text:
Career Center
JOB OBJECTIVE: To promote Little Rock as the ideal business and pleasure destination to the state, regional, national and international marketplace, targeting the multicultural and religious segments and positively impact the Little Rock economy through meetings, conventions and tourism visitors for the Little Rock Convention and Visitors Bureau (LRCVB).
Knowledge of a cross-section of business disciplines, including sales, marketing, hospitality industry operations, financial and accounting systems, e-commerce and business communications.
Knowledge of standard office practices and procedures.
Knowledge of computer fundamentals and business software, including database, internet, spreadsheet and word processing software.
Knowledge of computer filing, tracing and record-keeping systems.
Knowledge of market research methods and techniques.
Knowledge of hospitality/tourism industry.
Skill in the operation of a computer, utilizing database, spreadsheet and word processing software.
Skill in account management, direct sales and sales presentations.
Skill in the operation of an automatic transmission automobile.
Ability to perform mathematical calculations.
Ability to design and analyze communication platforms, marketing products, and events.
Ability to conduct market research and analyze and interpret research findings.
Ability to compile data and prepare narrative and statistical correspondence, reports, and presentations.
Ability to prepare bid proposals and conduct bid presentations.
Ability to provide lead working supervision to assigned personnel.
Ability to maintain computer files and trace and record-keeping systems.
Ability to communicate effectively, both written and orally, with individuals and groups.
These knowledge, skills, and abilities are usually, although not always, acquired through the completion of a Bachelor's degree in Business Administration, Marketing, Communications, Advertising, or a related area, and a minimum of four (4) years of demonstrated sales experience preferably in the hospitality/tourism industry or a related area. Equivalent combinations of education and experience will be considered.
Must possess a valid Arkansas Class D (Non-Commercial Vehicle) Driver's License before employment and maintain licensure for the duration of employment in this position. Must show proof of valid automobile insurance before being approved to drive any LRCVB vehicle.
Must have access to a properly licensed and insured, reliable automobile for reimbursable assigned business travel.
Must obtain a personal credit card for fully reimbursable business expenses.
Must be available to work evenings, holidays, and weekends as required.
This position may require one or more of the following pre-employment screenings: drug and alcohol screening, or background investigation.
Additional Salary Information: Hiring/Entry Salary Range: $46,997 - $59,921


text: Zealandia is a short drive out of the city, or you can take a free shuttle from the Botanical gardens. Free parking is provided. Zealandia offers plenty of opportunity to see a wise variety of birds and interesting flora / fauna. Walking trails are well marked and bird feeding stations are located in well marked areas. Take your time and... More

subset: commoncrawl


text:
Donate to Help Survivors Become Leaders
Your generous gift empowers survivors of rape, abuse and sex trafficking to overcome trauma and receive all they need to thrive. Thank you for taking a stand with us and showing the world that women and girls are worth fighting for. With your help, She *is* Rising.
-Brooke Axtell :: Founder
Who's donating
Cathy Studer
emily brearley
Scott Lesh
John Trefry


text:
Westmead Hospital Westmead
Westmead Hospital Westmead
Wong G.,Childrens Hospital at Westmead |
Wong G.,University of Sydney |
Wong G.,Westmead Hospital Westmead |
Lim W.H.,University of Western Australia |
And 11 more authors.
BMC Cancer | Year: 2015
Background: There is increasing evidence that vitamin D deficiency is a risk factor for cancer, however it remains uncertain whether vitamin D deficiency also predisposes to death from cancer. The aim of the study was to determine the association between serum 25-hydroxy-vitamin D (25 (OH) D) concentrations and cancer-specific mortality in a community-based cohort of older post-menopausal women. Methods: Cox proportional regression analyses were conducted to examine the association between serum 25 (OH) D concentrations and the risk of overall and site-specific cancer mortality in a cohort of elderly women. Results: Over a median follow-up time of 10years, a total of 84 cancer deaths were observed. Women with lower serum 25 (OH) D concentrations were at an increased risk of cancer death, but not for incident cancer. The excess risk for cancer death was observed with serum 25 (OH) D concentration less than 64nmol/L (the median value) [adjusted HR: 1.61 (95% CI: 1.02 - 2.54, p=0.04]. For every 30nmol/L reduction in serum 25 (OH) D concentrations, there was a 30% increase in the overall risk of cancer death [adjusted HR: 1.33; 95% CI: 1.03 - 1.72, p=0.02]. The excess risk appeared to be site-specific and greatest in those with haematological cancers [adjusted HR: 2.13: 95% CI: 1.0 - 4.55, p=0.05]. Conclusions: In elderly women, lower serum 25 (OH) D concentrations appear to be an independent risk factor for cancer-specific mortality, but not a risk factor for the development of cancer. © 2015 applies to the data made available in this article, unless otherwise stated.
Ramos E.M.,Massachusetts General Hospital |
Ramos E.M.,University of Porto |
Latourelle J.C.,Boston University |
Gillis T.,Massachusetts General Hospital |
And 41 more authors.
Neurogenetics | Year: 2013
Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and behavioral disturbances. It is caused by the expansion of the HTT CAG repeat, which is the major determinant of age at onset (AO) of motor symptoms. Aberrant function of N-methyl-D-aspartate receptors and/or overexposure to dopamine has been suggested to cause significant neurotoxicity, contributing to HD pathogenesis. We used genetic association analysis in 1,628 HD patients to evaluate candidate polymorphisms in N-methyl-D-aspartate receptor subtype genes (GRIN2A rs4998386 and rs2650427, and GRIN2B rs1806201) and functional polymorphisms in genes in the dopamine pathway (DAT1 3′ UTR 40-bp variable number tandem repeat (VNTR), DRD4 exon 3 48-bp VNTR, DRD2 rs1800497, and COMT rs4608) as potential modifiers of the disease process. None of the seven polymorphisms tested was found to be associated with significant modification of motor AO, either in a dominant or additive model, after adjusting for ancestry. The results of this candidate-genetic study therefore do not provide strong evidence to support a modulatory role for these variations within glutamatergic and dopaminergic genes in the AO of HD motor manifestations. © 2013 The Author(s).
PubMed | Westmead Hospital Westmead
Type: Journal Article | Journal: Journal of medical radiation sciences | Year: 2015
Radiotherapy is an indispensible part of the management of all stages of breast cancer. In this article, the common indications for radiotherapy in the management of early breast cancer (stages 0, I, and II) are reviewed, including whole-breast radiotherapy as part of breast-conserving treatment for early invasive breast cancer and pre-invasive disease of ductal carcinoma in situ, post-mastectomy radiotherapy, locoregional radiotherapy, and partial breast irradiation. Key clinical studies that underpin our current practice are discussed briefly.


