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Mu-opioid receptor (MOR) agonists profoundly influence digestive and other autonomic functions by modulating neurons in nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). Whole cell recordings were made from NTS and DMV neurons in brain stem slices from rats and transgenic mice that expressed enhanced green fluorescent protein (EGFP) under the control of a GAD67 promoter (EGFP-GABA neurons) to identify opioid-mediated effects on GABAergic circuitry. Synaptic and membrane properties of EGFP-GABA neurons were assessed. The endogenous selective MOR agonist endomorphin-1 (EM-1) reduced spontaneous and evoked excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) in both rat and mouse DMV neurons. Electrical stimulation of the solitary tract evoked constant-latency EPSCs in ∼50% of EGFP-GABA neurons, and the responses were reduced by EM-1 application. EM-1 reduced action potential firing, the frequency and amplitude of synaptic inputs in EGFP-GABA neurons and responses to direct glutamate stimulation. A subset of EGFP-GABA neurons colocalized mRFP1 after retrograde, transneuronal infection after gastric inoculation with PRV-614, indicating that they synapsed with gastric-projecting DMV neurons. Glutamate photolysis stimulation of intact NTS projections evoked IPSCs in DMV neurons, and EM-1 reduced the evoked response, most likely by activation of MOR on the soma of premotor GABA neurons in NTS. Naltrexone or H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), MOR antagonists, blocked the effects of EM-1. Our results show that GABA neurons in the NTS receive direct vagal afferent input and project to gastric-related DMV neurons. Furthermore, modulation by EM-1 of specific components of the vagal complex differentially suppresses excitatory and inhibitory synaptic input to the DMV by acting at different receptor locations.
The dorsal vagal complex (DVC) forms a sensory-motor circuit that provides parasympathetic control of the thoracic and abdominal viscera through the vagus nerve. Viscerosensory information carried by vagal afferents is initially processed by neurons in the nucleus tractus solitarius (NTS). These neurons in turn make abundant glutamatergic and GABAergic synaptic connections with neurons in the dorsal motor nucleus of the vagus (DMV), which are the major source of preganglionic vagal motor control of the gastrointestinal tract and other subdiaphragmatic viscera. In particular, GABAergic connections between the NTS and DMV exert important control over vagal motor neuron activity and gastric function (Davis et al. 2004; Travagli et al. 1991, 2006).
Opioid peptides have inhibitory effects on synaptic activity in the DVC (Appleyard et al. 2005; Browning et al. 2002, 2004; Glatzer and Smith 2005; Rhim et al. 1993). Electrophysiological recordings in vivo and in vitro have shown that μ-opioid receptor (MOR) agonists reduce solitary tract input to the NTS in rats and mice (Appleyard et al. 2005; Glatzer and Smith 2005; Rhim et al. 1993; Sapru and Chitravanshi 2002). Endomorphins are endogenous neuropeptide agonists for the MOR and are prominent in the spinal cord and brain stem, including the NTS (Martin-Schild et al. 1999). Endomorphin-1 (EM-1) potently inhibited solitary tract and other synaptic input to NTS neurons in the rat (Glatzer and Smith 2005). MORs are found in abundance in the DVC, and their activation has been shown to affect gastric functions acting at the level of DMV motor neurons (Burks et al. 1985, 1987; Del Tacca et al. 1987; Ding et al. 1996).
In the DMV, previous studies analyzed effects of the MOR agonist met-enkephalin on synaptic input evoked after electrical stimulation of the NTS (Browning et al. 2002). Whereas evoked excitatory postsynaptic currents (EPSCs) were suppressed by activating MOR on glutamatergic terminals, there was no effect of met-enkephalin on inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of the NTS under normal recording conditions (Browning et al. 2002). However, pretreatment with forskolin (Browning et al. 2004) or vagal deafferentation (Browning et al. 2006) induced a presynaptic inhibition of GABA release in response to MOR agonists, presumably caused by insertion of the receptors into the terminal membrane. However, effects in the DMV that are attributable to actions at the soma of afferent NTS neurons have not been determined.
Inhibitory neurons in the NTS potently regulate premotor output to the DMV in the vagal complex. Identification of this population of GABAergic NTS neurons in a slice preparation presents many technical challenges. To overcome this problem, we used the GIN (GFP-expressing inhibitory neurons) mouse line that contains a gene for enhanced green fluorescent protein (EGFP) under the control of a GAD67 promoter, which expresses EGFP at visibly detectable levels in known GABAergic neurons (Oliva et al. 2000, 2002) to target GABAergic NTS neurons in medullary slices prepared from these mice. We studied the effects of EM-1 on GABAergic synaptic transmission to the DMV and also on identified GABAergic NTS neurons. We tested the hypotheses that 1) EM-1 modulates inhibitory synaptic input to rat and mouse DMV neurons; 2) EM-1 modulation of NTS neurons decreases overall synaptic inhibition in the DMV; and 3) EM-1 modulates GABAergic NTS to DMV connections within the slice.
Gastric injection of PRV 152 and PRV 614
The gene encoding EGFP or monomeric red fluorescent protein (mRFP1, a generous gift of Dr. Roger Tsien) was inserted in the gG gene of the Bartha strain of the transneuronal viral tracer pseudorabies virus (Banfield et al. 2003; Smith et al. 2000). Male Sprague-Dawley rats 2–6 wk old (Harlan) or 3–9 wk old transgenic mice expressing EGFP under the control of the mouse GAD67 promoter [FVBTgN (GAD67GFP) 45704Swn; Jackson Laboratory; Oliva et al. 2000] were housed with a 12-h light/dark cycle, with food and water provided ad libitum. Breeding pairs of transgenic mice were purchased from Jackson Laboratory, and pups were raised in the animal facilities of Tulane University and the University of Kentucky.
Details of methods regarding use of viral constructs to label gastric-related brain stem neurons for electrophysiological and morphological analyses have been reported previously (Glatzer et al. 2003). Under sodium pentobarbital anesthesia (50 mg/kg, ip), animals received three to four injections (1 μl each) into the ventral stomach wall of PRV-152 (a generous gifts of Dr. Lynn Enquist) or PRV-614 over a 1-min interval using a 10-μl Hamilton syringe fitted with a 26-gauge needle. A fresh stock of virus was thawed for each injection. Animals were maintained in a biosafety level 2 facility for 60–72 h after injection.
Slice preparation
Electrophysiological experiments were performed on brain stem slices from 3- to 6-wk-old adult male Sprague-Dawley rats (Harlan) or 3- to 9-wk-old transgenic mice [FVBTgN (GAD67GFP 45704Swn]. All the animals were treated according to the rules of the Tulane University or University of Kentucky Animal Care and Use Committee. The animals were deeply anesthetized with halothane (Sigma-RBI, St. Louis, MO) inhalation and decapitated. The brain was removed and blocked on an ice-cold stand, and the medulla was glued to a stage and mounted on a Vibratome. Slices (300–400 μm thick) were prepared in the coronal plane (i.e., transverse), cutting the brain stem from dorsal to ventral in 0–2°C artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 3 KCl, 1.3–2 CaCl2, 1.3 MgCl2, 1.4 NaH2PO4, 26 NaHCO3, and 11 glucose, pH 7.2–7.4; osmolarity between 300 and 320 mOsm/kg. Slices were incubated for ≥1 h in 35°C oxygenated (95% O2-5% CO2) ACSF. After incubation, the slice was transferred to a superfusing recording chamber on a fixed stage under an upright microscope (Olympus, Melville, NY), where the slice was continually superfused by room temperature ACSF. Neurons expressing EGFP or mRFP1 were visualized in living tissue under epifluorescence by using fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC) filter sets (Smith et al. 2000). Once the cell of interest was targeted, infrared/differential interference contrast (IR/DIC) optics were used to obtain recordings.
Voltage-clamp recordings made in whole cell mode with an Axopatch 200B or 700A amplifier (Axon Instruments, Union City, CA), digitized at 88 kHz (Neurocorder; Cygnus Instruments, Dorchester, UK), and low-pass filtered at 2–5 kHz were recorded onto videotape and a PC running Clampex 8.0 software (Axon Instruments). Patch pipettes (4–7 MΩ) were pulled from borosilicate glass (1.65 mm OD, 0.45 mm wall thickness; Garner Glass Company, Claremont, CA) and filled with (mM) 130 potassium-gluconate (or Cs-gluconate), 10 HEPES, 1 NaCl, 1 CaCl2, 3 KOH (or CsOH), 5 EGTA, 2–4 Mg-ATP, and 0.1% biocytin. d-2-Amino-5-phosphono-pentanoic acid (AP-5; 50 μM; Sigma), picrotoxin (50 μM; Sigma), 6-cyano-7-nitroquinoxaline-2,3-dionedisodium (CNQX; 10 μM; Sigma), endomorphin-1 (Tyr-Pro-Trp-Phe-NH2; EM-1; 10 nM to 10 μM; Sigma), naltrexone HCl (20 μM; Sigma), and H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 1–3 μM; Sigma) were bath applied with the ACSF. The criteria for detecting synaptic currents were fast 10–90% rise time (<1 ms) and exponential decays. Electrical stimulation was performed using a platinum-iridium concentric bipolar electrodes (125 μm diam, FHC, Bowdoinham, ME) placed in either the NTS or solitary tract (ST). For electrical stimulation of the NTS, the NTS was stimulated by paired current pulses (2–100 μA; 300–400 μs; A.M.P.I., Jerusalem, Israel) at pairing frequencies of 10–20 Hz at a cycle rate of 0.2 Hz. For electrical stimulation of the solitary tract, sets of two to five current pulses (2–25 μA; 300–400 μs) were applied at 40 Hz. Resting membrane potentials, assessed by removing the voltage clamp, were corrected for junction potential (approximately −8 mV). Action potential frequency was measured over a 2-min period of continuous recording. Frequency and amplitude changes for PSCs within a recording were examined over 2 min of continuous recording before, during, and after a drug application using a Kolmogrov-Smirnov test for frequency and an unpaired Student's t-test for amplitude, and across recordings with a paired Student's t-test. The concentration-response curve and IC50 were calculated as previously described (Glatzer and Smith 2005), by fitting a sigmoidal curve to the points of the concentration response plot using Prism 6.0 (Graphpad, San Diego, CA). All drug-induced changes were considered significant at P < 0.05. All values are shown as ±SE.
Chemical stimulation with caged glutamate photolysis
Similar to previous descriptions of glutamate photolysis in the vagal complex (Davis et al. 2003, 2004; Glatzer and Smith 2005; Williams et al. 2007), γ-(-carboxy-2-nitrobenzyl) ester, trifluoroacetic acid salt (i.e., CNB-caged glutamate, 250 μM; Molecular Probes, Eugene, OR) was added to recirculating ACSF and uncaged using brief pulses of UV light directed into the slice. Fluorescent light (UV filter, Chroma Technology, Rockingham, VT) was directed onto the slice through the ×40 objective used to obtain the recording, which was moved progressively further away from the recorded cell until a photolysis-mediated increase in synaptic events was found. Exposure time was electronically controlled using a shutter (Vincent Associates, Rochester, NY). Opening the shutter with no UV filter or with other filters in place (e.g., FITC) did not result in uncaging. Uncaging glutamate directly onto the recorded neuron (9-s interval, 20- to 30-ms exposure) resulted in a fast inward current (50–200 pA at a holding potential of –60 mV; n = 4), or a large depolarization (n = 7), which response was repeatable in single cells in >100 consecutive applications (n = 4). Photic stimuli were applied on the ST and at sites just outside the NTS for negative controls (i.e., no synaptic responses were observed). The effective diameter of the uncaging (∼50 μm) was set by apertures in the light path and measured by moving the center of the illumination away from the cell and testing for a direct inward current after uncaging. The distance moved was analyzed posthoc by comparison with a scale slide.
