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It has been suggested that glutamatergic system hyperactivity may be related to the pathogenesis of Parkinson's disease (PD). Vesicular glutamate transporters (VGLUT1-3) import glutamate into synaptic vesicles and are key anatomical and functional markers of glutamatergic excitatory transmission. Both VGLUT1 and VGLUT2 have been identified as definitive markers of glutamatergic neurons, but VGLUT 3 is also expressed by non glutamatergic neurons. VGLUT1 and VGLUT2 are thought to be expressed in a complementary manner in the cortex and the thalamus (VL/VM), in glutamatergic neurons involved in different physiological functions. Chronic high-frequency stimulation (HFS) of the subthalamic nucleus (STN) is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced PD. STN-HFS is highly effective, but its mechanisms of action remain unclear. This study examines the effect of STN-HFS on VGLUT1-3 expression in different brain nuclei involved in motor circuits, namely the basal ganglia (BG) network, in normal and 6-hydroxydopamine (6-OHDA) lesioned rats.
Here we report that: 1) Dopamine(DA)-depletion did not affect VGLUT1 and VGLUT3 expression but significantly decreased that of VGLUT2 in almost all BG structures studied; 2) STN-HFS did not change VGLUT1-3 expression in the different brain areas of normal rats while, on the contrary, it systematically induced a significant increase of their expression in DA-depleted rats and 3) STN-HFS reversed the decrease in VGLUT2 expression induced by the DA-depletion.
These results show for the first time a comparative analysis of changes of expression for the three VGLUTs induced by STN-HFS in the BG network of normal and hemiparkinsonian rats. They provide evidence for the involvement of VGLUT2 in the modulation of BG cicuits and in particular that of thalamostriatal and thalamocortical pathways suggesting their key role in its therapeutic effects for alleviating PD motor symptoms.
Keywords:High frequency stimulation; Subthalamic nucleus; Parkinson's disease; Basal Ganglia; 6-OHDA-lesion; Rat; Glutamate; Vesicular glutamate transporters
It is long recognized that the degeneration of dopaminergic neurons induces an abnormal activation of glutamate systems in the basal ganglia (BG) that is central to the pathophysiology of Parkinson's disease (PD) [1-4]. Glutamate mediated mechanisms are also thought to play a role in the development of dyskinesias with long-term administration of L-3,4-dihydroxyphenylalanine (L-DOPA), the most efficient treatment for PD. Many experimental studies also evidence that dopamine denervation induces an increase in corticostriatal glutamate [5-11] and that L-DOPA-induced dyskinesia (LID) are linked to BG network glutamate transmission abnormalities [12,13]. Microdialysis studies have suggested that dopamine lesion may also increase glutamate transmission in the BG output structures, substantia nigra pars reticulata (SNr) [5,14-16] and entopeduncular nucleus [6], presumably as a result of the abnormal activation of the subthalamic nucleus (STN) [17].
Three subtypes of vesicular glutamate transporters have been identified: VGLUT1, 2 and 3 [18]. These transporters mediate glutamate uptake inside presynaptic vesicles and are anatomical and functional markers of glutamatergic excitatory transmission [19-25]. VGLUT1-3 are very similar in structure and function, but are used by different neuronal populations. VGLUT1 and VGLUT2 are expressed by the cortical and subcortical neurons respectively. VGLUT3 is expressed by nonglutamatergic neurons, such as cholinergic striatal interneurons, a GABAergic interneuron subpopulation from the cortex and hippocampus and serotoninergic neurons from the dorsal and medial raphe nuclei [22,26].
Since the 1990s, High Frequency Stimulation (HFS) of the STN has become an effective surgical treatment of late-stage Parkinson's disease (PD), improving all motor symptoms in PD patients, particularly in those who experience motor fluctuations [27-29]. However, the mechanisms underlying the improvement in symptoms remain unclear [30-32]. Beyond its local effect on STN activity, we know that, by activating axons, STN-HFS may generate widespread and heterogeneous distal effects throughout the BG network [32,33]. Indeed, we have already reported in previous studies that in intact or 6-OHDA (6-hydroxydopamine)-lesioned rats, STN-HFS increases extracellular glutamate in the striatum, the globus pallidus and the SNr [14-16,34].
The present study analyzed the effects of DA depletion and for the first time those of STN-HFS on VGLUT1-3 expression in several BG nuclei, by using immunoradioautography with affinity-purified rabbit VGLUT1, VGLUT2 or VGLUT3 antiserum.
We found that DA-depletion did not affect VGLUT1 and VGLUT3 expression in almost all BG structures studied while that of VGLUT2 significantly decreased. Interestingly, STN-HFS did not affect VGLUT1-3 expression in normal rats, but systematically increased their expression in most of the BG nuclei studied in DA-depleted animals.
According to the changes of VGLUT1-3 expression observed and to their known anatomical localization, we suggest that STN-HFS may achieve its therapeutic effect, at least in part, through normalization of the thalamostriatal and thalamocortical pathways.
Adult (5 to 7 weeks old) male Sprague–Dawley rats (Janvier, Le Genest St Isle, France), weighing 180 to 270 g, were housed in an animal room on a 12-hour light/dark cycle, with food and water supplied ad libitum. This study was carried out in strict accordance with the recommendations of the European Community Council Directive of 24 November 1986 (86/609/EEC) concerning the care of laboratory animals, French Ministry of Agriculture regulations (Direction Départementale de la Protection des Populations, Préfecture de l'Isère, France, Grenoble Institute of Neuroscience, agreement number: A 38-516-10-008; Marc Savasta, permit number 38-10-08, Carole Carcenac permit number 38-10-23) and French guidelines for the use of live animals in scientific investigations. The protocol was approved by the Committee on the Ethics of Animal Experiments of the "Grenoble Institute of Neuroscience ethical committee" agreement number 04. All surgery was performed under a mixture of xylazine and ketamaine and all efforts were made to minimize the number of animal used and their suffering. All operated rats were intraperitoneally treated with Rimadyl (1 ml.kg-1) to prevent post-surgery suffering.
Lesion procedure
Forty rats (n = 40) were anesthetized with a mixture of xylazine (10 mg.kg-1, intraperitoneal) and ketamine (100 mg.kg-1, intraperitoneal) and secured in a Kopf stereotaxic apparatus (Phymep, Paris, France). All animals received desipramine (25 mg/kg s.c.) pretreatment, to protect noradrenergic neurons. Lesioned animals (n = 20) received a unilateral injection of 9 μg of 6-hydroxydopamine (6-OHDA) (Sigma, St. Quentin-Fallavier, France) dissolved in 3 μl of 0.9% sterile NaCl supplemented with 0.2% ascorbic acid, administered at a flow rate of 0.5 μl · min-1 to the left SNc. An identical procedure was used for controls (n = 20) but with the injection of NaCl 0.9%. The stereotaxic coordinates for the injection site relative to the bregma were as follows: anteroposterior (AP), -5.3 mm; lateral (L), +2.35 mm; dorsoventral (DV), -7.5 mm, with the incisor bar at 3.3 mm below the interaural plane, according to the stereotaxic atlas of Paxinos and Watson [35]. After injections, animals were kept warm and allowed to recover from the anesthetic before being returned to the animal house for three weeks until the stimulation experiments. This time interval was left to allow the DA system degeneration induced by the neurotoxin to stabilize.
Implantation of the stimulation electrode
Rats from the two experimental groups (sham-operated controls, n = 20, and 6-OHDA lesioned, n = 20) were first anesthetized by the inhalation (1 l.min-1) of a mixture of 3% isoflurane in air (the air used being composed of 22% O2, 78% N2) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). The dorsal skull was exposed and holes were drilled for the implantation of the stimulation electrode into the left STN. During the implantation and stimulation procedure, anesthesia was maintained with an inhaled mixture of 1% isoflurane in air (1 l.min-1) and body temperature was maintained at 37°C with a feedback-controlled heating pad (Harvard Apparatus, Edenbridge, UK). Stereotaxic coordinates were chosen according to the atlas of Paxinos and Watson [35] and were as follows relative to the bregma: AP, -3.7 mm; L, +2.4 mm; and DV, -7.8 mm as previously described [14-16,34,36].