text:
chloe7022 Review
5 light chandelier
I purchases this chandelier along with two matching pendant lights for my daughter's new kitchen. When she saw them she was thrilled. Since then she has purchased several items from your site. Thanks for making it so easy to shop. I give Overstock.com 5 stars.
- How did the color on site compare with the actual product? Spot on
- Please tell us about the quality of the product. Beautiful
- How accurate was the on site description of the product? Excellent
Was This Review Helpful?


text:
Centralne Laboratorium Akumulatorow i Ogniw
Centralne Laboratorium Akumulatorow i Ogniw
Grzeczka G.,Maritime Academy of Gdynia |
Swoboda P.,Centralne Laboratorium Akumulatorow i Ogniw
Przemysl Chemiczny | Year: 2015
NaBH4 was converted to H2 in reaction with H2O in presence of catalysts. Co(NO3)2 was the most efficient catalyst of the reaction.
Lota G.,Poznan University of Technology |
Lota G.,Centralne Laboratorium Akumulatorow i Ogniw |
Baraniak M.,Centralne Laboratorium Akumulatorow i Ogniw |
Wasinski K.,Centralne Laboratorium Akumulatorow i Ogniw |
Jankowska E.,Centralne Laboratorium Akumulatorow i Ogniw
Przemysl Chemiczny | Year: 2013
(CH2=CHCH2)2Me2N +Cl- was polymerized in aq. soln.,converted to hydrogen sulfate(VI) with H2SO4 and added (7.5%) to H 2SO4 used in Pb battery. The electrochem. and electrical properties of Pb\ionic liq.\PbO2 model system showed applicability of the polymeric ionic liq. as a new component of gel electrolyte.
Szwarc-Rzepka K.,Poznan University of Technology |
Walkowiak M.,Centralne Laboratorium Akumulatorow i Ogniw |
Osinska-Broniarz M.,Centralne Laboratorium Akumulatorow i Ogniw |
Dutkiewicz M.,University Im ckiewicza znaniu |
And 2 more authors.
Polimery/Polymers | Year: 2013
Hybrid SiO2/POSS fillers were obtained using the method based on solvent evaporation. Octakis({1,1,2,2,3,3,4,4-octafluoropentyloxypropyl} dimethylsiloxy) octasilsesquioxane (F-SF POSS) was used to modify the surface of silica, hydrated or prepared via emulsion method. The effectiveness of modification was evaluated by FT-IR spectroscopy and nuclear magnetic resonance (29Si and 13C CP MAS NMR). The parameters of porous structure of the prepared hybrid fillers, such as surface area BET, mean pore diameter and pore volume were determined. The hybrid products were subjected to dispersive analysis (non-invasive backscattering method, NIBS) and morphological characterization (using transmission electron microscopy - TEM). The obtained hybrid systems were used as additives in gel polymer electrolytes, prepared by activation of dry composite membranes in a liquid solution of lithium salt. The electrolytes were subjected to electrochemical studies, including the measurements of specific conductivity by impedance spectroscopy in a wide range of temperatures and the determination of potential windows by the linear cyclic vol-tammetry. The addition of POSS composites had a beneficial effect on the transport of lithium cations and the room temperature conductivity of gel electrolytes based on PVdF/HFP membranes was enhanced by an order of magnitude. The observed increasein gel conductivity was poorly correlated with the ability of liquid phase absorption. The anodic stability of gels only slightly increased upon the addition of POSS to PVdF/HFP membranes. The fillers studied show promising properties in the context of future research and development studies on new solid electrolytes for lithium cells with increased safety.


text:
Note 1:If you are not satisfied with the item or the shipping time, please email us BEFORE you leave a feedback.We will try our best to HELP you.Thanks!
Note 2:Please leave us FIVE STARS rating and POSITIVE FEEDBACK if you are satisfied with the item.Because neutral feedback and 4 stars are regarded as negative feedback.
Note 3:Plz leave your FULL NAME and detailed Address if your address is in Russia.
Buy 2 pcs can get 5% OFF discount~~
Product Description>>>
1. New Brand Fashion Soft TPU Phone Case 2.Luxury back Cover for iphone 5 5s SE 6 6s 6Plus 6s Plus 7 7Plus 3. High Quality with low Price 4.Protects your phone from dirt,scratches,dings and shock.
NOTE: NO tracking Number for small orders(orders price < $3), cause it will increase your shipping cost.
NO Retail Box. Packed Safely in Bubble Bag and Envelope.
Delivery time Note: If your Confirmation Time is coming, you still not receive the goods, please contact us.
If after 60 days you still do not receive the goods, please contact us, we will give you a full refund.
No Dispute please: Please do not apply dispute within 60 days, let's keep good business relationship.
Thank you for your support and understanding, I wish you a happy shopping!


text:
Fourteen PLACE members and friends enjoyed a splendid afternoon in Millington Wood on Saturday 7th May, led by local naturalist Roy Crossley. A report will appear in the next PLACE newsletter. Meanwhile, here are some pictures of the trip.
Search Place.uk.com
Subscribe to PLACE posts
Posts by Category
- Conferences (8)
- Field Trips (28)
- General News (55)
- Newsletters (21)
- Publications (29)
- Research Projects (7)
- Short Courses (17)
Recent Posts
Previous Posts


text:
Recent Posts
Recent Comments
Sanford Groch on X-COM: Enemy Unknown Revi… janus32 on X-COM: Enemy Unknown Revi… janus32 on Games of 2013 Amedar Consulting Gr… on X-COM: Enemy Unknown Revi… NyNy on Games of 2013
Author Archives: janus32
The Ramblings of a…somewhat progressed man
It has been a few months since the last time I have written here. and strangely enough, I'm writing at work on my lunch break, squeezing what i can in a 40 minute time frame. With music in my ears … Continue reading
The Ramblings of a Dejected Man
Previously on my first/last time blog, I went through ramblings of how confused I was with the state of my life. As the time has progressed since then, I would say that I am still within that standstill. You could say … Continue reading
The Ramblings of a Confused Man
It's hard to say without equivocation what thoughts can go through ones mind when a lot has happened to them. I know what everyone says these days, including myself. "It all depends"And i would agree with that phraseLike the rapper Eminem, the … Continue reading
Tomb Raider Review
Tomb Raider PS3, XBOX360, PC By Janus 32 Introduction The Tomb Raider franchise has always had a female protagonist. Never has it changed with all its games, and still remains to this day the buxom brunette, Lara Croft. Since her … Continue reading
Posted in Video Game Reviews Tagged 3rd Person, Action, Brutal, Crystal Dynamics, Eidos Montreal, Lara Croft, Multiplayer component, New, Pure Survival, Reboot, Roth, Salvaging, Stealth, Survivalk, tomb raider, Tomb Raider 2013, Violence, Violent Leave a comment
Aliens: Colonial Marines Review
Aliens: Colonial Marines Review PS3, XBOX360 By Janus 32 Introduction For years since James Cameron's Aliens movie, there was an unspoken awe and admiration, predominantly for the male audience that witnessed the movie, of the badass Colonial Marines that went … Continue reading
Posted in Video Game Reviews Tagged Aliens colonial marines, apone, bishop, game over, gamer over man, gearbox software], hicks, hudson, lance hendrickson, michael biehn, ripley, sega, vasquez, weyland yutani Leave a comment
Dead Space 3 Review
Dead Space 3 Review PS3, XBOX360, PC By Janus 32 Introduction Electronic Arts's Visceral Games is a name that most gamers should know, whether they are veterans of the Survival Horror genre or just newbies stepping in for the first … Continue reading
Posted in Video Game Reviews Tagged Action, Darkness, Dead Space, Dead Space Franchise, Elli Langford, Isaac Clarke, Necropmorphs, ps3, Robert Norton, Survival Horror, Thriller, xbox360, XBOX36o Leave a comment