After recording, brain slices were fixed in 4% paraformaldehyde in 0.15 M sodium phosphate buffer overnight at 4°C (pH = 7.4) and processed as previously described (Glatzer et al. 2003). After three rinses with 0.01 M PBS, whole mount slices were immersed in Avidin conjugated to AMCA or Texas Red (1:400; Vector Laboratories) in PBS containing 0.5% Triton X-100 and incubated overnight at 4°C to identify biocytin-filled neurons and confirm they were labeled with EGFP or mRFP1. Slices were rinsed 2–3 times with PBS and mounted on slides to air dry for 10 min. The slices were covered in Vectashield (Vector Laboratories; Burlingame, CA). Cells labeled with biocytin during a recording and/or with EGFP or mRFP1 or in transgenic mice were identified with a Leica DMLB microscope, and images were captured with a Spot RT CCD camera (Diagnostic Instruments) using filters for the Avidin-conjugated fluorescent dyes and EGFP or mRFP1. Neurons were required to be positively identified and documented by one or both of these methods to be considered gastric-related DMV neurons or EGFP-GABA neurons.
Recordings in DMV neurons
Whole cell patch-clamp recordings were made from 52 neurons in the rat DMV and 7 DMV neurons from transgenic mice. Qualitative or quantitative differences were not seen between infected and uninfected DMV neurons, so the data were pooled unless otherwise specified. Using a potassium gluconate intracellular solution (K-gluconate), average resting membrane potential was −57 ± 3 mV, and input resistance averaged 466 ± 96 MΩ (n = 16, including 5 PRV-152–labeled neurons). Consistent with previous findings (Browning et al. 2002), EM-1 caused an outward current in 7/16 neurons, including 4/5 PRV-152–labeled DMV neurons. The average amplitude of the outward current was 13.6 ± 3.6 pA at a holding potential of −65 mV (n = 7). Neurons that responded to EM-1 application with an outward current also showed a decrease in input resistance that averaged −54 ± 20 MΩ (n = 7; P < 0.05; paired t-test). No significant change to input resistance was observed in response to EM-1 application using intracellular solution with Cs+ as the primary cation (425 ± 73 MΩ in control ACSF, 455 ± 92 MΩ in EM-1 1 μM; n = 21; P > 0.05).
Effects of EM-1 on excitatory synaptic inputs to DMV neurons
Electrical stimulation of the NTS using a concentric bipolar stimulating electrode resulted in excitatory and inhibitory synaptic inputs to DMV neurons. Pharmacological blockade of the inhibitory inputs (picrotoxin, 50 μM) resulted in expression of only excitatory glutamatergic inputs (Vm = −65 mV); all EPSCs were blocked by N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists. Analogous to other MOR agonists (Browning et al. 2004), application of EM-1 (10 nM to 10 μM) resulted in a concentration-dependent reduction in the amplitude of the evoked EPSC (IC50 = 136 nM). The reduction was blocked by the opioid receptor antagonist naltrexone HCl (25 μM) and was reversible after 15–20 min of wash in normal ACSF. Bath application of EM-1 (1 μM) reduced the average NTS-evoked EPSC amplitude from −38.0 ± 6.0 to −20.0 ± 4.2 pA (n = 7; P < 0.05; paired t-test). The paired-pulse ratio (PPR) of two consecutive stimuli was measured to see if EM-1 may have caused a change in the presynaptic glutamate release. The PPR significantly increased in the presence of EM-1 from 0.70 ± 0.09 to 1.00 ± 0.09 (n = 7; P < 0.05; paired t-test). Application of EM-1 (1 μM) reduced mean spontaneous EPSC frequency from 4.2 ± 1.3 to 1.3 ± 0.4 Hz (P < 0.05); EPSC amplitude was not significantly affected (15.7 ± 4.4 pA, control; 12.6 ± 2.4 pA in EM-1; P > 0.05; n = 6). To determine whether the reduction in EPSC frequency by EM-1 could occur independently of Na+-dependent synaptic release (i.e., action potentials in afferent intact neurons), the effects of EM-1 on miniature EPSCs (mEPSCs) were measured in the presence of TTX (2 μM) in five neurons (including 2 EGFP-labeled neurons). The mean frequency of mEPSCs was 2.1 ± 0.5 Hz, and amplitude was –8.5 ± 0.8 pA in control ACSF. The mEPSC frequency was significantly reduced to 1.2 ± 0.5 Hz in EM-1 (1 μM; n = 5), whereas amplitude was unaltered (–7.8 ± 0.7 pA; n = 10; P > 0.05, paired t-test). The reduction in mEPSC frequency was blocked by CTOP or CTAP, MOR-selective antagonists (1–3 μM; 96 ± 2% of control frequency; n = 5; P > 0.05).
Effects of EM-1 on inhibitory synaptic inputs to DMV neurons
We tested the effects of EM-1 on spontaneous IPSCs (sIPSCs) in gastric-related and unlabeled DMV neurons. Spontaneous IPSCs were recorded at −10 to 0 mV, and fast glutamate currents were blocked using CNQX (10 μM) and AP-5 (50 μM). In the rat DMV, the average frequency of sIPSCs was 1.3 ± 0.2 Hz, and average amplitude was 25.5 ± 6.5 pA (n = 9). After application of EM-1 (1 μM), sIPSC frequency and amplitude were significantly reduced to 0.8 ± 0.2 Hz and 22.9 ± 6.0 pA, respectively (Fig. 1; n = 9 including 6 PRV-labeled neurons; P < 0.05; Student's paired t-test). In the presence of TTX, the average mIPSC frequency was 0.9 ± 0.3 Hz, and the average amplitude was 27.6 ± 2.0 pA. After application of EM-1 (1 μM), mIPSC frequency was 0.8 ± 0.3 Hz, and average amplitude was 25.6 ± 2.6 pA, with no significant change to either measurement (Fig. 1; n = 6, including 2 PRV-152–labeled neurons; paired t-test; P > 0.05). EM-1 reduced sIPSC frequency in rat DMV neurons, but the effects were not seen when action potentials were blocked with TTX.
Spontaneous IPSCs were also recorded in unlabeled DMV neurons in slice preparations from GAD67-EGFP mice to verify that MOR activation affected inhibitory synaptic input in a manner similar to the rat. Average frequency of sIPSCs was 3.9 ± 1.8 Hz, and average amplitude was 32.3 ± 2.7 pA (n = 5) in this set of cells. After application of EM-1 (1 μM), sIPSC frequency and amplitude were significantly reduced to 1.8 ± 0.7 Hz and 25.7 ± 4.3 pA, respectively (n = 5; Kolmogrov-Smirnov; P < 0.05). The effects of EM-1 were prevented by CTAP (n = 4; P > 0.05). The effects of EM-1 on sIPSCs in DMV neurons in the GAD67-EGFP mouse were similar to the effects of EM-1 on DMV neurons in the rat.
Effects of EM-1 on IPSCs evoked after electrical stimulation of the NTS
Electrical stimulation of the NTS evoked constant latency GABAergic IPSCs in DMV neurons (Davis et al. 2004; Travagli et al. 1991). The average amplitude of the IPSCs was 73.3 ± 12.9 pA (Vm = 0 mV; n = 7). EM-1 (1 μM) caused a reduction in the amplitude of electrically evoked GABAergic IPSCs, reducing the average IPSC amplitude to 28.4 ± 6.9 pA (n = 6, including 2/2 PRV-152–labeled DMV neurons; Fig. 2). The effect of EM-1 was blocked by application of CTAP (n = 4; Fig. 2). The PPR did not change in the presence of EM-1 (1 μM; 0.90 ± 0.06 in control, 0.97 ± 0.08 in EM-1; n = 6; paired t-test; P > 0.05). That the PPR was not altered by EM-1 suggested that the reduction in amplitude of the first response was not caused by the effects of EM-1 on receptors located on terminals contacting DMV cells under normal conditions, as previously shown (Browning et al. 2004). However, unlike that study, the amplitude decreased significantly in our hands, and sIPSCs but not mIPSCs were reduced in frequency. We therefore reasoned that the suppression of IPSCs was caused by inhibition of intact GABA neurons by EM-1.
Recordings from GABAergic NTS neurons in transgenic mice
EM-1 reduced electrically evoked IPSCs, but did not seem to involve MOR activity at GABAergic terminals in the DMV under normal conditions. We therefore tested the hypothesis that identified GABA neurons in the NTS were directly inhibited by EM-1. Recordings were made from 26 EGFP-labeled NTS neurons from a transgenic mouse line that expresses EGFP under the control of a GAD67 promoter (Oliva et al. 2000).
Neurons within the NTS that expressed EGFP were widespread throughout the rostral-caudal and dorso-lateral NTS (Fig. 3). Although the neurons were distributed throughout the NTS, there was a relatively dense cluster found in the interstitial nucleus in the dorsolateral NTS (Paxinos et al. 1999; Stornetta and Guyenet 1999). Ten of 14 GABAergic NTS neurons fired spontaneous action potentials at >0.5 Hz, and only one neuron did not fire action potentials at rest, although it did fire in response to current injection (Fig. 4). Membrane properties of GABAergic NTS neurons are shown in Table 1.
GABAergic NTS neurons innervated gastric-projecting DMV neurons
Injection of the retrograde viral tracer PRV-614 (see methods) in the wall of the stomach of GAD67-EGFP mice resulted in transsynaptic labeling of parasympathetic preganglionic motor neurons in DMV and gastric-related putative premotor neurons in the NTS (Fig. 3), similar to other PRV constructs (Glatzer et al. 2003; Glatzer and Smith 2005; Rinaman et al. 1999). PRV-614 expresses mRFP1 (Campbell et al. 2002) and can be used for multicolor labeling with other fluorophores such as EGFP (Banfield et al. 2003). The number of NTS neurons labeled by PRV-614 was quantified in a previous study in rats and shown to be between 10 and 20% of NTS neurons at 60–72 h after infection (Glatzer et al. 2003). Our study showed a subset of the GABAergic neurons were also labeled with PRV-614 (Fig. 3, C–E). Some of these neurons, as well as EGFP-GABA neurons that were not retrogradely labeled transneuronally from the stomach, were targeted for recording (Figs. 3 and 4).
Effects of EM-1 on solitary tract input to GABAergic NTS neurons
The synapse of the central primary afferents from the vagus and glossopharyngeal nerve has characteristic properties that distinguish it from other inputs received in the NTS. Notably, there is a frequency-dependent depression of the input and an almost total blockade by CNQX near resting membrane potential (Doyle and Andresen 2001; Miles 1986; Smith et al. 1998). Previous studies in rats indicated that MOR activation reduced ST-evoked input to a phenotypically unidentified subset of NTS neurons (Rhim et al. 1993; Glatzer and Smith 2005). Of 14 EGFP-GABA NTS neurons tested, 10 received EPSCs that followed at constant latency (i.e., <400-μs variability in response latency over 10 stimuli) after electrical stimulation of the solitary tract (Figs. 4 and 5). At a Vm of −65 mV, the average amplitude of the ST-evoked EPSC was –52.5 ± 10.9 pA and was blocked completely by CNQX (10 μM; Fig. 4). EM-1 (1 μM) reduced the average EPSC amplitude in to –27.9 ± 9.4 pA (n = 5; P < 0.05). In the presence of CTAP (1–3 μM), a MOR antagonist, the effect of EM-1 on ST input to EGFP-GABA NTS neurons was blocked (102 ± 5% of control ESPC amplitude, n = 5; Fig. 5B). The PPR increased in the presence of EM-1 (58 ± 6% in control; 108 ± 20% in EM-1; P < 0.05; 1 μM, n = 5; Fig. 5C). These data indicated direct ST input to identified GABA neurons was commonly observed. This input was inhibited by EM-1, and MORs were likely present on ST terminals contacting GABA neurons.
Effects of EM-1 on inhibitory synaptic transmission to GABAergic NTS neurons
Spontaneous IPSCs recorded in EGFP-labeled NTS neurons were blocked by application of picrotoxin (n = 3). Spontaneous IPSCs were recorded either using Cs-gluconate electrode solution at a depolarized holding potential (−10 to 0 mV) or using KCl-containing electrode solution in the presence of AP5/CNQX, which blocked all EPSCs (Derbenev et al. 2004; Glatzer et al. 2003; Smith et al. 1998), and revealed reversed IPSCs. EM-1 reversibly reduced the frequency and amplitude of sIPSCs in NTS neurons. Average frequency of sIPSCs was 2.0 ± 0.5 Hz, and average amplitude was 26.0 ± 3.8 pA. After application of EM-1 (1 μM), sIPSC frequency was reduced to 0.7 ± 0.2 Hz and amplitude to 16.8 ± 4.7 pA (n = 6; P < 0.05, Student t-test). The effects of EM-1 were blocked by CTAP (n = 4) and TTX (2 μM; n = 4; P > 0.05; paired t-test), indicating an effect on MORs that reduced action potential firing in GABAergic neurons afferent to the recorded neurons. In addition to decreasing ST input and hyperpolarizing GABA neurons, inhibitory input to GABA neurons was also suppressed.
Effects of EM-1 on membrane potential of GABAergic NTS neurons
A hyperpolarization from resting membrane potential (–8.2 ± 2.3 mV; range, –4 to –18 mV) was seen in six of eight GABAergic NTS neurons in response to 1 μM EM-1, recorded using a K-gluconate electrode solution. No hyperpolarization was seen in any of five GABAergic NTS neurons recorded with Cs+ in the recording pipette. Spontaneous action potential firing was reduced from an average of 2.4 ± 0.7 Hz in control to 0.2 ± 0.1 Hz in the presence of EM-1 (Fig. 6; n = 5; P < 0.05; t-test), including in GABAergic NTS neurons that were also labeled with PRV-614 after gastric inoculation (n = 2). In addition to modulating endogenous action potentials, the effect of EM-1 on glutamate-evoked action potential firing was examined. Caged glutamate was photoactivated at the site of the recorded neuron to cause a glutamate-evoked depolarization of the cell (n = 7). EM-1 (1 μM) suppressed the activity evoked by uncaging glutamate directly on the recorded NTS neuron (n = 5 GABA neurons and 2 unidentified neurons; Fig. 6). EM-1 therefore hyperpolarized the membrane potential of GABA neurons, including those related to motor output to the stomach, and reduced responsiveness to glutamate-induced depolarization.