Electrical stimulation
For electrical stimulation, we used a concentric stimulating bipolar electrode (SNEX 100, Rhodes Medical Instruments, Woodland Hills, CA), with an outer diameter of 250 μm and a distance between the poles of 1 mm. Stimuli were delivered under anesthesia during 4 hours with a World Precision Instrument (Stevenage, UK) acupulser and stimulus isolation units giving a rectangular pulse. This duration of stimulation (> 1 h) was chosen to be sure that the proteic expression of VGLUTs can be detected and stabilized and almost corresponds to that used in previous studies analyzing mRNA levels of different target proteins of basal ganglia circuits [37]. As previously reported, the stimulation parameters (130 Hz, 60 μs, 200 μA) matched those routinely used in Parkinsonian patients [14,34,36]. At the end of each experiment, an electrical lesion was created in the STN so that the position of the electrode could be checked post-mortem. In control rats (sham-operated and 6-OHDA-lesioned) the stimulation was never switched "on".
At the end of the electrical stimulation, all animals were perfused transcardially with 0.9% saline, under chloral hydrate anesthesia. Brains were rapidly removed and frozen in cooled (−40°C) isopentane, then stored at −20°C. Serial frontal sections (14-μm thick) were cut with a cryostat (Microm HM 500, Microm, Francheville, France), collected on microscopic slides and stored at −20°C. Tissue sections from different BG nuclei and related structures (striatum (caudate-putamen), nucleus accumbens, motor and somatosensory cortices, thalamus (VL/VM), subthalamic nucleus, globus pallidus and substantia nigra pars reticulata (SNr)) were selected to analyze changes in VGLUT expression.
The correct location of the stimulation electrode was checked by collecting several subthalamic tissue sections (n = 12 sections per stimulated rat) (14 μm thick from AP, -3,6 to −4,3 mm relative to the bregma, Paxinos and Watson, [35]) and counterstaining with cresyl violet. The tip of the electrode was systematically implanted directly in the STN at the top of its dorsal part. These histological controls were systematically carried out for all the animals in each experimental group. All animals with incorrectly positioned stimulation electrodes were excluded (controls, n = 3 and 6-OHDA lesioned, n = 4).
We assessed the extent of the dopaminergic denervation induced by nigral 6-OHDA injection by TH immunostaining on striatal and nigral sections from the fixed brains of lesioned animals. TH immunostaining was carried out as previously described [14]. Briefly, striatal and nigral tissue sections from 6-OHDA-lesioned rats were mounted on silane-coated microscope slides. Tissue sections were postfixed in 4% paraformaldehyde, thoroughly washed with Tris buffered-saline (TBS, 0.1 M, pH 7.4) and incubated for 1 hour in 0.3% Triton X-100 in TBS (TBST) and 3% normal goat serum (NGS, Sigma-Aldrich, St Quentin Fallavier, France). They were then incubated with primary antisera diluted in TBST supplemented with 1% normal goat serum (NGS) for 24 h, at 4°C. The antiserum was diluted 1:500 for TH staining (mouse monoclonal antibody; Chemicon, Temecula, CA). Antibody binding was detected with avidin-biotin-peroxidase conjugate (Vectastain ABC Elite, Vector Laboratories, Burlingame, CA), with 3, 3'-diaminobenzidine as the chromagen. The detection reaction was allowed to proceed for one to three minutes, as previously described. Sections were dehydrated in a series of graded ethanol solutions, cleared in xylene, mounted in DPX (DBH Laboratories Supplies, Poole, UK) and covered with a coverslip for microscopy.
VGLUT 1–3 immunoradioautography
Tissue sections were air-dried, post-fixed by immersion in fixative (4% PFA), and then washed in PBS. Nonspecific binding sites were saturated by incubation with 3% bovine serum albumin (BSA) in PBS, 1% NGS and 2 mM NaI (buffer A). Sections were incubated overnight at 4°C in buffer A supplemented with affinity-purified rabbit VGLUT1, VGLUT2 or VGLUT3 antiserum (dilution 1/10000 for VGLUT1 and VGLUT2, 1/5000 for VGLUT3, from Dr Salah El Mestikawy), and then for 1 hour with an affinity-purified goat anti-rabbit [125I] IgG (0.25 μCi/ml, Perkin Elmer, Paris, France) in buffer A supplemented with 0.02% sodium azide. The sections were rinsed in water, dried and placed against X-ray films (Biomax MR, Kodak) for 9 to 11 days.
The specificity of all antisera used in this study have been previously validated by our group (Gras et al. [22], [38]; Herzog et al. [23], [26]). For each labeled section, a background value was estimated by measuring optical density in the corpus callosum, since this structure is devoided of specific staining for VGLUT 1–3 antibodies. This background value was then systematically subtracted from the optical density values obtained for each corresponding section.
Quantification and statistical analysis
For the evaluation of the extent of DA-denervation, striatal and nigral TH immunostained sections were directly processed by using the Calopix software of the computerized image analysis system (TRIBVN, 2.9.2 version, Châtillon, France). Six TH-immunostained sections from each structure (striatum and SNc) and for each rat were used for quantification. The loss of TH immunostaining in the SNc or in the striatum was evaluated by comparing the total surface of both structures, as revealed by the TH immunolabelling, in normal and lesioned animals.
For quantification of VGLUT1-3 contents, four AP levels (+1, -0.92, -3.8 and −5.5 mm relative to bregma (Paxinos et Watson, [35])) were choosen. For each rat, three stained sections of the same AP level were used for quantification and the triplicate OD values obtained for each structure analyzed were averaged. Immunoradioautograms obtained from X-ray films were analyzed with Autoradio V4.03 software (SAMBA Technologies, Meylan, France). Values of optical densities measured from each structure analyzed are expressed as a mean ± standard error (SEM) in Table 1. Histograms presented in figures show the mean ± standard error of the mean (SEM) of optical densities expressed as a percentage of control values. Data were analyzed for each brain structure by Kruskal-Wallis tests with SigmaStat 3.1 software. Post-hoc analyses were carried out with the Dunn's method.
Table 1. Effect of 6-OHDA-lesion and STN-HFS on bilateral changes of optical density measurements of immunoreactive signals for VGLUT1-3
Results and discussion
Histological controls of the extent of the dopamine lesion and of electrode location
Three weeks after the unilateral injection of 6-OHDA, all lesioned animals presented a substantial loss of TH immunostaining in the ipsilateral SNc and the striatum (caudate-putamen nucleus), as shown by comparison with the contralateral side (Figure 1A, B) or with control animals. An analysis of densitometric measurements of TH immunostaining showed an absence of statistical difference between the two lesioned groups (non stimulated and stimulated).
Figure 1. Photographs of TH-immunostained coronal rat-brain sections at the nigral (A) and striatal (B) levels and of cresyl violet-stained coronal rat-brain sections at subthalamic (C, D and E) levels in 6-OHDA-lesioned rats. Note, on the lesioned side (left), the loss of dopaminergic cells in the SNc (A) and the loss of dopaminergic terminals in the striatum (B). Note also the correct implantation of the stimulation electrode within the STN (C, D, E). C, The arrow indicates the electrode track. E, The asterisk indicates the point of stimulation. CPu: Caudate Putamen; Hip, Hippocampus; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; STN, Subthalamic nucleus; VTA, Ventral Tegmental Area. Scale bar, 0.75 mm.
In DA-depleted animals, the loss of SNc TH + neurons was evaluated by comparing the total SNc surface on the intact side with the homologous area on the lesioned side. A loss of 92 ± 5% (p < 0.001) of TH immunolabeled surface was measured. In the striatum of the same rats, the loss of DA nerve terminals, as revealed by TH immunostaining mainly affected the dorsal part of the striatum (Figure 1B). This loss affected around 83 ± 4% of the striatal surface as compared to the total striatal surface of the control side. In this denervated striatal area, TH immunolabeling, as evaluated by a mean of densitometric values, was decreased by 85 ± 5% (p < 0.001) when compared to the controlateral intact side.
The correct implantation of the stimulation electrode in the STN is illustrated in Figure 1C-E. Figure 1E shows, at a higher magnification, the small electrical lesion (asterisk) created at the end of the experiment, indicating the point stimulated.
Regional distribution of VGLUT1-3 in control rats (without lesioning and stimulation)
VGLUT1-3 expression was qualitatively analyzed in control rats that had been neither lesioned nor stimulated, to ensure the validity and specificity of the immunoradioautographical staining. Immunoradioautograms from the different sections showed a distribution of VGLUT1-3 similar to that previously reported [26,39], confirming the validity of our VGLUT1-3 staining procedure and the lack of cross-reactivity between the antibodies used.
VGLUT1 immunostaining was dense in almost all the structures studied, including, especially, the striatum, nucleus accumbens, cortex, the motor part of the thalamus (VL/VM) and hippocampus. By contrast, no VGLUT1 labeling was found in the globus pallidus, the substantia nigra and in most of the brainstem (Figure 2 E-H).