text:
SOLFEX energy systems Underfloor Heating Introduction
Underfloor heating has now become the preferred form of space heating within the UK due to its increased efficiency and even thermal comfort. Underfloor heating systems work by distributing lower temperature warm water through a network of pipe work within the floor construction and beneath the finished floor level, this heat is carefully monitored and
controlled by an advanced range of modern room thermostats to keep a constant ambient temperature throughout the property & individually for each zone.
Underfloor heating is now a complete solution for both existing and new construction homes, with a selection of systems to suit all scenarios; suspended floors, floating floors, low profile over floor and screed applications.
SOLFEX NEO app
With Solfex NEO you can control your underfloor heating from anywhere via your Android or iOS device. More information on SOLFEX NEO
Features of WARM-BOARD
WARM-BOARD Video Installation Guide
Press Releases
Download Our Underfloor Heating Product Guide & Price List 2017
Our new Underfloor Heating Product Guide and Price list is now available. Packed with information on our entire range of underfloor heating systems and accessories, it's the only underfloor heating catalogue you'll nee...
City Plumbing Supplies hosts Underfloor Heating Training in Leeds
Join us for a morning of Underfloor Heating Training with City Plumbing Supplies. Limited spaces available, please contact either of the following people to reserve your seat; Paul Hea...


text:
An Analysis Of The Conditions For The Validity Of Modigliani-Miller Theorem With Incomplete Markets
In this paper we investigate the consequences of the firms' financial decisions in the framework of a perfectly competitive general equilibrium model with incomplete markets. When markets are complete or there are no derivative securities (such as options, forwards or futures) written on the firms' shares, these decisions are irrelevant. This result reaffirms and qualifies the original claim by Modigliani and Miller. On the other hand, if markets are incomplete, we show that in the presence of any type of derivative security a change in the capital structure of a firm will modify, generically, both the real equilibrium allocation and the value of the firm. The reason is that the payoff of the derivative securities is affected in a non-linear way by changes in the firm's financial policy; thus the set of the agents' insurance opportunities is also modified.
(This abstract was borrowed from another version of this item.)
To our knowledge, this item is not available for
download. To find whether it is available, there are three
1. Check below under "Related research" whether another version of this item is available online.
2. Check on the provider's web page whether it is in fact available.
3. Perform a search for a similarly titled item that would be available.
|43 pages
|Date of creation:
|Contact details of provider:
| Postal: UNIVERSITY OF CAMBRIDGE, RESEARCH PROJECT ON RISK, INFORMATION AND QUANTITY SIGNALS IN ECONOMICS(E.S.R.C.), DEPARTMENT OF APPLIED ECONOMICS, SIDGWICK AV. CAMBRIDGE CB3 9DEDE U.K..
Web page: http://www.econ.cam.ac.uk/
More information through EDIRC
No references listed on IDEAS
You can help add them by filling out this form.


text:
Laura Mullen and Jonathan Stalling's Phonotaxis: The Way We Travel Through Each Other
Hover your cursor over the embedded chapbook and press "expand" to view the chapbook full size. It may take several seconds for the chapbook to load.
The Conversant is pleased to publish Phonotaxis: The Way We Travel Through Each Other, a collaborative project by Laura Mullen and Jonathan Stalling. Continue reading


text:
Neymar Jr Magic Skills & Tricks 2016-2017
The Brazilian Kid Has Some Insane Skills Specially This Year.
Comments for this video
Related Football Pages
Related Videos
Watch Live Football Streams at bet365!
UK customers at bet365 can watch live streams of thousands of matches from the world's top football leagues including La Liga, Bundesliga, Serie A, MLS and Ligue 1 and many more.
A funded account is required, so open a free account today, fund your account and start watching!


text:
Division of Biology and Biomedical Sciences Campus Box 8226 660 S. Euclid Ave. St. Louis, MO 63110-1093
Main Phone Numbers: (314) 362-3365 (800) 852-9074
Associate ProfessorDevelopmental Biology
Developmental, Regenerative and Stem Cell Biology ProgramNeurosciences ProgramMolecular Genetics and Genomics Program
McDonnell Science Building, 360
glia, mouse models, multiple sclerosis, nerve regeneration, neurobiology, zebrafish
Molecular and genetic mechanisms of myelination, myelin maintenance, and remyelination
The myelin sheath surrounding axons is an exquisite specialized membrane found in the vertebrate nervous system. Myelin is formed by glial cells called oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. The importance of myelin is underscored by diseases that compromise the integrity of myelinated axons, including multiple sclerosis and Charcot-Marie-Tooth disease. Despite its importance, however, relatively little is known about how the myelin sheath is formed and maintained. We are interested in the mechanisms that govern the development, maintenance, and regeneration of myelinated axons using both zebrafish and mouse models. We seek to uncover regulators of nervous system development, homeostasis, and repair.
Last Updated: 8/26/2016 1:59:20 PM
Follow us:
© 2011 Washington University in St. Louis DBBS


