, which include skin, muscle, bone, tendons and visceral organs. Many of the non-peptidergic, IB4-binding or MrgprD-positive neurons innervate superficial skin [3], [11], [12], [14], [42]. Because the plantar skin of the hind paw is a common region examined in behavioral assays and is an area used for ex vivo skin-nerve electrophysiological preparations, we retrograde-labeled DRG somata that project to this skin region with WGA-Alexafluor594. After 7 days, neurons from ipsilateral lumbar 3–5 ganglia were dissociated and cultured. Neurons that were strongly labeled with distinct, bright red fluorescence were imaged. The mean diameter of retrograde-labeled (cutaneous) neurons was smaller (20.8±0.5 µm) than those of mixed lumbar 1–6 DRG cultures (23.7±0.2 µm; compare Figs. 2B, C; t-test, p<0.0001). The difference in cell sizes was expected since most afferents innervating the superficial skin correspond to non-peptidergic (IB4-positive), small-diameter neurons [3], [9], [11], [12], [43]. Similar to lumbar DRG neurons with mixed peripheral targets, 38% (42/112) of all small-diameter cutaneous neurons responded to 100 µM CINN. Additionally, the mean response amplitudes were similar between cutaneous (159.4±16.9; n = 42) and mixed target responders (155.7±8.7; n = 154). As in our results with mixed target DRG cultures, significantly more IB4-positive cutaneous neurons (50%, 34/68) responded to CINN than IB4-negative neurons (18%, 8/44; Fisher's exact, p = 0.0007). Out of all cutaneous neurons responding to CINN, 81% (34/42) were IB4-positive. IB4-positive cutaneous neurons responded with an average amplitude (171±19%) that was not different than IB4-negative neurons (110±33%; t-test, p = 0.1588). These data indicate that TRPA1 is functionally present in neurons that innervate plantar hind paw skin and, as with DRG somata with mixed peripheral targets, most cutaneous neurons that express functional TRPA1 are IB4-positive.
The Pattern of TRPA1 Expression Differs between Mouse and Rat DRG Neurons
A frequent question is whether rat and mouse DRG neurons are similar. An optimal approach to compare species is to use identical techniques in the same laboratory to determine whether there are inter-species differences in the distribution of TRPA1 in sensory neurons. Therefore, we conducted calcium imaging experiments on lumbar 1–6 DRG neurons isolated from rat. The same culturing techniques and media were used, except twice the concentration of collagenase was used for chemical dissociation for rat DRGs than for mice (see Methods). We found a number of differences between rat and mouse sensory neurons. First, as expected, the distribution of soma diameters for all lumbar DRG neurons is larger in rat than mouse (Figs. 2D, B). The range of cell sizes for rat was 15–47 µm (median: 25.8 µm), whereas the size range for mouse neurons was 12–41 µm (median: 22.5 µm). Further, the size of neurons responding to CINN was significantly larger in rat than mouse (Figs. 2D, B). The rat neurons that responded to CINN had a mean soma diameter of 24.4±0.4 µm (median: 24 µm; n = 80), whereas the mean diameter of CINN responders from mouse was 20.6±0.2 µm (median: 20.4 µm; n = 160; t-test, p<0.0001). Based on the size of CINN responders in rat, we considered small-diameter neurons to be those less than 30 µm in diameter, compared to 27 µm in mouse. Mouse and rat neuronal populations had different percentages of responders and mean response amplitudes to CINN (Figs. 5A, B). Fewer total small-diameter neurons from rat responded to CINN, but the responsive neurons in rat had greater response amplitudes than those from mouse (Figs. 5A, B). In contrast to mouse where a larger percentage of IB4-positive neurons functionally express TRPA1 than the percentage of IB4-negative neurons, the percentage of IB4-positive and IB4-negative neurons responding to CINN was similar for rat (Fig. 5C). However, of the responding neurons in rat, the majority (68%; 51/75) were IB4-positive. The peak magnitude response for IB4-positive and IB4-negative neurons in rat was also similar (Fig. 5D). Interestingly, these differences between mouse and rat did not appear to result from fewer small rat neurons labeled with IB4 since significantly more small neurons from rat were IB4-positive compared to mouse (Fig. 5E; p<0.0001 Fisher's exact). Despite these details, the overall data indicate that most neurons that functionally express TRPA1 are IB4-positive in rat as in mice.
A. Percentage of small-diameter mouse and rat DRG neurons responding to 100 µM CINN. Small-diameter neurons for mouse were defined as less than 27 µm, whereas those for rat were defined as less than 30 µm in soma diameter. Significantly more small-diameter mouse neurons responded to CINN than rat neurons (***p = 0.0096, Fisher's exact). B. Peak amplitude of responses for mouse and rat small-diameter neurons to 100 µM CINN. Rat neurons had a greater response amplitude to CINN than did mouse neurons (***p = 0.0039, t-test). C. Percentage of mouse and rat neurons responding to 100 µM CINN defined by IB4 staining. For mouse, significantly more IB4-positive neurons responded than IB4-negative neurons (left bars). However, for rat there was no difference between IB4-positive and IB4-negative neurons (overall effect: Chi square p<0.0001; ***p<0.0001, Fisher's exact). Nonetheless, among the rat neurons that responded to CINN, the majority were IB4-positive (68%; 51/75). The mouse data set (left two bars) is the same data as previously shown in Fig 3B (middle two bars). D. Amplitude of responses to 100 µM CINN of mouse and rat neurons defined by IB4 binding. In mouse, the IB4-positive neurons responded with greater amplitudes than IB4-negative neurons. In contrast to mouse, there was no difference between the amplitudes of IB4-positive and IB4-negative neurons from rat (overall effect: ANOVA p<0.0001; *p<0.01, Tukey post hoc test). The mouse data (left two bars) is the same as shown in Fig 3C (middle two bars). E. Distribution of IB4 binding among all small-diameter neurons from mouse and rat. Significantly more neurons were IB4-positive in rat than in mouse (p<0.0001, Fisher's exact).