Figure 2. Regional distribution of VGLUT1-3 proteins in control (without lesion and stimulation) rats. A-D, Schematic diagrams adapted from the stereotaxic atlas of Paxinos and Watson [1982]. E-P, Photographs of immunoradioautograms obtained by incubating coronal rat-brain sections of control rats (non lesioned and non stimulated) with affinity-purified anti-VGLUT1 (E-H), anti-VGLUT2 (I-L) and anti-VGLUT3 (M-P) antisera and then with anti-rabbit [125I] IgG. Note the different distributions of the three VGLUTs in the brain structures studied. Acb, Accumbens nucleus; Amy, Amygdaloid nucleus; CPu, Caudate Putamen; Cg, Cingulate cortex; GP, Globus Pallidus; Hip, Hippocampus; PM Cx, Premotor Cortex; SNr, Substantia nigra pars reticulata; SS Cx, Somatosensory Cortex; STN, Subthalamic nucleus; Tha, Thalamus (VL/VM). Scale bar, 0.4 mm.
VGLUT2 proteins were detected in almost the same set of structures as VGLUT1 although the density of VGLUT2 immunostaining was slightly lower than that for VGLUT1 in striatal, cortical and thalamic areas, whereas the opposite was observed in many sub-cortical structures. These data are consistent with the well-described complementary pattern of expression of VGLUT1 and VGLUT2 in the rat brain. VGLUT2 staining, unlike that for VGLUT1, was detectable in the substantia nigra pars reticulata, hypothalamic nuclei and midbrain, which displayed widespread staining. Different, complementary patterns of immunostaining for VGLUT1 and VGLUT2 were observed in the hippocampus. The density of VGLUT2 proteins was highest in layers IV and VI of the cortex and in the hypothalamus, the central gray matter and the superior colliculus (Figure 2 I-L).
VGLUT3 staining was weaker than that for VGLUT1 and VGLUT2, but was also observed in many different areas. VGLUT3 levels were moderate in the striatum, but high in the hippocampus, with a complementary distribution for VGLUT1 and VGLUT2 (Figure 2M-P).
Effects of 6-OHDA-lesion and/or STN-HFS on VGLUT1-3 expression
DA lesion and STN stimulation were unilaterally performed in this study. However, we found similar changes of VGLUTs expression on both sides, as revealed by the optical density measurements of immunoreactive signals for VGLUT1-3 in all structures examined (see Table 1). In order to simplify the presentation of our data, we decided to only show the results obtained from the ipsilateral side (the lesioned and/or stimulated side) on Figures 3 and 4. As precised in materials and methods, changes in VGLUTs expression induced by 6-OHDA lesion and STN stimulation were first analyzed by Kruskal-Wallis tests with SigmaStat 3.1 software. Results of these tests for each brain structure are presented in legends of Figures 3 and 4. Post-hoc analyses were then carried out with the Dunn's method.
Figure 3. Effects of 6-OHDA-lesion and STN-HFS on striatal and cortical VGLUT1-3 expression. A-C, Histograms show the mean ± standard error of the mean (SEM) of optical density values expressed as a percentage of values of control rats (non lesioned and non stimulated). Data were analyzed for each brain structure by Kruskal-Wallis tests with SigmaStat 3.1 software. Post-hoc analyses were carried out with the Dunn's method. Kruskal-Wallis tests (VGLUT1, CPu, p = 0.024; Acb, p = 0.05; PMCx, p = 0.049; SSCx, p = 0.02), (VGLUT2, CPu, p = 0.009; Acb, p = 0.002; PMCx, p = 0.012; SSCx, p = 0.002), (VGLUT3, CPu, p = 0.003; Acb, p<0.001; PMCx, p = 0.043; SSCx, p = 0.002). a-l, Photographs of immunoradioautograms obtained by incubating coronal rat-brain sections with affinity-purified anti-VGLUT1 (a-d), anti-VGLUT2 (e-h) and anti-VGLUT3 (i-l) antisera and then with 125I-labeled anti-rabbit IgG. Acb, Accumbens nucleus; CPu, Caudate putamen; PM Cx, Premotor cortex; SS Cx, Somatosensory Cortex. *, controls vs 6-OHDA rats; #, controls vs 6-OHDA + STN-HFS rats; Δ, 6-OHDA rats vs 6-OHDA + STN-HFS rats: p < 0.05. Scale bar, 0.4 mm.
Figure 4. Effects of 6-OHDA-lesion and STN-HFS on thalamic, pallidal and nigral VGLUT1-3 expression. A-C, Histograms show the mean ± standard error of the mean (SEM) of optical density values expressed as a percentage of values of control rats (non lesioned and non stimulated). Data were analyzed for each brain structure by Kruskal-Wallis tests with SigmaStat 3.1 software. Post-hoc analyses were carried out with the Dunn's method. Kruskal-Wallis tests: (VGLUT1, Thalamus VL/VM, p = 0.036; STN, p = 0.536), (VGLUT2, Thalamus (VL/VM); p = 0.036; STN, p = 0.015; SNr, p = 0.049; GP, p = 0.048), (VGLUT3, Thalamus (VL/VM), p<0.001. a-l, Photographs of immunoradioautograms obtained by incubating coronal rat-brain sections with affinity-purified anti-VGLUT1 (a-d), anti-VGLUT2 (e-h) and anti-VGLUT3 (i-l) antisera and then 125I-labeled anti-rabbit IgG. GP, Globus Pallidus; SNr, Substantia nigra pars reticulata; STN, Subthalamic nucleus; Tha, Thalamus (VL/VM). *, controls vs 6-OHDA rats; #, controls vs 6-OHDA + STN-HFS rats; Δ, 6-OHDA rats vs 6-OHDA + STN-HFS rats: p < 0.05. Scale bar, 0.4 mm.
Effect of the 6-OHDA-lesion on VGLUT1-3 expression
DA depletion did not affect VGLUT1 and VGLUT3 expression whatever the brain structure analyzed. Slight changes were observed in the striatum, nucleus accumbens, somatosensory cortex and thalamus (VL/VM), but they were not statistically significant (Table 1, Figures 3 and 4, white versus dark grey histograms).
By contrast, 6-OHDA lesioning induced a significant decrease in VGLUT2 expression of nearly 50% with respect to that in sham-operated rats (Figures 3 and 4). This decrease was particularly strong in the STN, thalamus (VL/VM) (Figure 4B), and cortical areas (Figure 3B) which displayed decreases of 55%, 48% and 42% with respect to control (non-lesioned) rats, respectively (p < 0.05, n = 6).
Effect of STN-HFS on VGLUT1-3 expression in sham-operated control (non-lesioned) rats and in 6-OHDA-lesioned animals
No significant change in VGLUT1-3 expression was detected after four hours of STN-HFS in sham-operated control rats (non-lesioned) rats. Levels of VGLUT1-3 expression were similar between the two experimental groups (non-lesioned rats with and without STN stimulation) in all different structures studied, as show in Figures 3 and 4.
On the contrary, STN-HFS induced a marked increase in VGLUT1-3 expression in 6-OHDA-lesioned rats. This increase affected almost all the brain structures studied (Table 1, Figures 3 and 4). VGLUT1 levels were always higher for all brain areas studied in stimulated 6-OHDA rats when compared to non-stimulated 6-OHDA-rats except for the nucleus accumbens (Acb) and STN (Figure 3A, a-d, for examples of autoradiographs and Figure 4A, a-d). The largest differences were found in the somatosensory cortex (+52%) and the motor part of the thalamus (VL/VM) (+41%) (p < 0.05, n = 10). For many brain structures analyzed, VGLUT1 levels measured in stimulated 6-OHDA-rats were comparable to those detected in control (without lesion and stimulation) rats. Interestingly, for the striatum and the thalamus (VL/VM), VGLUT1 levels remained moderately overexpressed versus controls (+27%, p < 0.05 and +17%, p < 0.05, respectively, n = 10) (Figures 3A, a-d, and 4A, a-d).
STN-HFS induced a significant increase in VGLUT2 expression in all structures studied in 6-OHDA-lesioned rats when compared to non-stimulated 6-OHDA-rats. However this increase did not affect the globus pallidus. (Figures 3B, e-h and 4B, e-h). Thus, STN-HFS more or less completely reversed the decrease in VGLUT2 expression induced by the DA-depletion in all structures analyzed.
STN-HFS induced a strong upregulation of VGLUT3 expression in 6-OHDA-lesioned rats (Figures 3C, i-l and 4C, i-l). Interestingly, this effect was particularly marked in the thalamus (VL/VM) (+106% versus non-stimulated 6-OHDA-lesioned rats, p < 0.05, n = 10) and the somatosensorial cortex (+99% versus non-stimulated 6-OHDA-lesioned rats, p < 0.05, n = 10).