text:
It has been demonstrated that stimulation of protein kinase A (PKA) results in enhanced synaptic transmission in the hippocampus and other brain areas. To investigate mechanisms of the PKA-mediated potentiation of synaptic transmission, we used rat hippocampal embryonic cultures. In low-density cultures, paired recordings under the perforated patch demonstrated that 15-min forskolin treatment produced long-lasting potentiation of evoked excitatory postsynaptic currents (eEPSCs) mediated by the cAMP/PKA pathway. eEPSC amplitudes increased to 240 ± 10% of baseline after 15 min of forskolin treatment (early). After forskolin washout, eEPSCs declined to a potentiated level. Potentiation was sustained for ≥85 min after forskolin washout and, 60 min after forskolin washout, constituted 152 ± 7% of baseline (late potentiation). Disruption of presynaptic processes with the whole cell configuration and internal solution containing PKA inhibitor peptide did not affect forskolin-induced potentiation. Disruption of postsynaptic processes, in contrast, impaired early potentiation and abolished late potentiation. Study of mEPSCs confirmed the contribution of postsynaptic mechanisms. Forskolin-induced enhancement of mEPSC frequency observed under the perforated patch was attenuated by the whole cell configuration. Forskolin also induced an increase of mEPSC amplitudes in the perforated patch, but not in the whole cell, experiments. Potentiation of eEPSCs was not activity dependent, persisting in the absence of stimulation. NMDA receptor blockade did not abolish forskolin-induced potentiation. In summary, we demonstrate that forskolin-induced potentiation of eEPSCs was mediated by postsynaptic mechanisms, presumably by upregulation of AMPA receptors by phosphorylation.
Long-term potentiation (LTP) is a long-lasting, use-dependent increase of the effectiveness of synaptic transmission. Because LTP is believed to mediate memory formation, activity-induced LTP and its underlying mechanisms have been subjects of intensive research efforts. A substantial body of evidence implicates cAMP-dependent protein kinase (PKA) in the production and maintenance of LTP. For example, LTP induced by repeated tetanic stimulation of hippocampal neurons is accompanied by PKA activation (Huang and Kandel 1994; Roberson and Sweatt 1996). Inhibitors of PKA block or decrease LTP, and activators of PKA facilitate LTP in the CA3 and CA1 areas of the hippocampus (Frey et al. 1993; Huang and Kandel 1994; Huang et al. 1994; Matthies and Reymann 1993; Otmakhova et al. 2000). The role of PKA in LTP and memory has also been demonstrated using genetic approaches (Abel et al. 1997; Huang et al. 1995; Qi et al. 1996).
It is widely known that not only is PKA involved in LTP, but, when directly stimulated, it also produces a marked enhancement of synaptic efficacy (Carroll et al. 1998; Chavez-Noriega and Stevens 1992, 1994; Chen and Roper 2003; Greengard et al. 1991). Because of the involvement of PKA in LTP and memory, numerous attempts have been undertaken to elucidate mechanisms through which PKA might modify the synaptic strength. A large body of evidence suggests that the change of synaptic efficacy might be contributed by modulation of both presynaptic and postsynaptic PKA substrates. Early studies have demonstrated that PKA modulates the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function (Banke et al. 2000; Chavez-Noriega and Stevens 1992; Greengard et al. 1991; Roche et al. 1996; Wang et al. 1991, 1993). Activation of PKA is associated with an increase of the number of functional release sites in cultured neurons isolated from a dissociated dentate gyrus, and in the CA1 area of the hippocampus (Kohara et al. 2001; Ma et al. 1999). PKA also increased a kainate-sensitive transmitter release in mossy fibers (Goussakov et al. 2000).
Despite a potential involvement of AMPA receptors, electrophysiological studies failed to demonstrate a contribution of postsynaptic mechanisms to the effect of PKA activators on synaptic transmission. For example, Carroll et al. reported that forskolin dramatically increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in CA1 pyramidal neurons of the hippocampus but did not change mEPSC amplitudes (Carroll et al. 1998), leading to a conclusion about the presynaptic locus of forskolin action. A similar result has been reported by Chavez-Noriega and Stevens (1994).
Analysis of synaptic currents obtained during electrophysiological recording has being performed using postulates of the classical quantal theory, introduced by Del Castillo and Katz (1954) in their study of transmission at the neuromuscular junction. According to this theory, the frequency of mEPSCs at each synapse is determined by the probability of releasing a single quantum (interpreted as a vesicle) and the number of releasable quanta. At the neuromuscular junction, studied by Del Castillo and Katz, the number of quanta and active synapses remains unchanged throughout the experiment. Consequently, a change of mEPSC frequency has been thought to reflect a changed probability of neurotransmitter release.
The validity of some postulates on the nature of synaptic transmission, established by Del Castillo and Katz, remains questionable for central synapses. For example, the number of active synapses changes during some forms of LTP (Faber et al. 1991; Isaac et al. 1995). This change, by postsynaptic mechanisms, affects the frequency of mEPSCs, vitiating the conventional view that mEPSC frequency reflects only presynaptic changes. Thus conclusions about the presynaptic origin of PKA-mediated changes at central synapses, derived from the analysis of mEPSCs, should be reexamined.
To further investigate the locus of expression of PKA-mediated potentiation, we examined the effect of forskolin on eEPSCs and mEPSCs in rat embryonic hippocampal cultured neurons. By manipulating the method used to gain control over the cell membrane potential in patch-clamp experiments (perforated patch or whole cell voltage clamp), we demonstrate that forskolin-induced potentiation is mediated by postsynaptic mechanisms.
Neuronal culture
Rat hippocampal embryonic cultures were prepared as described (Li et al. 1998). Briefly, hippocampi isolated from Wistar rat embryos at day 18 of gestation (E18) were treated with papain (20 mM) for 15 min at 36°C, and then dissociated by trituration and plated on glass coverslips precoated with polyethyleneimine. Cells for mEPSC and paired recordings were plated at densities of 200 cells/mm2 (high-density cultures) and 140 cells/mm2 (low-density cultures), respectively. Cultures were maintained at 36°C in a 5% CO2 incubator and were grown in Neurobasal medium (Gibco) supplemented with B27 (a complex of vitamins and neurotrophic factors; Gibco), 0.5 mM each Glutamax (Gibco) and glutamate, and 5% horse serum. Low-density culture medium also contained 1.5 mM more Gln and 15 mM more KCl. All components of the culture medium were purchased from Invitrogen. As glia formed a monolayer 5–6 days after plating, an anti-mitotic agent 1-β-d-arabinofuranosylcytosine (Sigma) was added to the medium to 0.05% final concentration to inhibit further glial growth. Half of the medium in the dish was then substituted by fresh serum-free medium once every week. Cultures used for paired or mEPSC recordings were 18–20 and 23–25 days old, respectively.