TRPA1 Function and Distribution Changes with Duration in Culture
Time in culture is another factor that may potentially affect response to TRPA1 agonists. We therefore recorded from mouse neurons at 4.5–8.5 hr, 10.5–14.5 hr, and 18–24 hr after plating. As the culture duration progressed, we found that a greater percentage of small-diameter neurons responded to CINN. The increase in responders over time occurred specifically among IB4-positive neurons, and functional TRPA1 levels stabilized by 10.5 hr after the neurons were plated (Fig. 6A). Although there were fewer IB4-positive neurons responding to CINN at the earliest time point (4.5–8.5 hr), we still found significantly more IB4-positive neurons with functional TRPA1 than IB4-negative neurons (Fig. 6A). The response amplitudes also increased as culture duration progressed, and this effect was specific to IB4-positive neurons (Fig. 6B). We also observed greater response amplitudes for IB4-positive than for the IB4-negative responders at 18–24 hrs, but not at earlier time points (Fig. 6B). There was no difference in the percentage of neurons that stained positively for IB4 at any time point (Fig. 6C), suggesting that the increase in IB4-positive CINN-responsive neurons is not likely due to changes in IB4-binding.
A. Percentage of small-diameter L1-6 DRG neurons from adult mouse responding to 100 µM CINN defined by IB4 binding and duration between plating and imaging cells (5–7 cultures prepared from 5–7 animals). The percentage of IB4-positive responders significantly increased between the 4.5–8.5 hr and the 10.5–14.5 hr time points after plating (overall effect: Chi square p<0.0001; ***p<0.0001, Fisher's exact). At the earliest time point tested (4.5–8.5 hrs), although there was a smaller percentage of IB4-positive neurons responding to CINN than at the later time points, there were still significantly more IB4-positive than IB4-negative neurons responding at this time (**p = 0.0005, Fisher's exact). In contrast, there was no difference in IB4-negative responders across any of the time points. The data set for 18–24 hrs (right two bars) is the same as that shown in Fig 3B (middle two bars). B. Average amplitude of responses to 100 µM CINN defined by IB4 binding and duration between plating and imaging neurons. The amplitude of responses for IB4-positive neurons increases between 8.5 and 18 hrs (overall effect: ANOVA p<0.0001; *p<0.05, Tukey post hoc test). There was no overall difference in the amplitude of responses for IB4-negative responders. C. Distribution of IB4 staining among all small-diameter neurons defined by duration between plating and imaging cells. There was no significant change in IB4 binding across any of the time points tested (Chi square p = 0.0735). The data set for 18–23 hrs (right bar) is the same as shown in Fig 5E (left bar).
Exogenous NGF has No Effect on Distribution of TRPA1 Expression
In all of the previous experiments, we did not use exogenous growth factors in our culture media since nerve growth factor (NGF) can alter responsiveness of a number of TRP channels, including TRPA1 [34], [35], [44], and adult DRG neurons do not require growth factors such as NGF to survive in culture [33]. However, this is a factor that varies between studies and might affect expression of TRPA1. Therefore, we investigated the influence of exogenous NGF (100 ng/ml, overnight) on the functional distribution of TRPA1 in vitro (DRGs from 3 mice in 3 separate cultures). We found that significantly more total small neurons cultured with NGF responded to 100 µM CINN (53%; 115/216) than those cultured without NGF (42%; 155/366; Fisher's exact, p = 0.0126). Response amplitudes, however, were not affected (155.7±8.7% without NGF; 155.6±10.4% with NGF). When grouped by IB4 binding, we found that significantly more IB4-positive neurons (79%; 93/118) responded to CINN than IB4-negative neurons (22%; 22/98) after exposure to NGF. These results were not different than our results without NGF exposure (IB4-positive neurons: 72%; 129/180; IB4-negative neurons: 14%; 25/180). Further, IB4-positive neurons treated with NGF overnight still exhibited greater peak response amplitudes than IB4-negative neurons (IB4-positive: 174.1±11.5% n = 93; IB4-negative: 77.5±15.5% n = 22; t-test p = 0.0002). These responses were not different from untreated neurons (IB4-positive: 171.6±9.5% n = 129; IB4-negative: 74.1±12% n = 25). Therefore, although more neurons functionally express TRPA1 after treatment with exogenous NGF, the distribution of functional TRPA1 is not affected. Even with NGF treatment, more IB4-positive neurons functionally express TRPA1 than IB4-negative neurons.
Here we set out to resolve the contrasting claims regarding the populations of DRG neurons that express functional TRPA1 channels. Using standardized conditions, our results demonstrate that the majority of DRG neurons from mouse that respond to TRPA1 agonists are small-diameter, IB4-positive and CGRP-negative. Similarly in rat, most neurons that functionally express TRPA1 were IB4-positive. Very few large-diameter neurons from either species responded to TRPA1 agonists. In addition, we found that culture duration affects TRPA1 function in that responsiveness to TRPA1 agonists increases specifically in IB4-positive neurons despite the lack of added exogenous growth factors.
TRPA1 Functional Expression Correlates Extensively with IB4 Binding and Less with CGRP Expression
Contrary to a number of previous studies using in situ hybridization and immunochemistry that reported high TRPA1 expression among peptidergic, IB4-negative neurons [18], [21], [26], [27], we consistently found that the majority of small-diameter neurons that respond to TRPA1 agonists are IB4-binding and CGRP-negative. Our results agree with those of Hjerling-Leffler and colleagues [29], Kim and colleagues [28] and Caspani and colleagues [27] who reported high TRPA1 expression in IB4-positive neurons from mouse and rat. Disparities on this matter appear to be due to the markers used in each study. Those studies employing IB4 binding have all found high overlap between TRPA1 and IB4 binding, whereas studies that examined only CGRP or Substance P expression concluded that TRPA1 is expressed by peptidergic neurons. Our study is the first to employ both IB4 binding and CGRP expression in live neurons that are tested with TRPA1 agonists. We find that IB4 binding is a better indicator of TRPA1 expression than is CGRP expression in mouse. Surprisingly, we found that half of the CGRP-positive neurons in mouse bind IB4. Therefore, CGRP and IB4 do not label exclusive populations in mouse lumbar DRGs and caution should be exercised when using IB4 to identify "non-peptidergic" neurons.