The key findings of this study were: 1) DA depletion decreased VGLUT2 (−40 to -50%) in all brain structures studied; 2) STN-HFS did not affect VGLUT1-3 expression in control (sham-operated) rats whatever the brain structure analyzed; 3) STN-HFS increased VGLUT1-3 expression in 6-OHDA-lesioned rats in almost all structures analyzed. Thereby STN-HFS: i) normalized VGLUT2 levels after the decrease induced by DA depletion, and ii) significantly increased VGLUT3 levels above those detected in control animals. This was also true for VGLUT1 levels but only for the striatum.
This remodeling suggests that the mode of action of STN-HFS results from a global effect on basal ganglia network and related structures and that its therapeutic efficacy may to be linked, at least in part, to the normalization of thalamostriatal and thalamocortical neurotransmissions.
Bilateral effects of unilateral DA lesioning and STN-HFS on VGLUT expression
As stated above, unilateral DA lesioning and STN-HFS caused similar changes in VGLUT expression on both sides of the brain. Bilateral effects of unilateral DA lesioning have already been reported in the striatum for tissue concentrations of glutamate [9], extracellular glutamate content assessed by microdialysis or voltammetry [34,40], glutamate receptor mRNA [41] and the glial glutamate transporter GLT-1 [42]. Similarly, unilateral STN-HFS has been reported to induce bilateral increases in striatal and nigral glutamate content [14,15,34]. These bilateral effects may result from crossed glutamatergic projections from the cortex or the thalamus (VL/VM) innervating the BG on the contralateral side, consistent with cross-talk in cortico-BG-cortical loops [42].
Effect of 6-OHDA-SNc-lesioning on VGLUT expression
We found here that 6-OHDA-lesions had no effect on VGLUT1 or VGLUT3 levels in any of the structures studied. By contrast, VGLUT2 levels decreased significantly three weeks after lesioning. At first glance, our observations contrast with previous reported data showing that dopamine depletion is associated with an increase in synaptic glutamate release [43-45] and with high striatal extracellular glutamate levels and glutamatergic activity [9,11,34,44,46-48] and greater thalamostriatal activity [48,49], two to four weeks after lesioning of nigral dopaminergic neurons. However, other studies have reported an absence of change in glutamate levels [50]. These differences may be accounted for by differences in the extent of the dopamine lesion, lesion sites, methodologies and time courses. We cannot exclude the possibility that different cellular mechanisms underlie presynaptic glutamate processes and extracellular glutamate release after lesioning. Indeed, striatal extracellular glutamate levels have been reported to depend on a complex balance between vesicular release and non vesicular release via glutamate transporters on both neurons and glia and the cysteine-glutamine antiporter [50,51]. Dopamine depletion leads to complex, biphasic changes in striatal glutamatergic transmission over the first few weeks, possibly stabilizing over three months. Contradictory data have been reported, for the cysteine-glutamate antiporter [52] and glial transporters [42,53,54] for example.
Furthermore, the changes in VGLUTs expression induced by 6-OHDA lesions are also complex. VGLUT1 levels increase in the three weeks following the injection but then decrease, whereas VGLUT2 levels decrease and then normalize [42,55-57]. In monkeys, MPTP treatment increases VGLUT1 expression but does not affect VGLUT2 levels [53,58]. In postmortem samples of Parkinsonian patients VGLUT1 and VGLUT2 levels are increased in the putamen while VGLUT1 levels is lowered in the prefrontal and temporal cortex [56].
However, the decrease in VGLUT2 levels observed here in all the brain structures of 6-OHDA rats closely parallels the thalamic hypoactivity induced by the strengthening of GABAergic inputs from the SNr and EP/GPi by the STN overactivity observed in DA-depleted BG networks [59]. Furthermore, neuronal degeneration has been observed postmortem in the thalamic nuclei of PD patients [60] and in the parafascicular nucleus in 6-OHDA-lesioned rats [49]. VGLUT2 is massively expressed by thalamic nuclei [61] and have be postulated as selective marker of thalamo-striatal activity [62]. This observation support the notion of a potential decreased glutamatergic afferences from the thalamus (VL/VM).
Effect of STN-HFS on VGLUT1-3 expression in control and 6-OHDA-lesioned rats
STN-HFS had no effect on VGLUT1-3 expression in any of the brain structures studied in control rats. This suggests that in the absence of dopamine depletion, STN-HFS did not affect VGLUT1-3 expression. These data are rather surprising since we reported in previous microdialysis study that in intact rats STN-HFS increases extracellular glutamate in the striatum, the globus pallidus and the SNr [14-16,34]. However, the duration of stimulation used here was longer than that used in our previous studies. Thus, we can speculate that increase of extracellular glutamate levels induced by STN-HFS in physiological conditions mainly involves non vesicular release. By contrast, after 6-OHDA lesions, STN-HFS induced an increase in VGLUT1-3 expression in almost all the structures analyzed. In these DA depleted conditions, we cannot exclude the possibility that the balance between non vesicular and vesicular release of glutamate is disturbed, involving more glutamate transporters on both neurons and glia [50,51]. Indeed, it is well documented that following DA nigrostriatal lesion, there is an increase in the number of glial cells, including astrocytes and microglia. Therefore, the non vesicular release may be due to an increase in membrane transporters, such as glial glutamate transporters (GLT1 and GLAST) and the neuronal glutamate transporter EEAC1 [55,63,64]. Thus, our previous findings concerning increased extracellular glutamate levels in the striatum, globus pallidus and SNr of 6-OHDA-lesioned rats [15,16,34] are consistent and confirm that STN-HFS affects not only its direct targets, but also more distant structures of the BG network [32,33]. The mechanisms underlying the therapeutic effects of STN-HFS are not fully elucidated. STN neuron inhibition by HFS, with loss of the drive of the internal part of the globus pallidus and disinhibition of the thalamus (VL/VM), would be consistent with the classical BG model [59,65]. However, far more complex effects and circuitry are probably involved. For example, the direct activation of nearby thalamostriatal and pallidonigral fibres [16,66] or direct or antidromic cortex activation [67,68]. These mechanisms might lead to corticostriatal fiber activation and the observed increase in VGLUT1 levels.
Similarly, the increase in VGLUT2 levels induced here by STN-HFS may reflect a release of the classical thalamic inhibition induced by dopamine lesions. As VGLUT2 is found mostly in the thalamostriatal neurons specifically affected by DA depletion, these data strongly suggest a major role for this pathway in the therapeutic effects of STN-HFS in PD [69,70]. However, the normalization of VGLUT2 levels we observed in the somatosensory and premotor cortices may also play a non negligeable role in these effects of STN-HFS.
Interestingly, VGLUT2 was also overexpressed in the SNr following STN-HFS in 6-OHDA-lesioned rats. As VGLUT2 is the only VGLUT expressed by STN glutamatergic projections to the SNr [71], this confirms our hypothesis that information transmission via the transsubthalamic pathway is not completely blocked during STN-HFS [15,16,31,32,67,68] although the effect on VGLUT2 expression in the SNr might also be mediated by the modulation of thalamic afferents during STN-HFS.
VGLUT3 is expressed in striatal cholinergic interneurons linked to the dopaminergic, GABAergic and glutamatergic systems in the cortico-BG-cortical loop [38]. It is therefore unsurprising that STN-HFS, which affects the whole BG network, affects VGLUT3 expression in the striatum and related structures, such as the cortex, hippocampus, nucleus accumbens, thalamus (VL/VM) and amygdala. Indeed, STN-HFS has been reported to affect not only dopamine, glutamate and GABA [14-16,34,36,68] but also serotonin and cholinergic transmissions [72-74]. Consequently, it is difficult to explain the role of VGLUT3 in STN-HFS mechanisms. However, VGLUT3 in cholinergic interneurons has been shown to increase acetylcholine tone and to release both glutamate and acetylcholine [18]. This mechanism might explain the efficacy of STN-HFS, via complex modulation of the BG network [32,33].
In conclusion, this is the first study of the expression of the three VGLUT subtypes in various brain structures in control and 6-OHDA-lesioned rats subjected to STN-HFS. We know that all the three types of VGLUTs are expected to be found in axon terminals and that immunoradioautography cannot really disclose between neuronal vs. axonal vs. glial expression of VGLUTs. In situ hybridization looking for different VGLUT transcripts will probably provide some added morphological value to our present study. These experiments are planned in our lab. However, our present data can suggest that STN-HFS may achieve its therapeutic effect, at least in part, through normalization of the thalamostriatal and the thalamocortical pathways.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Conceived and designed the experiments: MF, SB, SEM, MS. Performed the experiments: MF, GD, CC. Analyzed the data: MF, CC, MS. Contributed reagents/materials/analysis tools: SEM, MS. Wrote the paper: MF, SB, SEM, MS. All authors read and approved the final manuscript.