Effects of EM-1 on glutamate photolysis stimulation of the NTS to DMV inhibitory circuit
Previous studies have focused on activation of MOR located on afferent terminals in the DMV, using mainly electrical stimulation of the NTS to measure evoked IPSCs (Browning et al. 2004, 2006), which can activate both somata and cut axons. Electrical stimulation and the physical disruption caused by introduction of the electrode may actually disrupt neuronal activity and result in preferrential activation of axons versus somata (Callaway and Katz 1993; Davis et al. 2004). To identify the effects of EM-1 on input from intact neurons within the slice, we used caged glutamate, which is activated by UV light, to depolarize intact NTS neurons while recording in the DMV (Callaway and Katz 1993; Davis et al. 2003, 2004; Glatzer and Smith 2005). Neurons were recorded at a holding potential of 0 mV so that evoked EPSC amplitude was minimal and IPSC amplitude was outward and relatively large. Focal, short-duration exposures to UV light directed through the microscope objective into the NTS (<50 μm diam; 15–30 ms) resulted in a glutamate uncaging that increased synaptic activity in DMV neurons (n = 8; Fig. 7). Moving the spot >50 μm from the area that generated a response resulted in loss of the stimulated response, suggesting an effective uncaging diameter of <100 μm. Stimuli directed onto the ST or to areas adjacent to the NTS were ineffective, suggesting that responses to NTS stimulation were caused by activation of neurons within the NTS that connected to DMV neurons. Application of TTX (2 μM) blocked the evoked IPSCs (n = 3), indicating that they were caused by actions potentials generated in intact, afferent neurons, as described in several previous studies (Callaway and Katz 1993; Dalva and Katz 1994; Davis et al. 2003, 2004; Katz and Dalva 1994). At a holding potential of 0 mV, uncaging glutamate in various areas of the NTS resulted in an increase in the number of IPSCs recorded during a variable period (100–500 ms) after the stimulation (Fig. 7). The average number of events recorded in the period before the stimulation was subtracted from the number after stimulation to measure the changes in the increase between the control condition and during EM-1 application. EM-1 significantly reduced the glutamate-stimulated increase in IPSC frequency in four of five DMV neurons (including each of 3 PRV-152–labeled neurons; paired t-test, P < 0.05). Inhibitory NTS input to DMV neurons was inhibited by EM-1 and appeared to involve suppression of action potentials in afferent GABA neurons rather than binding to MORs on GABAergic terminals.
Immunohistochemistry for endomorphins has shown they are present at sensory afferent terminals and in second-order sensory neurons within the NTS (Martin-Schild et al. 1999; Pierce and Wessendorf 2000). Robust staining for EM-1 in perikarya in the dorsomedial NTS suggests a role in gastric sensory regulation (Martin-Schild et al. 1999), because this is the area primarily contacted by gastric sensory afferents (Shapiro and Miselis 1985). MOR agonists, including EM-1, have consistently been shown to inhibit glutamatergic synaptic transmission in the NTS and DMV by acting at glutamatergic synaptic terminals (Appleyard et al. 2005; Browning et al. 2002; Glatzer and Smith 2005; Rhim et al. 1993). These results support the hypothesis that MOR activation in the DMV reduces excitatory glutamatergic inputs to DMV neurons and specifically to the gastric-projecting DMV motor neurons. The magnitude and consistency of the MOR inhibition of electrical and spontaneous EPSCs in this study were similar to that observed by Browning et al. (2002, 2004). This study also showed that MORs are functional on GABAergic NTS neurons and that they inhibit both local GABAergic transmission and GABAergic projections to the DMV. The glutamate photolysis experiments show that EM-1 reduces the IPSCs caused by inhibition of "premotor" GABAergic NTS neurons, suppressing intact inhibitory connections in the slice. Previous studies concluded that the effect of MOR agonists on electrically evoked IPSCs in the DMV could occur subsequent to activating receptors on presynaptic GABAergic terminals, but only after translocation of the receptors into the membrane (Browning et al. 2004, 2006). Our results further show that receptors on the soma-dendritic compartment of GABA neurons in the NTS can normally mediate an MOR-induced reduction in inhibitory drive to the DMV. EM-1 thus exerts a strong inhibitory effect on both glutamatergic and GABAergic synaptic input to NTS and DMV neurons, albeit by different means. This implies a high degree of specificity for opioideregic modulation of different neural components of the DVC.
Modulation of inhibition in the DVC by EM-1
Synaptic responses in the DMV to stimulation of the NTS reflected the complexity of the excitatory and inhibitory projections. Electrical stimulation produces a constant latency fast EPSC or IPSC and sometimes results in a transient increase in PSC frequency (Davis et al. 2004; Travagli et al. 1991). We used a model wherein a reduction in the presynaptic probability of release in response to electrical stimulation of the NTS in the presence of EM-1 would result in a greater reduction in the first PSC of the pair, whereas a change in postsynaptic response would result in equal reduction of both PSCs (Kline et al. 2002; Regehr and Stevens 2001). A change in evoked EPSC PPR was observed after EM-1 application, and mEPSCs were reduced in frequency. These results support the hypothesis that functional MOR are normally located in glutamatergic terminals in the DMV.
Electrically evoked responses occur subsequent to activation of neural elements originating in or passing through the NTS to contact the DMV neurons. Glutamate microstimulation of the NTS produces both excitatory and/or inhibitory responses in DMV neurons caused by depolarization and increased action potentials in intact NTS neurons projecting to the DMV (Davis et al. 2004) and not fibers of passage. The inhibitory response evoked by caged glutamate stimulation was reduced dramatically by application of EM-1. Previous reports indicated preterminal MORs require adenyl cyclase activity to be functional (Browning et al. 2004). Our data, which specifically identified responses originating in activity of afferent GABA neurons, indicate that MOR-mediated inhibition of intact GABA neurons (i.e., on the somatodendritic portion of NTS GABA neurons) suppresses inhibitory input to the DMV that arises in the NTS. In support of this, identified GABAergic NTS neurons were hyperpolarized by EM-1, and the response to glutamate-induced activation of GABAergic NTS neurons was suppressed. There was also an EM-1–induced reduction in input resistance in the DMV, consistent with a reduction in both sEPSC and sIPSC amplitude. Consistent with the findings of Browning et al. (2002, 2004), we found no evidence for preterminally located MORs under normal conditions. Thus neither the electrically evoked IPSC PPR nor the mIPSC frequency, measures of putative preterminal receptor activation, was altered by EM-1. Inhibition of action potentials in GABA cells afferent to the DMV could reflect functionally different MOR effects on inhibitory circuits in the DVC, depending on the location of the opioidergic input (NTS vs. DMV) and the functional state of the system (e.g., elevated cAMP).
GABAergic NTS cells are premotor and/or second order sensory neurons
We tested the hypothesis that MOR activation inhibited GABAergic NTS neurons using a transgenic mouse line (i.e., GIN mice) in which GABAergic neurons express EGFP. The transgene has been shown to label GABAergic neurons, although the subtypes of GABAergic neurons expressing GFP varies through different brain regions (Heinke et al. 2004; Oliva et al. 2000). The distribution of the EGFP-labeled neurons in the NTS appeared qualitatively similar to that of immunohistochemical staining for glutamic acid decarboxylase (GAD) (Blessing et al. 1984) and GABA (Izzo et al. 1992) performed in other studies. Use of this transgenic mouse allowed a straightforward method for identifying and targeting these neurons for recording a priori. The finding that most GABA neurons receive primary viscerosensory input, as shown by constant-latency EPSCs generated after stimulation of the TS, is suggestive of their position as second-order sensory neurons. As such, they could act as either a direct relays to the DMV or as interneurons, helping to coordinate afferent signals from various viscera at the level of the first central viscerosensory synapse. In addition, the NTS neurons double-labeled with both mRFP1 (through viral label from the stomach) and EGFP (through transgene expression) were considered to be GABAergic gastric-related premotor NTS neurons. Our findings regarding the membrane properties of GABAergic NTS neurons are consistent with previous conclusions from immunohistochemically identified cells (Kawai and Senba 1999).
Implications of EM-1 effects on GABAergic NTS neurons
Generalized administration of MOR agonists such as morphine can cause a pronounced reduction in all synaptic activity in the DVC. In contrast, spatially specific endogenous release of opioid agonists would presumably bind MORs to reduce specific aspects of synaptic input. If released in the DMV, opioids like met-enkephalin would normally cause an inhibition of the preganglionic neurons, through membrane hyperpolarization and/or inhibition of glutamatergic input to gastric-projecting DMV neurons (Browning et al. 2002). However, GABA release would not be reduced by MOR activation at terminals in the DMV under basal conditions because the MORs on GABAergic terminals are not active except under specific circumstances (Browning et al. 2002, 2004, 2006). Conversely, a disinhibition of DMV activity would occur if EM-1 were selectively released within the NTS because of the site of activity on glutamatergic synaptic terminals and GABAergic cell bodies and dendrites in the NTS. Application of MOR agonists to the NTS results in increased parasympathetic output (Kotz et al. 1997), which tends to be orexigenic, and this could be caused by inhibition of a tonically active inhibitory GABAergic projection to gastric-projecting DMV neurons. Thus activation of MORs in the DVC can result in variable responses in preganglionic motor neurons, which depends on the location of the release within the DVC in addition to the functional state of the system. Changes in preganglionic function can alter the excitatory/inhibitory balance in the enteric nervous system, causing up- or downregulation of digestive and other autonomic functions.
The results of this study indicate that EM-1 and other MOR agonists released in the NTS can have a net excitatory effect on vagal motor neuron output by suppressing excitatory solitary tract and other glutamatergic input to GABAergic NTS neurons and by hyperpolarizing them. MORs are present on glutamatergic terminals of NTS neurons in DMV and on glutamatergic terminals contacting NTS cells, and they are present on the somatodendritic region of NTS GABA neurons, including neurons projecting to the DMV. Activation of MORs in the NTS tends to decrease inhibition of DMV neurons, including gastric-projecting DMV neurons associated with digestion, whereas activation of MORs in DMV tends to decrease excitation of DMV neurons under normal conditions. These results support the hypothesis that spatially specific opioid peptide connections and cellular activity levels combine to produce precise regulation of gastric-related DMV neuron activity.
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-56132, National Science Foundation Grants IBN-0080322 and IOB-0518209, and American Heart Association Grant SDG-0030284N.
We thank L. Enquist for giving us the PRV-152 and R. Tsien for the mRFP1 construct used in making PRV-614.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society