As in mice, the majority of TRPA1-responsive neurons in rats were IB4-positive. However, while 80% of IB4-positive mouse neurons responded to CINN, only 30% of IB4-positive neurons from rat responded. This difference was not due to a lower IB4 binding as more neurons were IB4-positive in rat than mouse. The difference is likely due in part to a lower overall responsiveness to TRPA1 agonists in rat.
IB4 Positive Neurons and TRPA1 both Play Key Roles in Mechanical Pain Behavior
Accumulating evidence suggests that IB4-binding neurons play particular roles in somatosensation and pain. First, in non-injured skin, Mrgprd-positive afferents, which comprise approximately 90% of IB4 positive cutaneous afferents, are critical for transduction of noxious mechanical, but not thermal, stimuli [45]. Second, IB4-binding neurons are especially subject to plasticity after tissue injury. They become sensitized to mechanical stimuli in a chronic model of sickle cell disease pain [46]. Additionally, mechanical hypersensitivity after nerve injury and muscle inflammation depend on IB4-positive nerve fibers [45], [47]. IB4-positive neurons have been shown to play a key role in hyperalgesic priming[48]–[50], whereby the inflammatory mediators NGF and prostaglandin E2 (PGE2) initiate activation and membrane translocation of PKCε selectively in IB4-positive nociceptors[50]–[54]. Hyperalgesic priming in IB4-positive nociceptors has been proposed to mediate the transition from acute to chronic pain [50], [54]. Our finding that TRPA1 expression increases selectively on IB4-positive neurons with time in culture is consistent with the idea that dissociation of DRGs may simulate a type of "injury" that DRG neurons recover from over time.
The finding that functional TRPA1 is preferentially expressed by IB4-positive neurons suggests that TRPA1 may mediate specific contributions of IB4-positive neurons to mechanical pain. Growing evidence suggests that TRPA1 plays an integral role in both the development and maintenance of inflammatory mechanical hyperalgesia [18], [40], [55], [56], and TRPA1 inhibition at the peripheral terminal reverses the mechanical sensitization of C fibers after inflammation [57]. Thus, the TRPA1-expressing IB4-positive neurons may be particularly important for conveying mechanical pain from skin. TRPA1 is a promising target for therapeutic intervention of inflammatory pain, and therapies targeting TRPA1 on IB4-positive neurons may reduce mechanical hyperalgesia without abolishing normal tactile acuity for patients that suffer from inflammatory pain.
The authors thank Dr. Mark Zylka for the generous gift of CGRP-GFP mice and Dr. Quinn Hogan for critical comments on the manuscript.
Author Contributions
Conceived and designed the experiments: EAK MEB CLS. Performed the experiments: EAK MEB. Analyzed the data: EAK MEB CLS. Contributed reagents/materials/analysis tools: CLS. Wrote the paper: MEB CLS.
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text:
Harrison - 'Brothel' Apron Front Creeper
Style Number: Brothel
Last: Harrison - 'Brothel' Apron Front Creeper
The Harrison "Brothel" Apron Front in Polished Black with Creeper eyelets, contrasting red welt stitching, and the infamous Devil Head leather sole. Also emblazoned inside is the "Preoccupied by Sin" emblem.
To read more about George Harrison please visit our HEROES section.

subset: commoncrawl


text:
Lily's mum is very lonely since Paul died. Then she gets a new boyfriend and goes off on holiday with him, leaving Lily to look after 6-year-old twins Bliss and Baxter and little sister Pixie.
All alone. Lily is determined to show everyone that she can handle everything on her own, without ony parents. But when their secret is very nearly discovered, they need a hiding place and quick. What could be better than taking the kids to a secret campout in the park? Plenty of space, no house to look after, and hopefully no one will guess that they are there.
This is a thrilling book and i would reccommend for girls age 9-15. It is very exciting and you just want to keep on reading to find out what happens to them. I would give this book 9/10.
Want to tell the world about a book you've read? Join the site and send us your review!


text:
malekmusic wrote:may i ask why there is no stop key? its so important. yes i know you can press the play key again to stop, but with a stop button you are able to return to your last starting point. without that stop key the whole transport section is not useful for me. another question: why am i not able to open to channel editor with that app, or at least to select a channel ?????
that beeing said, im so happy that app finaly came. unfortunatly its very laggy on my s4, but i guess you will fix that.
ezaz61 wrote:Hi
I installed it all on my PC.
I have my PC connected to a wifi router through a lan-cable.
when I connect my android Phone with the IP adress, it makes connection, but then it asks for a password.
What could that be?
Andi wrote:Got IC Pro installed without issue (just had to restart Cubase 7.5 after adding the SKI Remote device). On a Nexus 7 the app acts like the iPhone app - so no split screens which is a bit of a disappointment. I also don't appear to be able to drag/zoom the project window vertically, nor the mixer horizontally which would have been useful. That said, for the cost it's probably worth having just for the remote transport and cmd keys.
Surely it would make more sense if this was determined by screen resolution, and not physical size? The Nexus 7 2013 has a resolution of 1920x1200, yet there is only the smartphone display? Another way to look at it is, how many people have 7" smartphones, seriously?crohde wrote:The Nexus 7 has a screen size of about 7". Up to about 8" the smartphone version is displayed
Another way to look at it is, how many people have 7" smartphones, seriously?
Soapmak3r wrote:I tried the demo last night on my Samsung S3 and it seems very underwhelming to me.