This work was supported by the Institut National de la Santé et de la Recherche Médicale, Université Joseph Fourier and Région Rhône-Alpes (ARC 2). We thank, Dr Emilie Lacombe for technical assistance and Dr Sebastien Carnicella for critical reading of this manuscript.
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MMA veteran Fabricio Werdum returned to the UFC against "Big Country" Roy Nelson, in what served as the co-main event of UFC 143 in Las Vegas, Nevada. Werdum defeated Nelson via Unanimous Decision after three grueling rounds, getting himself on track for a shot at the UFC Heavyweight title.
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Security and Other Technology Twitters for the Week Recently updated !
Another fine week passes with more and more interesting security issues arising, and not many success stories seen about how everything is nice and secure.
Chinese promotion company hijacks Android devices around the world – http://t.co/DHj4XMUQ2G – @FireEye pic.twitter.com/BSIvDXsadN
— Help Net Security (@helpnetsecurity) September 24, 2015
I don't want to look like some kind of Apple Lover mind you, and yes, Apple has had a major security breach this past little while as well, so don't get too smug all you hipster Apple lovers.
Biggest security breach in iOS store's history, hackers targeted Chinese developers. http://t.co/h1WY4JhbfX pic.twitter.com/EP3reaLqYV
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I always like it when hackers mix it up, and this story warms my heart, hackers fix 10,000 home routers to make them more secure? Thank you Hackers!
Someone has hacked 10,000 home routers to make them more secure: http://t.co/b0UGhcBaWS
— Forbes Tech News (@ForbesTech) October 2, 2015
Don't feel like doing anything much about security? You are not alone, evidently the Pentagon didn't really feel like doing much about things either.
Report: Pentagon made fewer than half of recommended cyber fixes last year: http://t.co/ddKlU2zNAS pic.twitter.com/ap9Um0U2a4
— Nextgov (@Nextgov) October 2, 2015


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Vote dooms 4-H: 'Lot of tears this morning'
EUGENE, Ore. -- Wednesday was Paisley Makinson's 16th birthday, but she's not in the mood to celebrate.
"I'm having a really hard time," Makinson said.
Makinson is one of 450 Lane County students active in 4-H, a program that will end Sept. 30. Voters turned down a tax measure, taking away all 4-H funding.
"When the results came out I bawled my eyes out," Makinson said.
The measure would have also funded the OSU Extension Service in Lane County for five more years. The Eugene office will now have to close. Steve Dodrill is the staff chair. He said it's been a rough day.
"Lot of tears this morning," Dodrill said.
Four faculty members will be re-located to other OSU offices. Locally supported staff may be laid off. Master Gardeners, Master Food Preservers, Climate Masters, Compost Specialist and the Extension Study Group will also end.
Dodrill said the Eugene office helps 65,000 people a year. Private funding is an option but even that has challenges.
"We have facilities here that serve all of those programs and have shared infrastructure costs," said Dodrill. "So if only one program for instance is saved, the costs of saving that program are going to be much higher if only one program is saved."
Makinson still has one more big event to prepare for, the 4-H Youth Fair in July.
"It's just going to be devastating," Makinson said. "Every corner I turn, I'm just going to remember, it's over this year."
To be clear, the federally funded nutrition education program will not end although it needs a new home now. The tax would have been ten dollars a year for every $200,000 of property value.


text:
Water Quality Products Current Issue
October 13, 2015
To help detect leaks, conserve water and assist with California's water mandate, Jones and Jones Management Group, one of the state's largest apartment management companies... more
October 13, 2015
B.F. Environmental Consultants, an environmental consulting firm providing services throughout the Northeast, announced the launch of a new mobile app that will make it... more
October 12, 2015
A material created by University of California, Riverside (UC Riverside) engineers is the key component of a swimsuit that won an international design competition... more
October 12, 2015
H.A. "Bill" Mills, CEO of 2M Co. Inc. in Billings, Mont., has received the Supplier of the Year Award from the National Ground Water Assn (NGWA). This award is presented to... more
October 13, 2015
Winter is nigh, and that means one thing in many regions: snow. While many dread the thought of winter snow due to the havoc it can cause in day-to-day life, snow is an... more
October 13, 2015
As an attendee of many conferences and networking events, I have found that a business card is regarded with pride as a first impression willingly given to anyone who will... more
October 13, 2015
Did you know that Section 32 of Quebec's Environmental Quality Act currently outlines requirements for potable water products that are beyond those of U.S. states and other... more
October 13, 2015
Both traditional and emerging treatment technologies can be effective at reducing or removing contaminants. There are four primary types of treatment modalities. Aesthetic,... more
October 13, 2015
Point-of-use (POU) and point-of-entry (POE) water treatment for microbial contaminants requires the end user and sales and service personnel to select their solutions wisely... more
Zurn PEX QickCap crimp rings with positioning end cap for crimp connections in plumbing and radiant heating applications virtually eliminate ring movement during installation, making it easier to accurately position the copper crimp rings onto tubing. The crimp rings are an ASTM F1807 fitting... more
Zurn PEX QickCap crimp rings with positioning end cap for crimp connections in plumbing and radiant heating applications virtually eliminate ring movement during installation, making it easier to...
The residential AquaCafé r is a single-unit water cooler that makes gourmet coffee and tea and dispenses purified hot and cold water for small offices and homes. It provides single-serve, freshly...
IntegraFlux UF modules feature XP fiber, which offers up to 35% higher permeability than previous-generation UF fiber. XP fiber provides higher flux, higher water recovery, lower waste, and reduced...
The AccUView LED online UV analyzer is a cost-effective, low-maintenance instrument for monitoring transmission and absorbance of drinking water sterilized by a UV system. It incorporates a UVC LED,...
Smart 'N Soft high-efficiency water conditioners use a metered control valve and "smart brining" technique to conserve salt and water. Resin tanks employ the dual-chamber Vortech distribution system...
The company offers thousands of fittings manufactured using FDA-compliant materials. Its extensive selection of products includes filters, check valves, plastic and push-in fittings, quick connectors...
The 304 SS three-stage wall mounted system provides particulate reduction, microbial retention and high-dose 186-mJ/sq cm UV. Its RLC monitor provides failsafe monitoring with alarms. It is useful...
The eXact iDip photometer with Bluetooth Smart technology is the first handheld photometer that pairs directly with a smart phone/tablet. It allows the ability to test more than 40 water...
The 5800 Fleck legacy continues with the new 5810 1-in. valve and 5812 1-¼-in. valves. Discover a common solution for a wide variety of filtration and softening applications with the Pentair 5800...
The company introduces the first Bluetooth-operated control valve. It features advanced diagnostics, simplified setup, water use history, a first-class Interface and Rental Lockout...


text:
April 27, 1934 - June 16, 2007
NORWALK - James L. Smith, 73, of Norwalk, died Saturday afternoon at Stein Hospice Care Center in Sandusky following a lengthy illness.
He was born April 27, 1934 in Norwalk.
He was employed for 43 years at the Whirlpool Corp., retiring in 1997. He was a member of St. Alphonsus Catholic Church, Knights of Columbus, St. John Newman Chapter No. 1117, Bronson Conservation League, Moose Lodge No. 1247, of Norwalk and the American Legion Post 547 of Monroeville, where he was one of the fish fryers. He was an avid euchre player, loved to go to auctions and garage sales, hunt and fish.
He is survived by his wife of 50 years, the former Theresa R. Ott, whom he married June 28, 1956; sons and daughters-in-law, Michael Smith of Norwalk, David and Jenny Smith of Willard, Joe and Jenny Smith of Monroeville; daughters and sons-in-law, Joann and Mike Meyer of Monroeville, Doris and Rick Whitehouse of Huron, Rose and Dave Stanley of Norwalk, Patty and Troy Wilhelm of Fremont and Karen and Steve Littlejohn of Monroeville; 22 grandchildren and seven great-grandchildren; six brothers and sisters-in-law, Alfred and Carolyn Smith of Columbia, S.C., Howard and Sharon Smith, Thomas Smith, both of Monroeville, Lawrence and Lisa Smith, William and Susanne Smith, both of Bowling Green, Va., and Steven and Debbie Smith of Perrysville; sisters and brothers-in-law, Sr. Frances Smith of Mexico, Bernadette and Jose Caramat of Dayton, Marcella and Don Schaffer, Theresa and Charlie Schaffer, both of Norwalk, and Marianne Brock of Chicago, Ill.; and nieces, nephews and other relatives.