A glass coverslip with cultured neurons was placed into a perfusion chamber with a solution volume 0.2–0.5 ml. Perfusion rate was set at 0.5–1 ml/min. All experiments were done at room temperature. The following is a composition of the bathing solution used in experiments (in mM): NaCl 145, KCl 5, MgCl 0.5, CaCl2 2, HEPES 5, glucose 10, pH 7.4. For mEPSC recordings the bath was supplemented with 0.5 μM tetrodotoxin (TTX). When indicated, the superfusion medium contained 50 μmol/l forskolin. The pipette solution used for perforated-patch experiments contained (in mM): potassium gluconate 150, NaCl 5, EGTA 0.1, HEPES 5, pH 7.2. Just before the experiment, Amphotericin B (Calbiochem) was dissolved in dimethylsulfoxide at a concentration 60 mg/ml. This Amphotericin B stock was added to the pipette solution to create 1.5 mg/ml final concentration. Electrode resistance was 4–6 MΩ. Access resistance stabilized during the first 10–15 min of experiments and ranged from 18 to 30 MΩ. Cells were voltage clamped at −60 or −65 mV using Axopatch 200 or Axopatch 200A. The contribution of inhibitory currents during recordings of the TTX-resistant spontaneous activity was negligible because the −80-mV membrane potential (−65 mV with the corrected liquid junction potential) was close to the reversal potential for chloride. In addition, picrotoxin (40 μM) did not significantly change the frequency of TTX-resistant spontaneous currents (four experiments, data not shown). Pipette solution for the whole cell experiments contained (in mM): potassium gluconate 150, NaCl 5, MgCl2 2, CaCl2 0.1, EGTA 5, HEPES 5, PKA inhibitor peptide 7–22 (PKI, Calbiochem) 0.015, pH 7.2. Access resistance in the whole cell experiments was in the range 15–25 MΩ. Access resistance did not change more than 15% in mEPSC experiments nor more than 20% during paired recordings.
Whole cell current in mEPSC experiments was filtered at 2 kHz and digitized at 10 kHz. mEPSCs were analyzed using a custom-made program. Asymmetric events with the rise time shorter than the decay time and amplitudes >4 pA (the threshold for event detection) were chosen for the analysis. mEPSC frequency and amplitudes were established by analyzing 90- to 300-s-long records in the high-density and low-density cultures, respectively. Extending the analysis for longer periods in a number of experiments did not significantly change the results. mEPSC frequency ranged between 1.3 and 19 Hz in high-density cultures and 0.15 and 2.6 Hz in low-density cultures. The average number of events analyzed for each record was 475 ± 66 (mean ± SE) in high-density cultures and 118 ± 25 in low-density cultures.
For paired recordings two connected neurons were voltage clamped using the perforated patch configuration. One of the neurons was stimulated at 30-s intervals by applying a 1-ms voltage step to +20 mV. Postsynaptic currents induced within 2–4 ms after the stimulus were recorded for 25–30 min before the forskolin treatment, to establish the baseline level of eEPSC amplitudes. The eEPSC signals were filtered at 1 kHz and digitized at 5 kHz. A series resistance compensation of 80% was used in all paired recording protocols. After forskolin washout, paired recordings continued for as long as the gigaseal remained intact. Baseline level was calculated as an average of eEPSC amplitudes during the 10 min preceding the forskolin application. During and after forskolin treatment eEPSC amplitudes in each experiment were normalized to the baseline level. In each group of experiments, results of paired recordings are presented as normalized eEPSCs averaged over a 2-min period.
Western blot
Cells were plated at a density of 200 cells/mm2 in 35-mm Biocoat plastic dishes (BD, Franklin Lakes, NJ). After 24 days in culture cells were incubated with either a solution containing TTX, or a solution with TTX and forskolin, depending on the experiment. Whole cell lysates were obtained by adding 400 μl lysis buffer to a dish. The following is the composition of the lysis buffer: 62.5 mM Tris pH 6.8, 1% SDS, 5% mercaptoethanol. PMSF (at a 1 mM final concentration) and Complete Mini Protease inhibitor cocktail tablets (Roche) were added to the lysis buffer just before the experiments to prevent protein degradation and inhibit phosphatase activity. After brief incubation in the lysis buffer, samples were collected and passed through a 26-gauge needle 10 times (to shear genomic DNA). Samples were then boiled for 5 min and placed on ice or stored in a −70°C freezer until use. A 25-μl aliquot of each sample was loaded onto a precast 4–15% gradient SDS-Tris HCl gel (Bio-Rad Laboratories). Proteins were electrophoretically separated at 120 V for 2 h, and then blotted onto a nitrocellulose membrane (Bio-Rad Laboratories) at 100 V for 1 h. Membranes were washed with Tris-buffered solution (TBS) and blocked with 5% fat-free dry milk (Carnation) in TBS for 30 min at room temperature. Blots were then incubated with rabbit anti-phospho-GluR1 (Ser845; Upstate, 1:4,000 dilution) or rabbit anti-GluR1 (Upstate, 1:10,000 dilution) antibodies in 0.1% Tween-TBS for 3 h at room temperature. For negative control, membranes were incubated with the Tween-TBS solution containing anti-phospho(Ser845)-GluR1 antibody and a saturating concentration (1 μM) of phospho-GluR1 immunizing peptide (Upstate). After two 5-min washes in Tween-TBS, blots were subjected to biotinylated anti-rabbit secondary antibodies (Vector Laboratories) for 1 h. After two additional washes HRP-conjugated streptavidin was applied (Vectastain ABC kit, Vector Laboratories) according to vendor instructions. The signal was detected using a Vector NovaRED substrate kit for peroxidase (Vector Laboratories).
All salts and forskolin were purchased from Sigma.
Perfusion with forskolin induced a long-lasting PKA-dependent potentiation of eEPSCs
To investigate the effect of forskolin on eEPSC amplitudes we conducted dual-cell voltage-clamp experiments in isolated pairs of neurons in a low-density culture (see methods), using the perforated-patch configuration. Perfusion with forskolin (50 μΜ) for 15 min substantially increased eEPSC amplitudes. The time course of the forskolin-induced changes of eEPSCs could be divided into three phases. The first, early phase of potentiation (EP) took place during forskolin treatment. It is characterized by a gradual rise of eEPSC amplitudes to 240 ± 10% of baseline 15 min after the start of forskolin treatment; the change was observed in all six pairs tested. The second phase of eEPSC changes consisted of a decline of eEPSC amplitudes during the first 10 min of forskolin washout. eEPSCs then stabilized–at a still potentiated level—thus marking the third, late phase of potentiation (LP). LP was sustained for ≥85 min after forskolin washout (five of six tested cell pairs). By 60 min after forskolin washout, eEPSC amplitudes averaged 152 ± 7% of baseline. The results of these experiments are summarized in Fig. 1A.
To test whether forskolin action was mediated by the adenylyl cyclase/PKA pathway, neuronal cultures were subjected to adenosine 3,5-monoaminophosphorothioate, Rp-isomer (Rp-cAMPS, 50 μM) 1 h before forskolin treatment. This drug was continuously present in a perfusion medium during the experiments. Rp-cAMPS is a membrane-permeable nonhydrolyzable cyclic adenosine monophosphate (cAMP) analogue that associates with the cAMP-binding site of PKA, thus blocking PKA activity. Rp-cAMPS abolished the LP (three cell pairs), indicating that forskolin acted through the PKA (Fig. 1A). However, despite the presence of the PKA inhibitor, there was a 36 ± 17% increase of eEPSC amplitudes during the EP (Fig. 1A).