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Anniversary Cake!
By Laurel Lakey
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- Find their other images
Here's my quick layout that includes sugar (button), butter (ribbon), eggs (SS Embellishment), vanilla extract (WordArt), flour (style), baking powder (brush), milk (frame), on a layered cake (Odd # photos), with blue (tag) and white (photo corner) butter cream frosting (1 patterned, 1 solid paper) and an edible image (something metal) with sprinkles (heart) on top!
Here are the delicious supplies I used:
Everyday Moments Collection Biggie
ScrapSimple Embellishment Template: Mesh
JIF 6 Plus: Today
ScrapSimple Digital Layout Album Templates: 12x12 On A Tilt Mini
Life Keeping Collection
Dynamic Brush Set: Straight Stitch 5001
Love Struck Collection
Storyteller Collection
Oh Snap Paper Mini
Click! Collection Super Mini
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subset: commoncrawl


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Besides the occasional movie I noticed I just missed the start time by 10 minutes or maybe some kids cartoons to pacify the monkeys need for SpongeBob during the one hour SpongeBob can't be found somewhere on the 20 versions of Nickelodeon, I don't use the VOD features of my expensive cable TV package. That changed this weekend in my attempts to avoid another MJ special attack on my sanity.
I found myself noticing that Showtime had Dexter seasons 1-3 available in anticipation of the show's return for the 4th season this fall. And all I can say is you had me at first slice!
While I admit I have no usual desire to watch slasher flicks - Horror just doesn't do it for me - I can't stop watching this show. The character just grabbed me and I can't get enough, watching Michael C Hall play a man void of emotion fake his way though the motions of being normal like the rest of us you think would bore the living snot out of me, but oddly not only does he have me believing every moment I find myself relating to him in a creepy sort of way.
No need to call the cops, I'll explain. Dexter is an extreme example of every one of us; how many times do we fake some emotion in our lives to be normal? While his darkness is defiantly something deeper and darker than anything I can imagine, there is something more to this character that I can't help but relate to. After finishing season 1 last night I found myself rooting for Dexter, hoping he doesn't get caught, hoping he kills that next scum bag who with the help of his slimy lawyer slipped right though the cracks of a broken justice system.
*cough* after being unable to stop the last 3 episodes no matter how hard I tried, staying up WAY past the time I should have been long asleep, I watched Dexter slash his way though Miami's most disgusting criminal element (not to spoil, but the Therapist - "I can't kill him yet...I need another session!" - wow that was genius - we'll just leave it at that).
If you are looking for a gripping character story that deviates completely from the norm then I suggest anyone who has access to Showtime OnDemand should check out a few episodes while they are offering the show's first 3 seasons as part of your subscription (or heck go get one for the month if you don't or pick them episodes up on DVD). I think after 3 episodes of the show you won't be able to stop!
I can't even start Season 2 tonight because I need to sleep thanks to Dexter grip on me the past 2 nights. How did I ever miss this show? I guess I really should check out some of these other shows I've missed, maybe squeeze a bit more out of my ungodly cable bill!