Some things that don't make sense:
No ability to change the time line to 'bars + beats'.
The loop indicators don't lock to anything, making the ability to adjust them useless.
You cannot create new midi/audio tracks.
You cannot arm, or select or even see midi/audio tracks in the track view.
There is no stop button to allow you to return to the start of your recording.
Hitting 'back' does not take you to the last window, it always takes you to the 'select computer' window for whatever reason.
To me, it seems like TouchDAW is just as full featured as this, and costs about a quarter of the price...I was looking forward to this software, but am failing to see the incentive to buy it here.
Lydiot wrote:Am I understanding it correctly that my computer requires a wireless device to be installed and that iC Pro only communicates via a "direct" ad hoc to the computer running Cubendo?
In other words no way of using this app by running the tablet through the wireless router and the host computer running wired to the same device?
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text:
I'll keep this short and simple.
I suggest you grab a City Life map to help you in looking for these spots.
Da Hood
Chatham Road store
TST store (Shop 2112, Harbour City)
Tsing Yi store (Maritime Square)
For a complete list of stores and other information, click on this link.
Marathon Sports
Hong Kong
Toronto Sports
If you ask me, Da Hood and Catalog are two of the best places to go to in Hong Kong for your sneaker needs. Both carry Nike SB and limited releases. Fa Yuen St. in Mong Kok is worth visiting if you've never been there but I find it a waste of time since 95% of the 30+ stores there carry the same stuff.
There are extensive Hong Kong Sneaker Guides but the stores I listed above will hopefully satisty the average sneakerhead.
More stores soon...


text:
Is the burqa an expression of faith or a form of enslavement? French legislators have decided it is the latter.
France's lower house of parliament on July 13 voted 335 to 1 to ban veils that cover the face in public. Experts say it is likely to pass the French Senate in September, but the country's highest administrative body, the Council of State, says it may be found unconstitutional.
In some conservative Islamic traditions, women wear garments such as the burqa and niqab that cover almost their entire bodies, leaving either a mesh window or small slit for the eyes, in order to preserve modesty.
Of French people polled, 82% approved of the ban, according to The Pew Global Attitudes Project. Smaller majorities in Germany, Britain, and Spain say that they would support a similar law.
It's estimated that fewer than 2,000 women in France actually wear face-covering veils, but that hasn't stopped the law from stirring up significant controversy.
Earlier today Egyptian Islamic scholar Samir Khalil Samir welcomed the ban as an ...
... invitation for Muslims to strive for integration and to marginalize Salafi trends of opposition and conflict. Moreover, the burqa has no justification in Islamic tradition, it is merely Saudi Arabian custom which confirms chauvinism and the 'the woman's grave.'
It must be said that in many Muslim countries the burqa has been banned because (as in Tunisia) 'it is not part of our tradition,' in Turkey it is forbidden in the name of secularism. In Egypt, in November 2009, the late Rector of the Islamic Al-Azhar, Muhammad Sayyed Tantawi, the highest religious authority in Egypt, banned it, saying to students: 'The niqab is only a custom, it has no link with Islam, neither close or distant!' In February 2010, Egyptian Prime Minister Nazif, called it 'a denial of woman!'
But others point to civil liberties. John Dalhuisen, Amnesty International's expert on discrimination in Europe, condemned the vote:
A complete ban on the covering of the face would violate the rights to freedom of expression and religion of those women who wear the burqa or the niqab in public as an expression of their identity or beliefs.
An editorial that appeared this morning in The Daily Star, a Lebanese newspaper, admonished the French decision:
Instead of taking the easy way out by pandering to local fears and prejudice, lawmakers and politicians in Europe need to take a good look in the mirror.
According to Pew, 65% of Americans disapprove of a ban on Burqas.
How do you feel about the burqa? Should it be banned or protected by freedom of religion in the USA?
--By Benjamin Soloway, USA TODAY
Cathy Lynn Grossman is too fidgety to meditate. But talking about visions and values, faith and ethics lights her up. Join in at Faith & Reason. More about Cathy.
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text:
Home Collection Red burnout leaf cushion
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Item No.3290010511
This red burnout leaf cushion is styled in soft chenille and has a plain red fabric reverse. This luxuriously comfortable cushion has a plump filling and a removable case that is suitable for dry clean only.
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text:
M ontreal, QC—April 30, 2012 – In response to industry demand, respected independent global research provider, BCA Research, will launch a Geopolitical Strategy service on May 8, 2012. This new service, believed to be the first of its kind, will be headed up by BCA Senior Strategist, Marko Papic.
"While the BCA tradition of rigorous macroeconomic analysis has always incorporated geopolitical analysis, this new service – led by the experienced Marko Papic, will encompass the knowledge and expertise of BCA's existing 60+ research staff – combining first class macroeconomic research and geopolitical risk analysis into a single framework", says BCA Director of Research, David Abramson. "The resources committed to the GPS service have been structured to evaluate the risks posed and opportunities presented by geopolitical developments and their impact on longer term investment trends".
In a world menaced by uncertainty and rising geopolitical risks, this new BCA Research service will provide actionable investment strategy against the backdrop of current geopolitical themes including unique and custom strategies adapted to specific tail-risk scenarios. With a background in private intelligence and academia, Senior Strategist, Marko Papic, will avoid bias – instead relying on the fundamental analysis based on constraints and necessity.
"Our market research has determined there is tremendous potential for this service. While 80% of clients surveyed responded that the impact of Geopolitics on their general investment decisions was "important" or "very important", over 65% confirmed "investment implications" were missing from their current source of Geopolitical analysis", states Nanci Murdock, BCA's Marketing Manager. "The feedback from March and April beta reports has been overwhelmingly positive – we are delighted to officially launch this product into the marketplace."
Click here to learn more about the BCA Geopolitical Strategy.