Forskolin increased frequency of mEPSCs
It has been demonstrated that activation of PKA upregulates mEPSC frequency in very young hippocampal cultures (Greengard et al. 1991) and in pyramidal neurons of the CA1 area of the hippocampus (Carroll et al. 1998; Chavez-Noriega and Stevens 1994). To verify that this is the case in our system, we recorded mEPSCs in high-density cultures using the perforated-patch configuration. To establish whether the increase of mEPSC frequency is long-lasting and whether it contributes to the forskolin-induced LP, we performed long-term recordings of mEPSCs. Baseline level of mEPSC frequency was measured just before forskolin treatment. Forskolin treatment for 15 min (50 μmol/l) resulted in an enhancement of mEPSC frequency to 445 ± 47% of the baseline (five experiments). This enhancement immediately after forskolin treatment was followed by a decline of mEPSC frequency to 163 ± 67% of the baseline during the first 15 min after forskolin washout. Subsequently, mEPSC frequency declined more slowly, reaching the baseline level 45 min after the start of forskolin washout. There was no detectable increase of mEPSC amplitudes (results not shown). The results of these experiments are shown in Fig. 1B.
In control experiments, the time course of changes of mEPSC frequency was monitored in untreated cultures. mEPSC frequency remained unchanged for the first 30 min of experiments, and then declined to 68 ± 5% of baseline 60 min after the start of experiments (five experiments, Fig. 1B). Because access resistance changes could contribute to the observed decline of mEPSC frequency, in all the analyzed experiments access resistance of perforated patches was monitored and changed ≤15% throughout the experiment.
Whole cell experiments reveal a postsynaptic locus of expression of the forskolin-induced potentiation
To determine whether presynaptic or postsynaptic events are the main contributing factors to the forskolin-induced potentiation, we uncoupled intracellular events in one of the neurons using the whole cell patch-clamp configuration. By including PKI (see methods) in an adenosine triphosphate (ATP)–deficient pipette solution, we attempted both to inhibit events that resulted from cytoplasmic factors and also to block the activity of the membrane-bound PKA.
In all six experiments done under the presynaptic whole cell configuration, the EP of the forskolin-induced potentiation remained unchanged. During this phase, eEPSC amplitudes rose to 244 ± 21% of baseline. In four of these six experiments neuronal pairs also displayed the LP at a level similar to that observed under the perforated patch (153 ± 9% of baseline 1 h after forskolin washout, four experiments; Fig. 2A). In the other two experiments, the EP was followed by gradual depression of eEPSC amplitudes on forskolin washout.
As opposed to the forskolin effect under the presynaptic whole cell, under the postsynaptic whole cell configuration both EP and LP were either impaired or abolished. During forskolin perfusion, eEPSCs rose to only 146 ± 11% (mean ± SE) of baseline (four cell pairs) in these experiments. Forskolin washout was associated with a decline of eEPSC amplitudes back to the baseline level soon after the start of the washout. By 60 min after forskolin washout, at times corresponding to LP, eEPSC amplitudes constituted 96 ± 6% of baseline (four experiments) (Fig. 2A).
In control experiments using either the presynaptic or postsynaptic whole cell configuration, we monitored eEPSCs in untreated cultures (two experiments in each configuration). These experiments demonstrated that a disturbance produced by the whole cell configuration, by itself, does not affect basal synaptic transmission, making it possible to conduct long-term experiments using the whole cell configuration (Fig. 2B).
Whole cell configuration attenuated forskolin-induced enhancement of mEPSC frequencies
To verify whether postsynaptic events contributed to the forskolin-induced upregulation of mEPSC frequency, we used the same strategy for disrupting postsynaptic events as in the previous set of experiments. A neuron in a high-density culture was voltage clamped in the whole cell configuration. The internal solution was supplemented with PKI. Under these conditions, a 15-min forskolin treatment induced an enhancement of mEPSC frequency to 275 ± 18% of baseline. mEPSC frequency then declined on forskolin washout, reaching the baseline level 45 min after the start of washout. Thus the time course of mEPSC frequency changes, produced by forskolin in these experiments, was similar to the one under the perforated patch. However, the increase of mEPSC frequency during forskolin treatment was significantly lower than that in perforated patch experiments (P < 0.01).
It was noted in earlier studies that, under the whole cell configuration, miniature events undergo a substantial rundown, presumably as a result of the washout of essential cytoplasmic components (Wang et al. 1991). This baseline mEPSC rundown under the whole cell configuration could contribute to an impairment of the forskolin-induced upregulation of mEPSC frequency. Indeed, control experiments demonstrated that mEPSC frequency decreased by 37 ± 8% of baseline during the first 30 min after formation of the whole cell configuration (eight experiments). During the next 15 min, however, the decline of mEPSC frequency was negligible (4.5 ± 8% of baseline, eight experiments; Fig. 3A). To minimize a contribution of mEPSC rundown under the whole cell configuration, forskolin was applied 30 min after the whole cell formation, when the level of AMPA receptor rundown was minimal. The mEPSC frequency just before forskolin application was considered a baseline. In these experiments the increase of mEPSC frequency after 15 min of forskolin treatment was 294 ± 25% (five experiments), similar to that in the previous set of experiments (275 ± 18%).
Factors other than differing patch-clamp configurations could also contribute to the differing effect of forskolin on mEPSC frequency. Such factors are developmental differences or variability of the cell density between dishes or batches of neurons. To avoid a contribution of these artifacts we examined the effect of forskolin on mEPSCs while recording simultaneously under the perforated patch and whole cell using dual-electrode voltage clamp. In these experiments 30 min was allowed between the whole cell formation and forskolin application. The resulting increase of mEPSC frequency in each individual experiment was consistently and significantly higher in neurons under the perforated-patch versus whole cell configuration (1.45 ± 0.11 times, P < 0.05, three experiments). Examples of traces recorded during these experiments are shown in Fig. 3B.
Paired-pulse facilitation