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NEW YORK (Aug. 31)
About two hundred schochtim employed in poultry markets in Harlem and the Bronx were ordered on strike by their manager, J. Rothman, who said the strike order was issued "reluctantly", following the refusal of the Poultry Market Owners Association to discharge all strikebreakers employed since the lockout of Union members about a week ago.
The poultry workers are affiliated with the American Federation of Labor, and the strike, according to Rothman, will affect about seventy poultry markets. Rothman said that in addition to the discharge of the non-union men the Union demands a wage increase of $5.00 a week and a 48-hour week in place of one of 52 hours.


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Yesterday, Sept. 4, the Food and Drug Administration ordered stronger warnings on four medications used to treat rheumatoid arthritis.
The drugs - Remicade, Enbrel, Humira, and Cimzia, - work by suppressing the immune system. According to the FDA this exposes some patients to serious, even fatal, fungal infections. As of Thursday, the FDA had received 240 reports of one such type of infection, called histoplasmosis. In at least 21 of the cases, doctors didn't initially recognize the problem, and 12 of the patients died.
The fungus that causes the infection is common in the Mississippi and Ohio river valleys. Patients should stop taking their medication and call the doctor if they develop persistent fever, cough, shortness of breath and fatigue.
Now would be a good time to consider alternatives to drugs that suppress immune function. It's not necessary to put your health at risk to address rheumatoid arthritis. Nutrition and homeopathy are 2 options that can help you get better without harming you.
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The luxurious Mercure Ardoe House modern Hotel is a brilliant marriage of Victorian opulence and contemporary elegance. Intricately carved staircases, enormous fireplaces and rich wood panelling are the hallmarks of a hotel which offers the tired traveller and the busy business guest a tranquil retreat from the pressures of the world outside. The Vital Health and Beauty facilities has a reputation second to none, for the more active person try the fully- equipped gym or you can choose to ease your cares away with a gentle swim or sauna.
South Deeside Road, Blairs , CP: AB12 5YP , Aberdeen , Reino Unido
Teléfono: No especificado
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text: USCIS announced on April 7, 2015 that they have received more petitions than the mandated cap of 65,000 for general category (including 5800 for Singapore and Chile) and 20,000 for US advanced degree. This is the third year in a row that USCIS has reached the cap within the first 5 days. Lottery selection will be done over the next couple of weeks.


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Download Calc Lab apk 1.0.3 free for Android smartphone
Posted by Boris Rayskiy in Education | Feb. 17, 2015 | 0 Comments
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Calc Lab is a graphing calculator (also graphics / graphic calculator) refers to a class of scientific calculators that are capable of plotting graphs (Cartesian two and three dimensional, Polar and Parametric) and performing calculations using BoaScript scripting language. BoaScript has a C-style syntax and allows to conduct the calculations in the wide range - from arithmetic and trigonometric calculations to complex numerical solutions.
Boris Rayskiy part of our Education and have average installs from 100 to 500. Last Update Feb. 17, 2015. Google play rating is 93.3333. Current verison is 1.0.3. Actual size 3.4 MB.
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text: Selfie with some of my favorite people, Me, Sister Hodson, Elder Bagley (Red Hair), and Elder Karlinsey (Behind). I have the best zone ever! There may only be 5 sisters, but we are the 5 best sisters in the MTC! Ima miss them so much! Following the example of the Elder in the bottom right corner (hehe)