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My father died February 26, 2013. His brokerage account was registered in the name of his revocable living trust for which he was sole trustee. I am the personal rep of the estate, the sole successor trustee of the trust, and sole beneficiary. Between the date of his death and the date when the stocks in the brokerage account were transferred to my name, the only income was $1,800 of dividends in the brokerage account and a $402 state income tax refund. The brokerage firm reported these dividends under my father's SS number and included them with $200 of dividends received in 2013 prior to his death. Is this proper? What income do I report on the 2013 income tax returns of my father, the estate and/or the trust? Thank you so much for your advice.
You really should see an accountant, who can determine which returns need to be whether they should be filed under his social security number or if there needs to be a separate Estate or Trust Tax ID number established. It's money well-spent.
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Here's the mission statement of San Fran's Honey Soundsystem crew: "We are a collective of disc jockeys, musicians, performers and designers. We bond over our shared love of timeless sound, iconic imagery, the complete look, indulgent behavior and most of all, attractive men." They've been stirring the Bay Area's underground pot via their long-running night at the Holy Cow for years, and tonight they're heading east to make their Bunker debut. Resident Mike Servito spins as well, as do Ryan Smith and Ron Like Hell from the super-fun Wrecked affair.
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Rewiring a replacement light fitting
Hi all,
I tried to replace a 6ft Florescent batten at weekend but ran into trouble when putting all the wires back.
Here is my setup.
1 Florescent batten on kitchen ceiling
2 spotlights on kitchen ceiling
4 terminal blocks
1 single switch - operates the 6ft light.
1 double switch - operates the 6ft light and the spot lights.
Coming from ceiling rose I have
5 red wires (live)
3 black (neutral possibly one switched also but isnt marked)
5 earths
1 cut of cable with L N and Earth on either side to finally wire into the 3 inputs in the 6ft fitting.
There is also a single black cable coming down into the 6ft fitting on its own. I appears to be coming across from the spotlights. Its definately not part of the ceiling rose bunch.
Any ideas on what colours/wires go into what connectors?????????
Is it possible to just have one black cable coming from the spolights????
Were is the neutral and earth gone for the spolights???????
Any advice is welcome
I think you are in UK possibly. This site mainly North America.
Yes I live in N. Ireland. I know that there is three other rooms looping from this kitchen fitting. Connected the terminals as follows:
5 reds into 1st terminal
5 earths into 2nd terminal
3 black into 3rd terminal
1 black (coming from spotlights) into 4th terminal.
When the power came back on I could turn on/off the spolights but not the 6ft fitting.
I think I have used the black spotlight cable as the live switched cable causing my problem.
If I knew were that seperate black cable coming from the spotlight goes and what one of the three blacks are the switched live I would be sorted.
Search for "frenchelectrician" on this site/forum and send him a PM. He can and will help you. He is our resident European expert.
Thanks J.V. for " hollering me "
Keep in your mind I will be using UK/ Ireland terms here.
Now let get to the bussiness how may one way switch and two way switches ?
that will make the diffrence of how you snagged the live and switched conductors.
Yes I am aware with old colour codes which you have them right now.
But I have a huntch you have two way switches for your kitchen luminaire but seems you got the live and switched conductor crossed.
so you have 5 red and 3 black that I am reading right on your ceiling rose ? if so then you will need to look for two more conductors I am pretty sure they are remaked or see a extra conductors running thru the ceiling rose.
Look at the flourscent luminaire and spotluminaire you should see total of 4 conductors I know red will show up there black ya freind it will be there for netural green/yellow stripe conductor that for earthing and a spare conductor possiblty brown or grey one of the two on older colour chart IIRC. unless it was double T&E { twin and earth } cable that can compound it a little.
At the same time look at the double swtiches check it out if you have switch loop there or not that can really ruin your day on that one.
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text:
Track My
UPS Order
How Does the Traverse Bay Farms' Buyer Reward Points Program Work?
The Traverse Bay Farms Buyer Reward program is
designed to reward our customers with free goodies.
You will earn Buyer Reward Points on every online purchase. Every dollar spent (not counting shipping or tax) earns one point. Every 350 points earned can be redeemed for $10.00 off any purchase. Points are earned on every product on our website.
Our points schedule:
350 point level - Earn a $10 coupon
700 point level - Earn a $20 coupon
1050 points level - Earn a $30 coupon
You earn free stuff faster with our Buyer Reward Points program because we round up to the nearest whole amount.
Here is an example:
You purchase two bottles of Fruit Advantage Tart Cherry Joint Formula for $31.77. Your account will be credited 32 points, not just 31.77 points. This means you earn free stuff faster!
If you have any questions about the Traverse Bay Farms Buyer Reward Points please contact us toll-free at 1-877-746-7477.
(The fine print.) Traverse Bay Farms reserves the right to modify or discontinue the Buyer Reward Points program at any time. Points earned on items that are later returned will be removed. Points will not be earned on the portion of an order paid for with redeemed points. Points will be earned only on the initial purchase (first month) of any of monthly express programs.
A portion of profits and products are donated to support non-profit organizations supporting arthritis research, diabetic research, Lou Gehrig's
disease and Epilepsy research.


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Saskatchewan Government Insurance signs an agreement with Canadian Business Intelligence leader SQLiaison
May 12, 2010
SQLiaison, the Canadian leader in Insurance Business Intelligence (BI), will be implementing a complete Insurance BI program with Saskatchewan Government Insurance (SGI), Saskatchewan's self-sustaining auto insurance fund and fully competitive property and casualty insurance supplier. Besides using the FellowDSS™ suite of analytic applications for the insurance industry, SGI also partners with SQLiaison for its BI expertise in implementing and supporting large scale Insurance BI Programs.
The FellowDSS™ solution is designed to adapt insurance industry BI concepts to the specific needs of clients such as SGI. In addition, users can compile relevant data and organize it in the form of reports or analytic applications. Such tools are essential for insurance decision makers and are used to drive competitive advantage.