Paired-pulse facilitation (PPF) was not systematically studied, mainly because the double-stimulus protocol occasionally led to the upregulation of baseline eEPSCs. In addition, during baseline recording, presynaptic neurons under the whole cell configuration did not express PPF. This contrasts with the neurons under the perforated patch, of which six out of eight expressed PPF. The average PPF in the perforated-patch experiments was 1.65 ± 0.24 (eight experiments, 50-ms interpulse interval). Similarly, in experiments with the postsynaptic neuron under the whole cell, the average PPF was 1.74 ± 0.22 (eight experiments). None of the six presynaptic neurons under the whole cell configuration expressed PPF. The average ratio of responses to the paired-pulse stimuli in these experiments was significantly lower than that in two other sets of experiments (0.85 ± 0.12, P < 0.01). Contrasting PPF in experiments using different patch-clamp configurations affirm that presynaptic terminals under the whole cell were affected by the dialyzing conditions of the experiment, presumably by PKI present in the pipette solution.
Forskolin-induced potentiation was independent of NMDA receptors but associated with upregulation of AMPA receptors
To verify that forskolin treatment produced phosphorylation of the GluR1 subunit of AMPA receptors, we used Western blot analysis. Western blots of the whole cell protein extracts, electrophoretically separated using SDS-PAGE, were immunostained with the anti-phospho-GluR1 antibody specific for phosphorylated Ser845 (anti-P-GluR1Ser845 antibody). High-density cultures were treated with forskolin for 15 min, and then lysed 0 or 45 min after the treatment. Control samples were obtained from untreated cultures. The 45-min period was chosen because it represents the approximate period of time that the mEPSC frequency, initially upregulated by forskolin, returned to the baseline level.
The presence of an extra band in the lane corresponding only to cultures lysed immediately after forskolin treatment indicated that forskolin produced a new, short-lived phosphorylation state of the GluR1 (Fig. 4A, top). Specificity of the forskolin-induced extra band was demonstrated by incubating the blots with the antibody-containing medium supplemented with the anti-Ser845 phospho-GluR1 immunizing peptide (at a saturating concentration, 1 μM). Under these conditions, the forskolin-induced extra band did not appear in the Western blot (Fig. 4A, middle).
It has been reported that PKA increased the peak open probability of the AMPA receptor channel opening (Banke et al. 2000). This effect is expected to upregulate macroscopic currents through AMPA receptors, including mEPSCs. Despite an apparent increase of GluR1 phosphorylation arising from forskolin treatment, the expected increase of mEPSC amplitudes was not observed in dense cultures (data not shown). We hypothesized that the increase of mEPSC amplitudes was undetectable because of the problems of dendritic filtering and a low signal-to-noise ratio in the dense cultures. To obviate these problems we monitored changes of mEPSC amplitudes in low-density cultures. The filtering effect in such a system must be negligible because a neuronal dendritic tree in low-density cultures is formed by few short dendrites.
Average mEPSC amplitude in low-density cultures in the perforated-patch experiments increased significantly as a result of forskolin treatment (by 29 ± 3%, P < 0.001, three experiments, two-sample t-test). This effect of forskolin on mEPSC amplitudes was disrupted by the whole cell patch-clamp configuration. Analysis of mEPSC amplitudes as cumulative probabilities also demonstrated that, under the perforated patch, mEPSC amplitudes were significantly upregulated after 15-min forskolin treatment (P < 0.05, Kolmogorov–Smirnov test, Fig. 4B). The change of mEPSC amplitudes was short term and reversed on forskolin washout.
To further investigate the mechanism of the forskolin-induced potentiation, we aimed at establishing whether synaptic activity during and after forskolin treatment was essential for the effect of the drug on eEPSCs. For this purpose, after about 0.5 h of baseline eEPSC recording, paired stimulation was paused and was not resumed until 40 min after forskolin washout. Under such conditions, eEPSC amplitudes, at times corresponding to LP, were even higher than those in experiments on continuously active neuronal pairs (211 ± 17% of baseline, three experiments, vs. 152 ± 7%, five experiments).
To decide whether N-methyl-d-aspartate (NMDA) receptors contributed to the forskolin-induced potentiation, we treated cultures with the NMDA-receptor blocker d-(−)-2-amino-5-phosphonopentanoic acid (AP-5, 50 μM) for ≥1 h before experiments. During the experiments cultures were superfused with forskolin (50 μM) as well as with AP-5 (50 μM). Both the EP and LP of the forskolin-induced potentiation of eEPSCs in these experiments were unaffected by the NMDA-receptor blocker. With the background of AP-5, eEPSC amplitudes were 162 ± 7% (three experiments) 60 min after forskolin washout. The results of these experiments are presented in Fig. 4C.
In the present study, by comparing patch-clamp results in perforated and whole cell configurations, we demonstrated that forskolin produced a PKA-dependent LTP of synaptic transmission by postsynaptic mechanisms.
Our strategy was to investigate the effect of forskolin in neurons minimally perturbed by experimental conditions, and to compare this effect to that in neurons dialyzed from the inside using the whole cell patch-clamp configuration. Under the perforated patch, most of intracellular molecules, including divalent ions, remain intact (Rae et al. 1991). The whole cell configuration, in contrast, is associated with the washout of intracellular components, including cytoplasmic proteins and ATP, and buffering intracellular calcium by EGTA present in the pipette solution (Marty and Neher 1995). Such conditions are expected to perturb, if not abolish, intracellular processes relying on cytoplasmic factors. To inhibit the activity of the membrane-bound PKA, which might not be washed out under the whole cell configuration, we used an ATP-deficient electrode solution supplemented with PKI.
It was essential to demonstrate that long-term changes of eEPSCs under the whole cell configuration were not associated with the absence of ATP and the presence of PKI in the pipette solution. In a control set of experiments, there was no significant change of eEPSC amplitudes for a prolonged period (≥80 min after the whole cell formation) when either the presynaptic or the postsynaptic cell was dialyzed by the whole cell configuration (Fig. 2B). This result does not support the idea that AMPA-receptor–mediated currents are stabilized by internal ATP. The role of ATP was emphasized in the study by Wang et al. (1991). That group reported a partial rundown of the whole cell currents evoked by pressure application of 250 μM kainate to the soma of hippocampal-cultured neurons. This rundown was prevented by supplementing the internal solution with ATP-regenerating agents. The difference in the stability of evoked currents in our experiments and those done by Wang et al. could be explained by the difference in the nature of the recorded currents. The relative contribution of the extrasynaptic and synaptic receptors in the study by Wang et al. is unknown.