text: Mossberg Patriot Night Train OD Green .300 WIN MAG w/ Scope 27925 For Sale from Ammo And Arms | Positive feedback: 98% View | Verified Seller | 4270 Completed Sales View Sellers Items /UserImages/5133/913365587 11532851.jpg Description: Brand New in the Box, Ready to Ship. Buy It Now!! The Mossberg Patriot Night Train is a fantastic long range choice when it comes time to put meat on the table. It is an accurate and versatile offering that provides excellent constancy with each and every round sent down range. The fluted barrel has a recessed target crown with a 1:10 twist. Mossberg included weaver style bases and a 6-24x50MM scope to get you on target right away! This lightweight long range rifle is sure to impress new and seasoned shooters alike with its reliability in form and function no matter what the conditions. Don't miss another opportunity to fill your freezer hunting season after hunting season with the Mossberg Patriot Night Train. Caliber: .300 WINCHESTER MAG Capacity: 5 ROUND Barrel Type: Fluted Barrel Length: 22" Sight: None (Picatinny Rail) Scope: 6-24x50MM Twist: 1:10 Length of Pull Type: Fixed Length of Pull: 13.75" Barrel Finish: Matte Blued Stock Finish: Synthetic (OD Green) Weight: 8.75" Length: 42.75" Shipping to Lower 48 Only Shipping $30.00 KNOW YOUR LOCAL AND STATE LAWS!! Read More Condition: New In Box Brand: MOSSBERG INTERNATIONAL UPC: 27925 Caliber: .300 Winmag Location: PA Trades Accepted: No Read More Please ask for Internet Sales when calling in. Shipping Notes: Shipping to Lower 48 Only!! Read More Share Item Item #: 913365587 Stock No.: 27925 Ammo And Arms Seller Since: Jan. 2004 Seller Level: Gold Seller + This seller is an FFL City: St. Marys State: PA Price: $609.99 Buy It Now Add To Cart (Price reflects 2% cash discount.) Please Login/Register to Order Login or Register to buy This Seller Accepts Online Payment Shipping: $30.00 (or Arrange local transfer) Accepted Payment Methods: Returns: No Returns Send Message to You must login or register to send a message to this seller. Contact Seller as Guest Message To Seller Email * That Email Address is already in use. If you typed your email correctly, you are already registered and you should login or click here for the password recovery system. First Name * Last Name * Phone * Address 1 * Address 2 City * State * -State- AK AL AR AZ CA CO CT DC DE FL GA GU HI IA ID IL IN KS KY LA MA MD ME MI MN MO MS MT NC ND NE NH NJ NM NV NY OH OK OR PA PR RI SC SD TN TX UT VA VI VT WA WI WV WY Zip * Already have an account? Login Your Recently Viewed Items


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Archives for Snacks
Trail Mix
Directions: 1 cup of almond 1 cup of cashews 1 cup of raisins 1 cup of sunflower seeds and pumpkin seeds 1 cup of dates mix in a bowl and…
Healthy Recipe Figs and Ricotta
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Cut 1 apple into slices and serve with 1 oz Brie Total calories: 150
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Directions: Skewer 5 cherry tomatoes, 1 mozzarella string cheese (cut into cubes) , 5 fresh basil leaves, 3 pitted olives. Total Calories: 150 calories
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Veggie & Dip Snack
Ingredients Slice 1 red bell into strips, and dip it 10 baby carrots into 1/3 cup hummus Total calories: 197, 15 g fiber


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Editorial: A sign of disapproval
Yet another hurdle has been placed in the way of the Government's plans to sell shareholdings in four state-owned energy companies and Air New Zealand.
The public will have an opportunity to reiterate their opposition, now that a petition to force a referendum - according to the campaigners who have collected signatures - has reached the required threshold.
It is not an insurmountable hurdle. It will give another measure of dissatisfaction with the sales programme, but the Government does not regard a referendum result as binding. The anti-sales campaigners needed 10 per cent of all registered voters - about 310,000 people - to sign their petition. The reported number of signatures collected is about 340,000, enough to trigger a referendum, but not to support Labour MP Chris Hipkins' claim that New Zealanders "overwhelmingly" oppose the asset sales. His insistence that "the Government is now panicking about it" looks fanciful too.
He was right to say the vote will give an opportunity to demonstrate the weight of opposition, but governments are apt to cock a snook at the results of citizen-initiated referenda. A formidable 85 per cent of New Zealanders voted in a referendum that a light parental smack for correctional purposes should not be a criminal offence, yet a few weeks later the Greens and the Labour Party - cheerleaders in the campaign for the referendum on asset sales - voted against a bill which would have translated that strong vote into law.
The state share sales face other obstacles. In the Supreme Court, the New Zealand Maori Council has been granted leave to appeal a High Court ruling dismissing its application for a review of Cabinet decisions relating to the sale of up to 49 per cent of Mighty River Power.
In the Treasury, advisers are warning against selling shares in three state energy companies all at once. One sale every six months is recommended, with the final sale next year, to ensure the price fetched is not eroded.
But the biggest obstacle was overcome at the 2011 general election. The Government mustered enough votes to remain in office for a second term. Voters had their opportunity to scuttle the partial-privatisation policy. They did not take it.
Waikato Times


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US public health service needs strengthening to fight infectious diseaseBMJ 2003; 326 doi: https://doi.org/10.1136/bmj.326.7391.680/d (Published 29 March 2003) Cite this as: BMJ 2003;326:680
- Charles Marwick
- Washington, DC
Infectious diseases are a global threat, and the United States should take the lead in addressing it, says a new report from the US Institute of Medicine. Such an initiative would mean that the United States would have to strengthen its public health service, the report says, and to help other nations monitor and respond to outbreaks of infectious disease, including enhancing global disease surveillance.
"Infectious diseases continue to burden populations around the world. Both natural infections and the possibility of a bioterrorist attack hold increasing potential to cause disease, disability, and death," said Dr Margaret Hamburg, co-chairperson of the committee that prepared the report. Dr Hamburg is vice president …
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Monday, 29 November 2010
more or less?
This thinking was brought about by a chat I had with the man who presses the apples from our apple trees every year. We discussed all sorts of subjects but the topic that got me thinking was about how companies (and perhaps people) can almost get too big. If everyone wants the biggest market share and competes aggressively to get it, at what point are they happy with that market share and if they never are, what happens if something goes wrong or if the market collapses?
Is this business practice just the same as trying to out-do your neighbours with your car or your lifestyle? I'm no economist (you might have guessed), but presumably there's only so much market to grab and there's only so much money in customers' purses. It doesn't matter if the shops are always open if we poor consumers only have so much cash. Surely we then just spend the same over a longer period of time?
I do know, however, that I'm over wanting 'more'. I think I've realised that unless the lottery god picks me out, I've neither the talent or the perseverance to strive for anything much more than I have. Of course, I like the idea of Birkins and Mulberrys and Cartiers but, let's face it, they wouldn't go with my New Look jeans.
PS: Who else is delighted that Wagner has been flung back to obscurity....could it be Ann's turn next?
4 comments:
- marigold jam said...
I am so with you on this - bigger isn't necessarily better. It's one of the reasons I refuse to worship at the altar of Tesco and prefer Waitrose who have said they are not interested in taking over the world! I don't know if it's true but certainly smaller is easier for me - I hate the fact that the supermarkets often have 50,000,000 pakcets of crisps but no cocoa powder or something and that to find anything requires a compass and map etc. No give me small and good any day. Like you I would be happy to be wrapped in cashmere and to have beautiful shoes on a whim but on the whole I am happy with my lot. Haven't been following Strictly so can't comment but a letter in the paper recently said that the public love Ann not because she can dance but because she is good entertainment value - as I haven't seen it I can't comment!
- 29 November 2010 at 17:34
- Sue said...
Big is definitely not beautiful. I'm glad that I've never been bothered about keeping up with anyone, as long as I've got the things I need I'm happy.
I have no idea what your last sentence means so can't possibly comment.
- 29 November 2010 at 21:36
- Only Me said...
I quite agree and having bigger and better doesn't necessarily make you happy or content if you are forever in pursuit of more.
And yes, I'm very pleased that Wagner has disappeared into the distance.
- 30 November 2010 at 09:42
- Sue Gee said...
There comes a time when you realise that less is more! Well done x
- 5 December 2010 at 08:58


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SIC signs agreements with private equity advisor Sun Mountain Capital
The New Mexico State Investment Council approved measures on Tuesday to increase the transparency and oversight of its private equity program advisor, Sun Mountain Capital.
The five-point agreement was drafted by the SIC's legal counsel to address concerns discussed at the council's meeting on March 27. The SIC agreed to hear a motion in April by council member Leonard Lee Rawson to terminate Sun Mountain because of investments in Growstone Inc., a startup created by political backers of former Gov. Bill Richardson.
Rather than fire Sun Mountain, the SIC's Private Equity Advisory Committee proposed to reinforce transparency, accountability, reporting and internal controls in its contract with Sun Mountain, said SIC spokesman Charles Wollmann.
Sun Mountain advises the SIC on private equity investments in venture capital funds in New Mexico, and it manages the SIC's $90 million Co-Investment Fund for investments in local startups.
"The council took that up at its meeting today and voted unanimously to accept amendments [to Sun Mountain's contract]," Wollmann said. "The amendments heighten standards to ensure that the right investments are being made."
The agreements commit Sun Mountain to:
- accept management oversight of Co-Investment Fund activities by an independent third party;
- provide an update on Fund activities next month or soon after;
- reinforce ethical standards by disclosing to the SIC information about investments that could reflect negatively on the state's image;
- outline exit strategies from the Co-Investment Fund's portfolio companies before the end of fiscal year 2013; and
- report twice annually on Sun Mountain expenses and management fees in fulfilling its contracts with the SIC.
Wollmann said some of the increased reporting that Sun Mountain has committed to could be done in executive session or some other confidential format to protect companies that have received state investments.
"The council is concerned about nondisclosure of sensitive information," Wollmann said. "It's adding transparency in a way that will not damage investments."