He was preceded in death by his parents, Clemons and Blandina (Schurr) Smith.
Friends may call from 2 to 4 and 6 to 9 p.m. today at the Pfeil Funeral Home, Monroeville Chapel, 109 Monroe St., Monroeville, where Christian Wake Services will be held at 4 p.m. today. A Mass will take place at 10:30 a.m. Tuesday at St. Alphonsus Catholic Church, Settlement Road, Norwalk. The Rev. Kenneth J. Lill will officiate. Burial will follow in the parish cemetery.
Memorial gifts may be sent to the St. Alphonsus Building Fund, Settlement Road, Norwalk, Ohio 44857 or to Stein Hospice Service, 1200 Sycamore Line, Sandusky, Ohio 44870.


text:
Lessons from a FinTech CEO
For some people, fear and money go hand in hand
Join the NASDAQ Community today and get free, instant access to portfolios, stock ratings, real-time alerts, and more!
Say so long to your aching back. Like Apple's razor-thin MacBook
Air, the new Windows machines called Ultrabooks are ultralight-most
weigh in at 4 pounds or less. Many eschew hard drives and DVD
drives, saving space, reducing noise and conserving power. Like
tablets and smart phones, they start up instantly and save files
and apps on a flash memory chip, also known as a solid-state drive
Ultra Thin Laptops Worth the Cost?
Before you buy, take the machine for a test-drive. Does it feel
solid, and is the keyboard big enough for your hands? Is the screen
big and bright enough? Make sure the laptop has at least two USB
ports-preferably ones that support the faster USB 3.0 standard-and
an HDMI port for connecting to an HDTV. And don't forget about
battery life. Some Ultrabooks promise to run for as long as 11
hours on a charge; others power down after five hours or less.
Powerful and thin.
Acer Aspire S5
($1,400) holds its own with a comparably equipped MacBook Air and
costs $100 less. It is amazingly thin-about a tenth of an inch
thinner than the Air-and has a sharp, 13.3-inch display. At 2.65
pounds, the Aspire weighs just slightly less than the 13-inch Air.
Plus, the Aspire S5 has a 256-gigabyte solid-state drive, same as
the Air.
The S5's most clever innovation is a motorized door on the back
of the system that opens to reveal four ports for connecting
peripherals. The Dolby sound system is crisp and clear for a
laptop, and the S5 comes with a wireless Bluetooth mouse-handy for
those of us who loathe laptop track pads. The Aspire S5 does have a
few shortcomings. Unlike the Samsung machine described below, its
keyboard isn't backlit. Plus, given the laptop's high price, I'd
have expected screen resolution better than the usual 1,366-by-768
Big-screen beauty.
Samsung Series 9
($1,400 to $1,800, depending on configuration) manages to squeeze a
big, 15-inch display into a 14-inch chassis that's only 0.58 inch
thick. Weighing 3.6 pounds, the laptop features a curvy, black case
made of dura�lumin (an aluminum alloy), and the screen
boasts a 1,600-by-900-pixel resolution that is better than the
MacBook Air's display. (Samsung also offers a 13-inch Series 9 for
$1,300 or $1,400.) Among the downsides: the price. Also, most
models have a 128GB SSD-half the size of the Aspire and high-end
MacBook Air SSDs and a bit skimpy for a high-end Ultrabook.
Bargain alternative.
Dell Inspiron 14z
($700) skirts the line between a laptop and an Ultrabook. It
includes both DVD and hard drives, and it weighs a relatively hefty
4.12 pounds. Still, it is only 0.83 inch thick, has a 14-inch
display and includes a 32GB SSD for fast start-ups. The 14z won't
win any design awards, but it's a solid, affordable laptop for home
and school use. Unlike some Ultrabooks, it has a dedicated graphics
card that enables it to play videos smoothly. And the spillproof
keyboard is a nice safety feature when you're sipping java in the
wee hours.
Looking ahead.
Ultrabook sales have been disappointing so far, largely because of
steep prices of $1,000 or more. But the average price is expected
to fall to about $800 by the end of the year, according to research
firm IDC. And dozens of Ultra�books are arriving in the
coming months from major vendors. The new Ultra�books
will be loaded with Micro�soft's Windows 8 software,
with its redesigned interface tailored for touch tablets and PCs
(See our story:
Review of Microsoft's Windows 8
). We'll report on them as they hit the market.
This article first appeared in Kiplinger's Personal Finance
magazine. For more help with your personal finances and
investments, please
subscribe to the magazine
. It might be the best investment you ever make.
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text:
11.26.10 Mark Boals Radio Interview – 11/27
Mark Boals will be interviewed on the Toronto Rock Radio Show at 9:00pm on Saturday November 27! Listen online at www.maximumthreshold.net
10.04.10 STHW Live Radio Performance!
Listen to Tony Palkovic's Radio Show on KSPC 88.7FM (heard in the greater Los Angeles area) on Tuesday 10/5/10 12 – 2PM (Pacific).
Guests will be Grammy Nominee Tony MacAlpine (CAB, Steve Vai, Planet X and Ring of Fire), Mark Boals (Yngwie Malmsteen, Ring of Fire, Ted Nugent, Uli Jon Roth and Royal Hunt) and Stefania Daniel of Seven The Hardway. The band will perform live and talk about their recently released self-titled album.
For more info visit: www.kspc.org
09.13.10 New Album Out Sept 14 in USA
Seven The Hardway's debut album will be released tomorrow – September 14th in the USA through iTunes, Amazon.com and other fine digital and retail music outlets.
Preview a stream of the entire album at iLike! Don't forget to share with a friend!
09.06.10 European Tour Cancellation
We regret to announce the cancellation of the Seven The Hardway tour of Europe.
Due to the delayed release of the album (August instead of April) and the booking of the tour immediately following European summer vacation, there has not been adequate opportunity to properly promote the record or the tour.
We apologize for the inconvenience. For ticket refunds, please visit your original place of purchase.
We plan to re-book in the future, once the new album has had a chance to be heard by our fans.
As always, we thank you for your continued support.
Tony, Mark, Virgil, Doug and Stefania
Seven The Hardway
08.23.10 New Album Available Now!
Seven The Hardway's debut album is released today in Europe!
The album is available through the EU iTunes Music Store, Amazon Europe (CD and download), and Mascot Records EU.
The album will be released in the USA on September 14.
Check out samples of every song on the album on The Album page!
08.23.10 Band Image Gallery Added
We've added a new image gallery in the Media section of the site! Check out photos of the band!
08.10.10 Tony MacAlpine Interview
Tony talks to IHeartGuitarBlog about the new Seven The Hardway album, the writing process, his gear and the upcoming tour!
Read more: http://iheartguitarblog.com/2010/08/tmac.html
07.27.10 Follow us!
Follow us and share with your friends on:
Thanks for your support! We hope to see you on the road!
Tony, Virgil, Mark, Doug and Stefania
07.27.10 Tour Dates Update
A date change for the upcoming tour dates in Lorsch, Roncade and Rome:
- Sept 29: Lorsch, DE – Musiktheater Rex
- Sept 30: Roncade, IT – New Age Club
- Oct 01: Rome, IT – Crossroads
Entry to the show at Crossroads in Rome is free!
For more tour dates, check the TOURS page. More dates will be added soon.
07.23.10 Liar
Preview the track "Liar" from Seven The Hardway's forthcoming album – due out August 30!Seven The Hardway - Liar
- 11.26.10Mark Boals Radio Interview – 11/27
- 10.04.10STHW Live Radio Performance!
- 09.13.10New Album Out Sept 14 in USA
- 09.06.10European Tour Cancellation
- 08.23.10New Album Available Now!
- 08.23.10Band Image Gallery Added
- 08.10.10Tony MacAlpine Interview
- 07.27.10Follow us!
- 07.27.10Tour Dates Update
- 07.23.10Liar
- 07.22.10European Tour Dates
- 07.01.10Welcome


text:
The Mentalist: Season 3, Episode 17
Bloodstream (10 Mar. 2011)
LaRoche demotes Agent Lisbon and puts Cho in charge of the team as they investigate the murder of a doctor.
Director:Bobby Roth
Writers:Bruno Heller (creator), Erika Green Swafford (as Erika Green Stafford)
Stars:Simon Baker, Robin Tunney, Tim Kang | See full cast and crew »
Watch Now
From $1.99 on Amazon Video


text:
Earn up to 5% on hotels and 1% on flights - It's free!
By clicking, I agree to the terms and conditions.