"SGI and SGI CANADA have been successful for many years, and we want to build on that success and position ourselves as an innovative, customer-focused company," says Don Thompson, Chief Financial Officer, SGI. "Collecting and analyzing information is critical to an insurance company's success. More effectively using this data in making business decisions will increase customer service and profitability. The innovative solutions offered by SQLiaison will play an integral role in this process, as well as contribute to our overall efficiency and productivity efforts to ensure we meet the growing needs of our customers."
SQLiaison's solutions allow companies to make tactical and strategic decisions using the large amount of data they gather on a regular basis. Insurance professionals need easy and timely access to accurate, complete and integrated information. "The complex environment insurers evolve in requires complete, credible and usable information in order to make strategic decisions," said Philippe Torres, President of SQLiaison. "Business Intelligence is a key solution because it enables access to information in any format, no matter where that information comes from."
"The FellowDSS™ suite is designed to be a customizable solution. SGI's version will be entirely built to meet their requirements and needs," said Mr. Torres. "We further continue our market penetration within Canada and we are proud that SGI, one of the country's biggest property and casualty insurers, trusts our solutions and services. No one has our experience and expertise in insurance BI."
About SGI CANADA
SGI CANADA has been in business for more than 60 years, selling property and casualty insurance in seven Canadian provinces. It operates as SGI CANADA in Saskatchewan; SGI CANADA Insurance Services Limited in Alberta and Manitoba; Coachman Insurance Company in Ontario; and the Insurance Company of Prince Edward Island (ICPEI) in New Brunswick, Nova Scotia and Prince Edward Island. Saskatchewan Government Insurance (SGI) is the province's self-sustaining auto insurance fund.
About FellowDSS™
FellowDSS™ is an enterprise-scale suite of analytic applications for the insurance industry. It is built around an integrated data warehouse that stores transactional data from a company's operational support systems. FellowDSS™ consolidates and integrates data from across the various systems of a company, and makes it easily available to users for consumption through report objects either pre-defined or in an ad-hoc mode.
About SQLiaison
SQLiaison is the Canadian leader in Insurance Business Intelligence (BI) solutions. Expert at quickly leveraging data, SQLiaison seamlessly and powerfully creates business advantage for its clients. In Canada, $4.88 billion Property & Casualty Direct Written Premiums are analysed through solutions designed and implemented by SQLiaison's experienced team. Its clients add up to an impressive roster of Canadian insurance companies, prominent banks, pharmaceutical companies and other large Canadian corporations. SQLiaison has offices in Toronto, Montreal and New York. Visit sqliaison.com for more information.
Media inquiries
Kelley Brinkworth
Manager of Media Relations
306-535-6207 (cell)
Pierre Breton
Director of Marketing
Corporate Communications
514-333-6600 ext. 122


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That's what a new Missoula-made T-shirt reads, along with an image of Max Baucus in a cowboy hat (see below).
The origins of the T-shirt are a bit clandestine. Ours was dropped off this morning with few details. The person responsible doesn't want credit and doesn't know yet where fellow anti-Baucusers can pick them up.
"Just tell people they'll see them around," he said vaguely.
Will do.


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Ben Affleck is overjoyed with the newest addition to the Affleck-Garner household - his 7-month-old son, Samuel.
Access Hollywood's Shaun Robinson caught up with actor-director while promoting his new movie, "Argo," where he beamed about his little boy.
PLAY IT NOW: Ben Affleck: Argo Is 'Really Special'
"I've just gotten to a place where it was hard to even imagine I would get to," the actor, who said he's "lucky" and "blessed" with his family and career, told Shaun.
"I've got a teammate," he said of his son, who wife Jennifer Garner gave birth to in February. The couple also are parents to daughters Violet, 6, and Seraphina, 3.
VIEW THE PHOTOS: Hollywood Dads & Their Adorable Little Ones!
"It's too early on to get a sense of [his] personality, but it's very exciting. It's a pretty cool thing. As I said, very lucky," he continued.
And does little Samuel have Red Sox pride like his dad?
"I try to resist the temptation to make my kids like props for sports teams' jerseys, but it's hard," he told Shaun.
VIEW THE PHOTOS: Jennifer Garner: From 'Alias' To Moviestar & Supermom
Ben also weighed in on his wife's recent "wonder sperm" comment on "The Ellen DeGeneres Show," something that took him by surprise.
"I like the role reversal now. It's usually me that says the outrageous thing, and they ask her the question and she has to justify it... is that what she said? Really? Wow!" he told Shaun.
VIEW THE PHOTOS: Ben Affleck & Jennifer Garner
"That's definitely... I'm going to put that on the poster for 'Argo,'" Ben joked about his '70s Iranian hostage-themed movie.
"Argo" opens on October 12.
-- Jesse Spero
Copyright 2012 by NBC Universal, Inc. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


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|Thread Tools
SBC Crate Motor
Hey Guys,
I will be putting in a SBC in my project soon and I wanted to get everyones opinion. I am planning on buying a Crate Motor, definitely a SBC. I used an old 350 engine block to mock everything in place. Considering I want 350-400 HP and I still want to get good enough mileage to drive everyday, what are some good options? Any thoughts on carb vs. FI? I have looked at the Ram Jet 350, but I have not heard enough about it. Are there any other FI options? What is the word on Strokers? Will it bolt in place using the motor mounts I used for the Mock up? Any contributions would greatly be appreciated.
Thanks for your help!
Brian Hix
Your small block chevy mount-up was a good start. from there you could go with a 383 if you wanted to.
In my personal opinion, I'd go with carburetion instead of injection, because it requires less "computer interaction" and its just generally easier to set up.
If you're insisting on buying a crate motor, you're gonna spend at least $4K to get 350 HP out of it.
Look at ZZ4's with upgrades to reach that kind of output.