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We fancied Pizza for lunch so went to Paradiso. Good selection of Pizza. What we had was very good. Very tasty cooked well and presented well. Good clean restaurant, friendly staff and good quick service. Value for money. Give it a try.
1 - 10 of 447 reviews
We fancied Pizza for lunch so went to Paradiso. Good selection of Pizza. What we had was very good. Very tasty cooked well and presented well. Good clean restaurant, friendly staff and good quick service. Value for money. Give it a try.
Nice spot at the heart of Senegambia. The spot is very lively and staff are friendly and helpful.We ordered for vegetable Pizza which was nice.
Excellent food, good range of choices on the menu. I have visited many times and never had cause to complain. The staff are extremely friendly and I am always made to feel welcome.
Amazing food, good quick service. A favourite in Senegambia The food is reasonably priced and your not hassled to go in like some places here
Cheap and cheerful for the Senegambia on the drag road, but not that cheap. A bulk tourist eatery rather quiet in early June, not so full so you notice the worn tiled floor damaged plastic seating and rather garish bright lighting and strong florescent colours....More
Clean, excellent service, very friendly staff, never had a bad meal, the lime drink with mint on the top with ice cubes so refreshing. We went there over and over during the month I was there and took different friends there too. Definitely coming back....More
Even in the off season, the place look really busy ، that's very good with high quality of food and very reasonable price.. excellent manager with top service ... the pizza is amazing, and it's the best in the Gambia so far , with a...More
Came with a few other people, we had just got into the country and wanted something simple. We all had pizzas, some small which I can tell you, small meant finished in 4 bites they were child pizzas if that! The rest of us had...More
Everything from the food to the team are fantastic ,we eat at paradise every time we visit,the pizzas are great lots to choose from they also do a varied menu the beers are ice cold and after a hot day it's a must, very popular...More
We have aeten several times in this restaurant. The food was from a high quality. The drinks are very good The sevice was excellent. Overall it is fantastic place to eat, great ambiance, always smiling waiters and the guy who runs the place is very...More


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Roosevelt Apartments
Aberdeen, SD 2013


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How far along: 24 weeks.
Total weight gain: Probably up from the 12 lbs last week. I'm not weighing myself though.
Maternity clothes: yep, not all the time though.
Stretch marks: nope! but I'm not looking!
Sleep: Still not sleeping great. I haven't been using the Snoogle very much because I like to use my own pillows for my head and with that thing I have to use it by itself basically. I find that if I go to bed a little hungry that I have less heart burn and sleep better.
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Miss anything: Sleep, energy
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Happy or moody most of the time: Happy!
Looking forward to: The weekend!


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Liposuction is the process of getting rid of stubborn fat in certain areas by getting the fat literally suctioned out of those areas by a doctor. Alternatives to this liposuction are juicing to get rid of stubbornly lodged fat in problem areas such as the stomach, thighs, hips, and butt area. Fat forms when bad foods are eaten such as fried foods, refined sugars, and lots of fat. One juice that helps to cut the fat is a fast of grapefruit juice, pineapple and lemons. Drinking this juice for 3 days will help to get rid of stubborn cellulite and the skin will also glow because this juice is full of enzymes.