= Cheapest round-trip flights
* Prices are round trip per person (conditions apply)
Earn up to 5% on hotels and 1% on flights - It's free!
By clicking, I agree to the terms and conditions.
...The hotel is located 7 miles from Eagle County Regional Airport and offers a free airport shuttle.... More
...A free shuttle takes guests to and from Eagle County Regional Airport (EGE) and Vail Valley Jet Center from 6am - 9pm to keep your travel costs low.... In addition, Century Link, Edward Jones... More
...This Central Colorado hotel also features business services, meeting and banquet facilities and a courtesy shuttle to/from the Eagle Vail Airport upon arrival and departure-reservations or... More
...Comfort Inn offers a free shuttle to and from Eagle Valley Airport.... More
...Transportation Vail/Eagle County Airport (6 mi/9.6 km).... More
Orbitz.com can help you find cheap flights from Columbus, Ohio, for your next trip. Columbus is served by the Port Columbus International Airport, located at 4600 International Gateway. It is about six miles east of the city center. It has three terminals and 44 gates. Domestic and international flights are provided by over 20 airlines, including the American Eagle Network, United, American Airlines and US Airways.
The Port Columbus Airport has three lots and a garage available for parking with complimentary shuttles. Fees are from $4 to $9 per day for long-term parking and $2 to $3 per hour for short-term parking. There is also bicycle parking and a cellphone lot.
Metered taxis are available to transport you to the airport. The fare from downtown Columbus to the airport is approximately $25. There are also daily shuttles from the downtown area. Columbus also has bus service. The fare is $2.00 one-way or $4.50 for an all-day pass. Not only can Orbitz.com help you plan your air travel, it can also help you plan your ground transportation.
There are many restaurant options available at the Port Columbus International Airport, so you can grab a bite to eat while waiting for your flight. Er King, Cold Stone Creamery, Tim Hortons and many more ensure that there will be something that everyone likes. There are also shops, so you can purchase anything you forgot to pack. Checking Orbitz.com for cheap flights to Columbus is your best bet for finding bargain tickets.
The best budget plans for flights to Eagle are made using Orbitz.com. You get to book low-cost flights to Vail Valley and ski resorts in Western Colorado via Eagle County Airport (EGE). The airport is four miles from Eagle and served by United, Delta and American. It has a single runway and one terminal with five gates.
Hertz, Budget and Avis are some of the car rental companies providing ground transportation services. Orbitz.com even offers advance booking services of the Eagle County Airport shuttles that operate between the airport and ski resorts in Vail, Aspen and Beaver Creek. You pay for your Colorado Mountain Express ticket at a counter near baggage carousel 3 and get to Vail in 45 minutes or Beaver Creek in 30 minutes.
Eagle experiences an annual average high of around 61 degrees Fahrenheit and a low of 27 degrees Fahrenheit. It also gets an average of 11 inches of rain each year. On average, the town experiences 244 sunny days annually.
If you love golfing, you need to visit Eagle Golf course, located at 50 Lime Park Drive. Other areas that you might want to visit include Eagle Dog Park, Sylvan Lake State Park and Adam's Golf Course. January is the best time to visit, as the snow conditions are perfect for skiing, and there are many seasonal flights at this time of year. Whether you seek cheap flights to Eagle or budget accommodation deals, Orbitz.com is your ultimate trip planner.
|Departing Arriving
|Carrier Flight #
|Flight time
DepartingCMH 4:45 PM
ArrivingEGE 7:31 PM
United Airlines/United Airlines
|Aircraft E70/DH4
|Flight time 4hr 46min
Book a qualifying 4-night flight + hotel vacation package between August 13, 2015 12:00AM CT and September 30, 2015 11:59PM CT, for $1750 or more for travel between August 13, 2015 and December 31, 2015 via Orbitz and instantly receive $150 off the hotel portion of your booking through the use of the promotion code. Limit one discount per hotel room and one promotion code per booking. Discounts are not redeemable for cash for any reason. Promotion codes cannot be resold. Any attempt at fraud will be prosecuted to the fullest extent of the law. Void where prohibited, taxed or restricted. Orbitz reserves the right to change or limit the promotion in its sole discretion. Promo code not valid for Las Vegas, NV bookings.
Fare, taxes & fees: Fares include all taxes and fees, except baggage fees. Rates shown reflect fares found by other Orbitz users over the past 48 hours. Due to rapidly changing airline prices & seat availability, these fares may no longer be available.
Refunds/changes/cancellations: Itinerary changes, if permitted for the fare, will have a $30 service fee charged by Orbitz. Additional airline charges may apply.
Other conditions: Schedules, fares and rules are subject to change without notice. Seats are limited and may not be available on all flights/dates or in all markets. Lower fares may be available. Fares will not be honored retroactively or in exchange for any wholly/partially unused ticket. Tickets are non-transferable and non-refundable. Fare rules are provided for the selected itinerary before booking.


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Free Shipping
Product Information:
Whether you're packing for an overnight trip or commuting into the city, these bags help keep you organized on the go. Zero Halliburton Profile bags feature durable Cordura nylon, leather trimmed handles and zipper pulls as well as padded electronic pockets.
Product Details
Strap Included
Weights & Dimensions
|Product Type: Briefcase
|Color: Black
|Primary Material: Fabric
|Top Loading: Yes
|Laptop Compatible: Yes
|Wheeled: No
Strap Included: Yes
|Closure Type: Zipper closure
|Gender: Men's
|Country of Manufacture: China
About the ManufacturerSince the 1930s, Zero Halliburton has specialized in stylish aluminum cases with a classic design using compound curves, horizontal lines and simple arcs. These design elements, combined with the use of premium materials, make up the Zero Halliburton "Brand DNA"—the essence that inspires the brand's unique look and appeal.
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Ships via: FedEx/UPS
Enter your shipping zip or postal code to calculate shipping estimatesGet Delivery Estimate
We want you to love it. If you don't, you may return your purchase within 30 days of delivery. All you need to do is visit our Self-Service Return Center for instructions. Return credits are issued within 24 hours once the item is received and inspected. Please note that shipping charges are non-refundable. The cost of return shipping will be deducted from your refund. 30-Day Return Policy
Tell Us What You Think!
Tell us what you think about this product. It will help us provide you with better products in the future and help your fellow shoppers make better purchases.Write the first review


text:
Outage hits 168,000 customers from Camarillo to Ojai; restoration takes longer in Simi
About 168,000 utility customers from Camarillo to Ojai lost electricity Tuesday during a mostly brief but widespread power outage, authorities said.
Power in most areas returned within 10 minutes after the 11:28 a.m. outage, and electricity was fully restored by 2:17 p.m., said Patricia Bartoli-Wible, a spokeswoman for Southern California Edison.
The cause of the outage was unclear and remained under investigation, Edison officials said. Some reports blamed a transmission line disruption.
Emergency generators kept power running at facilities including hospitals, public safety agencies and the county jail during the outage, and there were no reports of interruptions to 911 service.
Authorities responded to some false fire alarms and minor traffic accidents during the outage, which are common in such situations, authorities said. Police briefly lost radio communication in Ventura, forcing officers to keep in touch by cellphone for a few minutes, but no major problems were reported.
The outage hit a wide swath of Ventura County, including Camarillo, Oxnard, Port Hueneme, Ventura and Ojai.
It affected 24 substations, 95 circuits and 168,000 utility customers in all, Bartoli-Wible said.
Cellphone service also died in some areas. Bartoli-Wible said that can happen if relay towers lose power, but many have emergency generators.
St. John's hospitals in Oxnard and Camarillo immediately use backup generators when the power goes out, said spokeswoman Rita O'Connor. No problems were reported Tuesday, O'Connor said.
Schools in Ventura, Oxnard, Camarillo and Port Hueneme briefly lost power, but officials reported no major problems.
"We were flooded with calls for probably a good 45 minutes," Ventura police Cmdr. Dave Wilson said.
Wilson said a police radio frequency in the city was lost for about 15 minutes, and officials were analyzing the problem to determine what went wrong.
Critical communications equipment is backed up with batteries, and 911 service was not interrupted, Wilson said. Internet access also was lost at one point, he said.
Emergency generators at the Hall of Justice, Ventura County Jail and Government Center immediately activated after the outage, according to Capt. Mike Aranda, a spokesman for the Ventura County Sheriff's Department.
The Oxnard Fire Department received several calls for elevator rescues in the downtown area, said Battalion Chief Mike O'Malia. He said he was not aware of any injuries.
Bill Nash, a Ventura County Fire Department spokesman, said his agency had a steady stream of calls for at least 15 minutes, including numerous fire alarm reports, which firefighters had to track down to make sure they were false.