I was torn between buying a crate and doing it the easy way, and trying to build my own. I chose to build, and it was the best decision I ever made in my life. I learned far more than I ever expected, and got way more than what I would have if I paid GM to put it together for me.
Remember a ZZ4's gonna cost you like $2500 and it doesn't even have forged pistons or a balanced rotating assembly!
My advice is to build, rather than buy. But I'm old fashioned like that.
Good luck, whatever your decision turns out to be!
It really depends on what your looking for as far as mileage. You can still get decent mileage on a carb that is tuned in. But if your truely concerned FI is the way to go. Done properly its actually quite easy to setup. Most the hardest way is to rip it off another car and retrofit it to yours. But many companies sell complete kits. These things can make some serious power and still maintain great MPG ratings. If you go FI tho, go with a multiport because in my opinion, TBI is a waste of time.
Like was said above, you will pay a lot for a crate engine in that power range. Depending on your level of knowledge and comfort with engine work, I'd build instead of buy. That way you can truely make it your unique setup.
Bringing history and technology together.
IF YOU need great milage and HP then you need overdrive
|Recent Hotrodding Basics posts with photos
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Seattle Likely To Get NHL Team Before NBA? KU Announces Memorial Stadium Plans Facility Notes Toyota Stadium Renovations Exceed $40M Budget Timbers Agree To Providence Park Expansion Deal DC United Gets $120M To Help Pay For Stadium Facility Notes New A's Ballpark Would Be Financial Boon For Oakland San Diego State Regroups On Football Stadium Talks Illinois Postpones Football Stadium Upgrades
Upcoming Conferences and Events
SBD/November 13, 2013/Facilities
Univ. Of Arkansas Announces Plans For New $25M Basketball Practice Facility
Published November 13, 2013
UCONN HO! In Hartford, John Altavilla notes UConn is "considering soliciting naming rights for its basketball practice facility, which is expected to cost" between $35-40M and open in the spring of '14. IMG would "solicit corporations to seek interest in those rights." This likely means the venue "won't be named for any of the private donors who contributed to it." If UConn "chooses a corporation, it would be a break in tradition" from some of the other names on campus athletic facilities (HARTFORD COURANT, 11/13).
STAYING CLASSY: In San Diego, Matt Calkins notes San Diego State Univ. yesterday "announced its plans to build a state-of-the-art practice center for the men's and women's basketball teams." The privately funded, $14.5M facility is "projected to be ready-to-use by 2015." The SDSU basketball teams currently share "with the students at the Aztec Recreation Center when Viejas or Peterson Gym is in use." Men's basketball coach Steve Fisher has been "pushing for such a venue since the turn of the millennium" (SAN DIEGO UNION-TRIBUNE, 11/13).
RAISING CANE: In Miami, Susan Miller Degnan reports the Univ. of Miami is initiating a $7M fundraising effort to "build a lighted artificial turf football practice field, dining hall for athletes and four cold pools for cooling down and recovering after workouts." The project is "being called the Football Victory Fund," and the construction timetable is not yet known (MIAMI HERALD, 11/13).


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Sept. 26, 2012
PITTSBURGH--Pitt students will have the opportunity to showcase their basketball skills when the Oakland Zoo and Pitt Basketball program host their first-ever 3-on-3 charity basketball tournament on Sunday, Sept. 30. Tipoff is set for noon at the Petersen Events Center. All proceeds from the tournament will benefit the local Big Brothers Big Sisters Club of Greater Pittsburgh. Pitt Head Coach Jamie Dixon and members of the Pitt men's basketball team will be present throughout the day.
"We were searching for an opportunity to promote wellness and healthy lifestyle among our students at Pitt," Dixon said. "This is also a great opportunity for us to raise money for charity, thank our student body and team with the Oakland Zoo on this fantastic event. Over the years, the Oakland Zoo has been one of the nation's top college basketball student cheering sections. Their support for us is unmatched and that's evident in this event."
"When the idea for the 3-on-3 tournament was developed, we could not have been more excited," Oakland Zoo leader Joe Lassi said. "It's the perfect way to raise awareness for the Big Brothers Big Sisters organization and play the game of basketball that we all love. We are happy to be working with Coach Dixon and the team and love the fact that they are assisting us on this event."
The 3-on-3 tournament will consist of 32, four player teams (three players, one sub to use interchangeably) in a double elimination bracket format. Teams will play throughout the day in the Petersen Events Center main floor and auxiliary gym. The two teams advancing to the championship game will have the opportunity to play the title game during halftime of a men's basketball contest. That date has not been determined.
Prizes for the winning team will include a tournament trophy, four free 2012-13 Oakland Zoo tee-shirts, autographed Jamie Dixon basketballs, a chance to enter a Pitt men's basketball game of their choice before other students and a behind the scenes Pitt basketball experience. The runner-up team will receive four free 2012-13 Oakland Zoo tee-shirts and have a chance to enter the Oakland Zoo prior to other students at a Pitt men's basketball game of their choice.
Doors open to the public (students and non students) at 11 a.m. on Sunday. General admission for the event is $1 per ticket. All collected admission fees will be donated to the Big Brothers Big Sisters of Greater Pittsburgh. The Oakland Zoo will also be there to collect any other donations to the charity.
All 32 team spots have been filled. Registration on the day of the tournament will begin at 11 a.m. and end at 11:30 a.m. for all participating teams.
George Signs Financial Aid Agreement with Pitt
Artis & Young Ink NBA Summer League Deals
Pitt Adds Combo Guard Stewart to 2017 Recruiting Class
Pitt Announces Basketball Staff Changes
Pitt Men's Basketball Season Tickets Now on Sale


text:
The press event kicking off the 2013 Nice Ride Minnesota expansion was a major success! Every news station and media outlet featured today's ride and rally! Check out the coverage on 5 Eyewitness News, FOX 9, Star Tribune, Minnesota Public Radio and CBS Minnesota. We're thrilled to be a part of the 4th season of Nice Ride Minnesota!