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Israel News
Middle East
American Politics
Edition française
Green Israel
Joint session of the US Congress 311 (R).
(photo credit:Jim Young / Reuters)
The US Senate was expected to vote early on Tuesday on a deal to avert $600 billion in automatic tax increases and spending cuts in the "fiscal cliff" that could hobble the economy if allowed to take effect this week.If this alternative to the fiscal cliff passes the Senate, the House of Representatives could also vote on the measure later on Tuesday.
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The Board of Directors of Gulf Investment Services Holding Co. S.A.O.G. is pleased to invite the shareholders of the Company to attend the Annual General Meeting (AGM) scheduled to be held Haffa House Hotel (Sohar Hall), Muscat on Tuesday, March
The Board of Directors of Gulf Investment Services Holding Company SAOG met on Thursday, 26th February 2015 to approve the draft audited accounts for the year ended 31 December 2014. The Parent Company has made a net profit of RO
Gulf Investment Services Holding Co. SAOG would like to inform the market that its subsidiary company, Gulf Baader Capital Markets SAOC (GBCM) has acquired the Oman based brokerage business of Bank Muscat which provides brokerage services for securities traded on
Total assets of Islamic banks and windows in Oman reached to RO3.4bn at the end of April 2017 compared with RO2.54bn a year ago, recording a robust growth of 34 per cent. Islamic banking industry now accounts...
Al Ahlia Insurance Company SAOG (under transformation) announced the opening of its initial public offering (IPO) for subscription. Al Ahlia's IPO, which opened for subscription on July 4, is the first IPO on the Muscat Securities Market...
Vision Insurance SAOG (under transformation) announced to launch its initial public offering (IPO) to raise nearly RO4mn from the primary market in Oman. The company is offering 25mn shares at a price of 162bz per share, according...
Fitch Ratings has revised the outlook on five Omani banks - Bank Muscat, HSBC Bank Oman, ahlibank, BankDhofar and Bank Sohar - to negative from stable. The outlook on National Bank of Oman (NBO) remained stable. Fitch...
The introduction of a harmonized Value Added Tax (VAT) and excise tax in the Gulf Cooperation Council (GCC) by 2018 constitutes an important policy reform that aims to reduce reliance on hydrocarbon revenues. According to the IMF,...
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In De Wolf Maritime Safety BV v Traffic-Tech International Inc ('The Cap Jackson') 2017 FC 23, the Federal Court in Canada has held that (1) undeclared on-deck carriage did not prevent the application of the Hague-Visby Rules to the bill of lading and (2) that the carrier was entitled to rely on the limitation provisions in art. IV(5) of the Hague-Visby Rules. The decision on both points is in accordance with English law on the Hague-Visby Rules and deck cargo.
Category: Sea Carriage
Something fishy in the containers. Package limitation under the Hague Visby Rules
The High Court has just tackled the thorny issue, raised in the Australian case of The El Greco [2004] 2 Lloyd's Rep 537 of what is the applicable limitation of liability for loss or damage of goods carried in a container under a contract of carriage subject to the Hague-Visby Rules. Kyokuyo v AP Moller –Maersk [2017] EWHC 654 (Comm) involved carriage of three containers from Spain to Japan. The contract of carriage initially provided for the issue of straight bills of lading, consigned to the claimants, but the carrier and shipper subsequently agreed to issue seawaybills which were handed over to the consignee. As the contract of carriage contemplated the issue of bills of lading and the contract of carriage was made in Spain the contract of carriage was subject to the mandatory application of the Hague-Visby Rules under Rule X(b), even though the shipping documents that were issued were seawaybills.
The goods in the three containers were frozen tuna, some of which were carried in packages, some in individual units. The individual tuna pieces constituted 'units' for the purposes of package limitation and constituted the 'packages or units' of the cargo as packed. By operation of Article IV rule 5(c) of the Hague- Visby Rules they were the 'packages or units' for the purposes of Article IV rule 5(a). It sufficed that the language of enumeration was consistent with the truth (something that had not been the case in The El Greco). The limitation figure was presumed to be a single one for each container, unless the claimant could prove that there had been enumeration of the packages or units as packed within the container. The waybills had referred to the number of individual tuna pieces, on a 'said to contain' basis, but had not referred to the packages of tuna. The limit for the damaged goods in each container was a separate limit of 666.67 SDRs for each enumerated unit, the individual tuna pieces, and a single package limit of the larger of 666.67 SDRs or 2 SDRs per kilo of the gross weight of the damaged packaged tuna.
Bulk Carriage: Package limit or no package limit: That is the question.
The decision of the Commercial Court (Sir Jeremy Cooke sitting as a Judge of the High Court) in The Aqasia [2016] EWHC 2514 (Comm); [2016] 2 Lloyd's Rep. 510 (noted swiftly in this blog; a picture of the vessel in question appears here) has clarified an issue that has been at the heart of cargo claim negotiations for decades, namely whether a carrier of a bulk cargo is entitled to limit his liability under the Hague Rules. Article IV Rule 5 of the Hague Rules provides that: "Neither the carrier nor the ship shall in any event be or become liable for any loss or damage to or in connection with goods in an amount exceeding £100 per package or unit".
Whilst there had hitherto been no English authority directly on point the Commercial Court adopted the approach that has been followed by courts in Commonwealth countries and by textbook commentators and held that the words "package or unit" could not be applied to bulk cargo as they were intended to refer solely to physical packages or other units such as cars. It should be appreciated in this connection that the Hague Rules were adopted in the 1920s at a time when bulk ships were not common. Consequently, it appears that a carrier of a bulk cargo may have no right to package limitation under the Hague Rules.
By the time that the Hague-Visby Rules were adopted at the end of the 1960s, bulk shipments had become common and it was recognised that the Hague Rules wording was no longer fit for purpose. Consequently, Article IV Rule 5 (a) of the Hague-Visby Rules provided that a bulk carrier could limit his liability to "666.67 units of account per package or unit or 2 units of account per kilo of gross weight of the goods lost or damaged, whichever is the higher". The highlighted words were intended to provide a bulk carrier with limitation rights (i.e. based on weight) that were not available under the Hague Rules.
It should also be appreciated that the version of the Hague Rules that the USA adopted in its Carriage of Goods Act 1936 is also worded differently and provides that a carrier may limit his liability to"$500 per package…or in the case of goods not shipped in packages, per customary freight unit…" The phrase "customary freight unit" has been construed to refer to the unit of measurement that is customarily used to calculate the freight for that particular type of carriage (e.g. so much per ton or US Barrel etc) and not to a physical unit.
Therefore, it is important for a bulk carrier (particularly in high value claims) to determine which version of the Rules is applicable in any particular case. This is also important when the Rules are adopted by means of a Paramount Clause since there are many different types of Paramount Clauses some of which refer to the Hague Rules, some to US COGSA 1936 and some to the Hague-Visby Rules. A reference to the "Vague Rules" could be expensive!
Costs of defending cargo claim. Recovery under Inter-Club Agreement.
In London Arbitration 30/16 the tribunal held that where a claim was made against owners, in circumstances in which they incurred no liability to the claimants under the bill of lading, the costs of defending the claim were recoverable under clause 3 of the 1996 Inter-Club Agreement. This had been incorporated into the time charter on NYPE 1993 form.
The claim had been brought in a foreign court against the registered owners, the master, and the charterers and judgment had been given against the time charterers. In doing so the tribunal departed from a previous arbitration award in London Arbitration 10/15 where it was held that such costs could not be recovered when there was in fact no liability to cargo owners. Alternatively, the tribunal found that the owners would be able to recover under an implied indemnity as the cause of the cargo damage was the charterer's order to wait outside the discharge port for 35 days.
Delivery without bills of lading. Enforceability of LOI.
The Zagora (Oldendorff GmbH & Co KG v. Sea Powerful II Special Maritime Enterprises) [2016] EWHC 3212.
Where bills of lading are not available at the discharge port, it is common practice for the cargo to be discharged to the receiver against a letter of indemnity which will be conditional on delivery of the cargo to the receiver specified therein. The Zagora involved an allegation that delivery had not been made to the nominated receiver and the LOI was therefore unenforceable.
A series of indemnities down the chartering chain were given in respect of delivery in China in December 2013. When the bank brought a claim for misdelivery the owners called on their indemnity from the charterers who made a similar claim on the indemnity from the receivers. The indemnities required delivery to Xiamen, the first buyer in a chain of sales, or "to such party as you believe to be or to represent Xiamen… or to be acting on behalf of Xiamen." Delivery was made to the agent of Xiamen's sub-purchaser, Sea Road, and it was argued that the indemnities were not enforceable as there had been no delivery to Xiamen.
Teare J held that the indemnities were enforceable and that the sub-purchaser's agent was also Xiamen's agent, and, if that were not the case, then the owners believed that they had been acting as such. The master's recollection was that the representative of Sea-Road who boarded the vessel stated that he was there to handle discharge on behalf of Xiamen. The inevitable inference to be drawn from Xiamen naming itself as the person to whom the cargo should be delivered in the absence of an original bill of lading was that Xiamen intended that the nominated agent Sea-Road would take delivery of the cargo on its behalf. Conversely, the shipowners had no interest in discharging the cargo into the possession of Sea-Road as their own agent, as this would not provide the protection of the LOI because the owners would not have delivered the cargo to Xiamen, but would have retained possession of the cargo through Sea-Road.
Charterer's use of vessel as a floating warehouse an 'act' under the Inter-Club Agreement
In the Yangste Xing Hua [2016] EWHC 3132 (Comm) Teare J has construed the reference to 'act or neglect of' charterers or shipowners in cl.8 (d) of the 1996 Inter-Club Agreement as encompassing any act whether or not culpable. The relevant provision reads:
(d) All other cargo claims whatsoever (including claims for delay to cargo):
50% Charterers
50% Owners
unless there is clear and irrefutable evidence that the claim arose out of the act or neglect of the one or the other (including their servants or sub-contractors) in which case that party shall then bear 100% of the claim."
Cargo damage arose due to overheating while the vessel waited off the discharge port in Iran for four months. The trip charterers had ordered the vessel to wait there as they had not been paid for the cargo. The resulting cargo damage fell under cl.8(d) of the ICA and was 100% for charterer's account as it had arisen from their 'act'.
How to read the Inter-Club Agreement: order a ship around at your own risk
Teare J today faced a neat point of interpretation of the ICA. In Transgrain Shipping (Singapore) PTE Ltd v Yangtze Navigation (Hong Kong) Co Ltd [2016] EWHC 3132 (Comm) Transgrain sold soya bean meal to Iranian buyers. To transport it they chartered 45000 grt bulker MV Yangtze Xing Hua on a NYPE time charter trip. The ICA was expressly incorporated. The vessel arrived: the buyers were decidedly leisurely about paying for the cargo or collecting it. Transgrain in response ordered the vessel to wait for four months, it being cheaper to pay hire and/or demurrage (which could then be billed to the buyer) than to warehouse the cargo ashore.
At the end of the period the cargo overheated. The owners settled a cargo claim by the consignees for about € 2.6 million, and then turned on Transgrain. In arbitration proceedings neither Transgrain nor the owners were found to have been at fault; the overheating had been caused, unsurprisingly, by sitting for about 18 weeks off the Iranian coast. As a result Transgrain argued that this was a case of "all other cargo claims" and argued for a 50-50 split under s.8(d) of the ICA. Owners countered that under the proviso to s.8(d) claims caused by the "act or neglect" of one or other party were for that party's account: Transgrain argued that "act", yoked as it was to "neglect", implied "negligent act" and that, there being no fault in anyone, the proviso fell away.
Teare J sided with owners: "act", he said, meant "act", no more and no less. Probably rightly, in our view. If you insist on using a vessel in a particular way – for example as a floating warehouse – and a third-party claim results, there is much to be said for the idea that this is something you do at your own risk. Mind you, this result now leaves plenty of work for lawyers in future cases in arguing about how far a given claim is caused by a given (faultless) act: but we can leave that for another day.
Limitation less limited
Limits of liability under the 1976 Convention go up in 2 days by just over 50%, no doubt to the resigned chagrin of P&I interests. Details here courtesy of our friends at HFW.
Volcafe reversed
No package limitation for bulk cargo under Hague Rules.
In The Aquasia [2016] EWHC 2514 (Comm) the English High Court has determined the effect of Article IV r.5 of the Hague Rules which provides that the carrier's liability for loss or damage to or in connection with goods shall not exceed £100 "per package or unit". The shipowners argued that Article IV r.5 can be applied to bulk or liquid cargo by reading the word 'unit' as a reference to the unit used by the parties to denominate or quantify the cargo in the contract of carriage, relying on the description of the cargo in the charterparty as "2,000 tons cargo of fishoil in bulk". The cargo claimants argued that the word 'unit' can only refer to a physical item of cargo, or to a combination of physical items bundled together for shipment.
Sir Jeremy Cooke, sitting as a judge of the High Court, found in favour of the cargo claimants' contention, concluding that "[t]he word "unit" in Article IV Rule 5 of the Hague Rules is not apt to apply to bulk cargoes and that even if it could apply, the only legitimate application would be by way of interpreting the word "unit" as "freight unit". This cannot be done in the present case in a way which gives rise to a lower limitation figure than the claim because of the lump sum nature of the freight."


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Round Locket Pearl Bracelet
A beautiful sterling silver locket is strung onto beautiful fresh water pearls either grey or pink. A lovely gift for a friend or loved one, especially if you want to keep precious photos of loved ones inside it! All lockets open. Could be a gorgeous gift for a bridesmaid or a bride on her special day. Each locket is a one off special item and individual to you. You can pick from either pink or grey fresh water pearls. Pink or grey 5mm fresh water pearls with round locket which is sterling silver and finished with our signature feather charm.


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BREAKING…..Iowa's Republican Governor to sign modernized HIV Law
The office of Iowa's Republican Gov. Terry Branstad has announced he will sign legislation to modernize that state's archaic HIV transmission law Friday in a public ceremony.
The bill will be signed at 2:15 p.m. CST in Branstad's formal office in Des Moines. The news comes as nearly 200 advocates from around the world descend on Grinnell College in Grinnell, Iowa next week for the first conference on HIV decriminalization. Source: hivplusmag.com