In Simi Valley ...
While the wide-scale power outage in west Ventura County was over, about 535 Southern California Edison customers remained without power in two Simi Valley outages early today. Most of those who were affected lost power in an outage that was reported early Tuesday, and several lost power in an outage that was reported Tuesday night, according to Edison.
Edison expected power would be restored from 5 to 5:30 a.m.
Equipment problems were blamed for the Simi Valley outages.
Subscribe to the Ventura County Star
Need Help? Call us at 1-877-710-6182.
Monday-Friday: 6am-3pm / Saturday: 6:30am-10:30am / Sunday: 7am-11am


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Long before taxpayer-sponsored bailouts or state-sponsored "resolutions" of financial institutions came to occupy such a prominent place in our collective consciousness, financial institutions relied on hybrid capital securities as an important component of their funding plans.
Hybrid securities have certain equity and certain debt characteristics and provided an attractive, cost-efficient capital-raising tool. Trust preferred securities, mandatorily convertible debt securities, and various other forms of preferred stock were viewed by issuers as non-dilutive and had tax and ratings advantages.
Originally published in Thomson Reuters: Business Law Currents on August 14, 2013.
Please see full publication below for more information.
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text:
"The evidence suggests that the resin production of the boreal conifers may be critically important for the survival of gray jays, especially at the southern limits of their range," Norris said. "This fits perfectly with our observation that the jay territories that are no longer occupied in Algonquin Park are the areas dominated by deciduous forest."
An important question still remains: why are gray jays declining in Algonquin Park?
"We think it may have something to do with climate change affecting the quality of cached food, and we are currently conducting a series of experiments to look at this," said Norris.
The Guelph study may help in understanding territorial limits for other boreal breeding animals.
"If other northern-breeding or alpine-breeding species rely on cached food, then their ranges may also be limited by the ability of certain habitat types to preserve food over the winter season."
|Contact: Ryan Norris
519-824-4120 x54582
University of Guelph


text:
Self | Illuminated: Industrial Museum Hosts Groundbreaking Technological Art Exhibit
Presented by Charles River Museum of Industry & Innovation at Charles River Museum of Industry & Innovation
July 15, 2012 - January 15, 2013
The Charles River Museum of Industry & Innovation will host Wayne Strattman, the world's foremost maker of kinetic plasma sculpture, in a major solo exhibition opening July 15. The six month exhibit, with almost one hundred individual pieces grouped into ten installations, will be complemented by several events celebrating glass and technology-based art.
The Charles River Museum of Industry & Innovation's main galleries tell the stories of innovators such as Francis Cabot Lowell, creator of the American system of manufacturing, and Arthur Denison, creator of the Waltham Watch Company; however, in the past four years the museum has focused its temporary gallery space on exhibits highlighting the creative side of the innovation / invention process.
Charles River Museum of Industry & Innovation
154 Moody Street
Waltham, MA 02354
Admission Info:
Admission is $7 for the general public and $5 for seniors / students; admission is free for children under six, for US Military both active and retired, and for museum members.
General Day and Time Info:
10 AM to 5 PM Thursday through Sunday
Phone: 781 893 5410
Accessibility Information: Currently, no accessibility information is available for this event.


text:
Breeders of purebred dogs speak a language all their own. Wherever they gather, at dog shows, seminars or in chat rooms, words and phrases are used that have very narrow definitions. Their usage makes it difficult for a novice to fully participate...Read more
The first night of judging at the 136th Westminster Kennel Club dog show on Monday, February 13, 2012, included the finals of the Hound, Toy, Non-Sporting and Herding Groups. Viewers of the television broadcast were no doubt delighted when the USA Network's...Read more
By Christi McDonald Editor's note: Determining the top AKC winners in any category is an inexact science. We explain why in a companion article: "Biggest BIS Winner Tough to Call." For many, many years, the Best in Show record in the Toy...Read more
AKC delegates will vote on March 13 whether to recommend to the board of directors that the current seven Groups be further split into 11. The realignment of breeds would make major changes to the Hound, Sporting and Working Groups. Hounds would...Read more
THE SPORTING GROUP Group Placements 1st Place – Cocker Spaniel Black – GCH CH Casablanca's Thrilling Seduction 2nd Place – Pointer – GCH CH Chesterhope Sharp As 3rd Place – Weimaraner – GCH CH Northwoods Send Money Honey 4th Place – Irish...Read more


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Recent Comments
it's about time
- Becky
Love it! Can an oven be added?
- Al
Can this motorhome support (or be modified to support) stackable washer/drier? Are there any small (30' or smaller) Class A motorhomes that are washer/drier-capable?


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Houston Catholic Homeschool
Join us to view curriculum, hear awesome speakers, buy used curriculum,
enjoy time with fellow homeschooling parents.
Join us to view curriculum, hear awesome speakers, buy used curriculum,
enjoy time with fellow homeschooling parents.
Since 1998, this annual two-day hotel getaway and get together has been a ray of light in the gray of winter for thousands of Christian momsread more
This will be one of the biggest conventions Texas has seen. With 9 pages of exhibitors, using up the whole Fort Worth Convention Center, if you have ever wondered what the rest of the country experiences for their conventions, then you want to see this one!read more
This is a great boon for all Texas Home Educators who have teens doing great work and ready for the next step in their education, but are not near a great engineering college.read more
"Our new organization is a non-profit, created to nurture and cultivate the families who constitute the grass roots of home education in the Lone Star State,"read more
Remembering Dr. King, reviewing what he stood for, and knowing that Peace is for all people, is a great way to celebrate Martin Luther King Day. Take time to review these sites, talk with your children, consider reading "King Rules", the newest book about Dr. King. Here are a few resources to help you with your unit study.read more
For one day only, we offer Home School students unlimited access to our 14 galleries of non-stop, action-packed fun. Home School students will explore science, technology, history, culture, health, environment and engineering hands-on at the ultimate Playground for your Mind™! Pre-registration is required.read more
There is a dress code, formal attire required. My boys went for the first time last year and enjoyed it. In fact, they couldn't wait until the next one. They do require the kids to take dance lessons, well worth it since my two had never danced before.read more


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This is a discussion on What martial art do you practice? within the Off Topic & Humor Discussion forums, part of the The Back Porch category; What kind of martial art do you train?...
What kind of martial art do you train?
A real man loves his wife, and places his family as the most important thing in life. Nothing has brought me more peace and content in life than simply being a good husband and father.
Right now I'm off (lack of sufficient funds). At any given time though I've trained Wrestling, Hapkido, a wee bit of Taekwondo(which turned into taekwonDOH because I found it useless), MMA(bjj, boxing, etc...), and Haganah. When I'm ready to burn money again I'll go back to Haganah for another 6months to a year, more for refresher than anything else. I had thought about doing more BJJ, but after careful thought on what I already know I decided against it. I figure the whole concept of bjj is great on a mat and it really works...but in a street fight rolling around on your backside is BAD bigtime. Asphalt, concrete, etc... all TEAR you up. It takes your mind off what you should be doing and instead you end up more concerned with all the beating you're giving yourself on the ground.
-The Mist (2007)"My God David, We're a Civilized society."
"Sure, As long as the machines are workin' and you can call 911. But you take those things away, you throw people in the dark, and you scare the crap out of them; no more rules...You'll see how primitive they can get."
Using materials i received from another to start doing a slow intro into tai-chi.
I suggest that if you can find in your inner self the will to inflict violence on someone who is trying to victimize you, and can back up that iron will with all the force that you can muster, you will most likely be successful.
Current Student of Goju Ryu
Former Student of Judo
Uhh, Poop, Pee, And throw up. If that doesn't work, Then Gun-Go-Pow, With
apoligies to Pgrass101.
quis custodiet ipsos custodes?
None anymore. Thinking about starting up Tae Kwon Do or Kickboxing again. Maybe find something else. Anyone know of a good instructor of something unique in South Florida?
Shotokan Karate.
Klik Pao.
Rak Chunk (12th gauge. I mean 12th Dan. No, I was right the first time)
"Deine Papieren bitte?" or "ΜΟΛΩΝ ΛΑΒΕ !"
(Choose only one)
NRA Endowment Member
"I bark at no man's bid. I will never come and go, and fetch and carry, at the whistle of the great man in the White House no matter who he is." -- David Crockett
Does wrestling with Crackheads, Drunks, Psychos and complete moron's in a county jail count? If so then I have a black belt in that....
"If you ladies leave my island, if you survive recruit training, you will be a weapon, you will be a minister of death praying for war. "R. Lee Ermey---FMJ"