Nice Ride Minnesota, the popular bike-share program, celebrated the addition of 24 new stations today with a rally and kick-off ride at the Historic Fort Snelling. The 2013 expansion brings the total system up to 170 bike share stations and 1,550 bright green bicycles.
Bill Dossett, Nice Ride Minnesota's executive director, kicked off the event along with speeches from Paul Labovitz, Mississippi River and Recreation Area superintendent, Dave Spalding, Blue Cross and Blue Shield of Minnesota senior vice president of business development, Peter McLaughlin, Hennepin County commissioner, Jayne Miller, Minneapolis Park and Recreation Board superintendent and Minneapolis Mayor R.T. Rybak.
Thank you to the National Park Service, Blue Cross and Blue Shield of Minnesota, the City of Minneapolis, Hennepin County, the Minneapolis Park and Recreation Board and countless others for the success of this expansion, which brought thirteen new stations along the Mississippi River.


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The Asian Development Bank - Japan Scholarship Program (ADB-JSP) provides support for graduate (post-baccalaureate) studies in ADB-approved fields of study at the University of Hawai'i at Manoa (UHM), and for participation in the educational, residential, and leadership development programs at the East-West Center (EWC).
The ADB-JSP was established to encourage and strengthen human resource development in ADB's developing member countries. After completing their studies, scholars are expected to return to their home countries to apply and share their new knowledge and skills to promote socioeconomic development.
To make certain you are eligible to apply for the ADB-JSP Scholarship please read carefully all of the ADB eligibility requirements, criteria for selection, and Frequently Asked Questions on the ADB-JSP website and the EWC website.
The deadline for the 2017-2018 fellowship application has passed.
Fellowships are awarded annually in an international competition. To apply for study beginning in the 2018-2019 academic year, the deadline is November 1, 2017. Please see below for the 2017-2018 application materials, which are archived for your reference. The updated application will be posted sometime in early 2017.
Application Packet (includes program information, eligibility requirements instructions and forms) [pdf]
To download the application forms as separate files, click below:
- Eligibility Requirements [pdf] [doc]
- Steps for Submitting an Acceptable ADB-JSP application [pdf] [doc]
- Overview and Application Information [pdf] [doc]
- Application Checklist [pdf] [doc]
- Application Form Instructions [pdf] [doc]
- Application Form [pdf] [form-fillable doc]
- Cover Letter and Essays [pdf] [doc]
- Letter of Reference Form (Academic or Professional) & Instructions [pdf] [form-fillable doc]
- Letter of Reference Form (Civic Engagement) & Instructions [pdf] [form-fillable doc]
- Verification of Funds [pdf] [form-fillable doc] - provide only if you also want to be considered for the East-West Center Graduate Degree Fellowship
The forms below should be submitted with the East-West Center ADB-JSP application forms; see the UHM Office of Graduate Education website for these and other forms for the 2017-2018 academic year.
Attention: Applicants seeking admission to any Business program or to the Law program should not use the Graduate Admission Application Form. The application forms for these fields must be obtained from the respective programs.
- UHM Notice to Applicants
- UHM Graduate Admissions Application Instructions
- UHM Graduate Admissions Application Form (includes the UHM Credit Card Payment Form for those applying for independent consideration)
- UHM Statement of Objectives Form
Area of Study and Required Test Scores:
- Business Administration (Global MBA) --- GMAT and TOEFL or IELTS
- Business Administration (Global MBA - Japan Track) --- GMAT and TOEFL or IELTS
- Economics (MA) --- GRE and TOEFL or IELTS
- Geography (MA) --- GRE and TOEFL or IELTS
- Law (LLM Degree, a one-year program) --- TOEFL or IELTS
- Natural Resources and Environmental Management (MS) --- GRE and TOEFL or IELTS
- Ocean and Resources Engineering (MS) --- GRE and TOEFL or IELTS
- Oceanography (MS) --- GRE and TOEFL or IELTS
- Pacific Islands Studies (MA) --- TOEFL or IELTS
- Public Administration (MPA) --- TOEFL or IELTS
- Sociology (MA) --- GRE and TOEFL or IELTS
- Tropical Plant and Soil Sciences (MS) --- GRE and TOEFL or IELTS
- Urban and Regional Planning (MURP) --- TOEFL or IELTS
Note: As part of current efforts supporting human resource development in Myanmar, the ADB has approved an additional ADB scholarship for a finalist from Myanmar to pursue a Master's in Public Health. The MPH degree requires the following exams: TOEFL or IELTS and GRE).
Check the University of Hawai'i at Manoa website for details about the programs and areas of study listed above.
The basic eligibility requirements for the ADB-JSP scholarship are as follows. Applicants must:
- Be a citizen of a developing member country of the Asian Development Bank.
- Have a 4-year bachelor's degree or the equivalent of a 4-year Bachelor's degree, at the time of application. (An equivalent could be a 3-year Bachelor's degree plus a 1-year Master's degree, or a 2-year bachelor's degree plus a 2-year Master's degree. This is subject to review.)
- Have at least 2 years of full-time professional work experience after a university degree.
- Take one of the English proficiency tests; the minimum acceptable scores are 550 for the paper-based TOEFL; 213 for the computer-based TOEFL; and 79-80 for the internet-based TOEFL; or 6.5 for the IELTS.
- Applicants living or working in a country other than his/her home country are not eligible to apply for this scholarship.
Applicants with the following experience are not eligible for the ADB-JSP Scholarship:
- Applicants living or working in a country other than his/her home country
- Applicants who have already studied abroad, or may be seeking a second Master's degree
Carefully review the ADB-JSP Overview and Application Information document before beginning the application process.
Award Services Office/ADB-JSP
East-West Center
John A. Burns Hall, Room 2066
1601 East-West Road
Honolulu, Hawaii 96848-1601
Email: email@example.com