It is worth pointing out the difference between experimental conditions in the present study and other studies involving forskolin. First, 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor that enhances the effect of forskolin, was omitted from our experiments. Second, the experiments were conducted in the absence of a γ-aminobutyric acid type A (GABAA) receptor inhibitor, routinely used in other studies (Carroll et al. 1998; Chavez-Noriega and Stevens 1994; Chen and Roper 2003; Duffy and Nguyen 2003). Previously, our group demonstrated that picrotoxin, presumably acting through the GABAA receptors, reversed a facilitating effect of Sp-cAMPS on synaptic transmission in the CA1 area of hippocampal cultured slices (Yu et al. 2001). Sp-cAMPS is a membrane-permeable analogue of cAMP that activates PKA by directly binding to its regulatory subunit.
In experiments using the perforated patch, 15-min forskolin treatment (50 μM) consistently produced an enhancement of eEPSC amplitudes. Each of the six tested neuronal pairs exhibited EP of eEPSCs, and five of six tested pairs exhibited LP of eEPSCs after drug application. That the LP was mediated by the cAMP/PKA pathway is indicated by the fact that it was blocked by Rp-cAMPS, a membrane-permeable nonmetabolizable analogue of cAMP that prevents PKA activation. In contrast to LP, forskolin-induced EP was not completely abolished by Rp-cAMPS (36 ± 17% increase of eEPSC amplitudes when both forskolin and Rp-cAMPS were present in the perfusion solution, Fig. 1A). Upregulation of eEPSCs during forskolin perfusion, despite the presence of Rp-cAMPS, could be explained by the PKA-independent effect of forskolin on synaptic transmission. Such an effect might be attributed to the ability of forskolin, a lipophilic substance, to incorporate into the plasma membrane and/or directly effect certain types of membrane ion channels (Ono et al. 1995; Su et al. 2001).
Results of experiments with Rp-cAMPS were used to quantify a PKA-mediated component of the forskolin-induced changes of eEPSCs during EP. The PKA-mediated component was determined by subtracting a PKA-independent effect of forskolin from the total effect of the drug. Results of the effect of forskolin on eEPSC amplitudes and mEPSC frequency in experiments using different patch-clamp configurations are summarized in Table 1.
Data presented in the table indicate that forskolin-induced EP and LP of eEPSCs are contributed by modulation of postsynaptic proteins. Indeed, when presynaptic events were uncoupled by the whole cell configuration, both EP and LP of eEPSCs were equivalent to those in perforated patch experiments [144 ± 21% (EP) and 53 ± 9% (LP) vs. 140 ± 10% (EP) and 52 ± 7% (LP) increase of eEPSC amplitudes under the presynaptic whole cell and perforated patch, respectively]. Postsynaptic whole cell, to the contrary, abolished both the PKA-dependent component of EP and LP [10 ± 20% (EP) and −4 ± 6% (LP) vs. 104 ± 20% (EP) and 52 ± 7% (LP) PKA-dependent potentiation of eEPSC amplitudes under the postsynaptic whole cell and perforated patch, respectively]. A similar conclusion about the mechanism of the late potentiation of eEPSCs by forskolin in hippocampal slices was made by Duffy and Nguyen (2003).
It is highly improbable that the ineffectiveness of the presynaptic whole cell configuration in disrupting eEPSC potentiation arose from the inability of PKI to reach presynaptic terminals as a result of its limited diffusion. In the low-density culture used for paired recordings, neuronal network is formed by a few closely situated neurons with short processes. A time of 25–30 min, allowed from the moment of the whole cell formation to the start of forskolin application, is sufficient for dialyzing even extensive axonal arbors or long dendritic trees in slice experiments. That the forskolin effect remained unchanged by the presynaptic whole cell could not be explained by merely the ineffectiveness of PKI. Even if the drug was not included in the pipette solution, the lack of ATP and substrates for the endogenous ATP-regenerating machinery in the pipette solution is expected to uncouple, if not impair, phosphorylation events. The most convincing evidence of the effectiveness of the presynaptic whole cell in dialyzing presynaptic terminals is the changed pattern of synaptic responses to the paired-pulse stimulation. Presynaptic whole cell configuration disrupted PPF, which was consistently present in neuronal pairs under other patch-clamp configurations.
In a minority of experiments (one of six experiments under the perforated patch and two of five experiments under the presynaptic whole cell configuration) forskolin failed to produce LP. This variability in the long-term effect of forskolin might be explained by the differential effect of the drug on various types of neurons present in culture. Indeed, similar to the mature hippocampal formation, developed neuronal cultures might contain excitatory neurons of at least two types: pyramidal neurons and granule cells of the dentate gyrus. Thus variability of the long-term effect of forskolin on eEPSCs could be ascribed to the differences in regulation by PKA of the synaptic transmission in these two neuronal phenotypes.
The conclusion about the postsynaptic nature of the forskolin-induced EP is supported by experiments on mEPSCs. The increase of mEPSC frequency during perfusion with forskolin was attenuated when the postsynaptic cell was under the whole cell configuration. We attempted to address the concern that this attenuation could be attributable to the rundown or loss of sensitivity of AMPA receptors during the whole cell recordings (Wang et al. 1991), rather than to a disruption of postsynaptic phosphorylation. To avoid distortion by mEPSC rundown under the whole cell configuration, forskolin was applied 30 min after the whole cell formation, at which time the decline of mEPSC frequency was minimal (Fig. 3A). The upregulation of mEPSC frequency by forskolin applied 30 min after the whole cell formation was still less pronounced than that in perforated-patch experiments (194 ± 25 vs. 345 ± 47% increase under the whole cell and perforated patch, respectively; Fig. 3A). Such an impairment of the forskolin effect would not be expected if the increase of mEPSC frequency took place exclusively as a result of the presynaptic processes because presynaptic neurons remained intact during mEPSC recordings. The results of these experiments suggest that almost half the increase of mEPSC frequency was contributed by postsynaptic mechanisms.
From the perspective of classical quantal theory, existence of the postsynaptic component of the forskolin-induced enhancement of mEPSC frequency seems paradoxical. Classical quantal theory is based on the assumption that the total number of active synapses in a preparation remains unchanged. Thus variability of mEPSC frequency would originate only from a changed probability of transmitter release. The assumption about the constancy of active synapses, however, has been disproved for central synapses. For example, in the hippocampus, one of the mechanisms of synaptic potentiation is induction of postsynaptically silent synapses (Faber et al. 1991; Isaac et al. 1995).
The same mechanism might have contributed to the forskolin-induced potentiation of mEPSC frequency observed under the perforated patch. Although the role of PKA in the induction of silent synapses has not been demonstrated, its role in membrane targeting of AMPA receptors is well established. For example, phosphorylation by PKA of the GluR4 AMPA receptor subunit was necessary and sufficient for its incorporation into synapses (Esteban et al. 2003). In addition, PKA regulates GluR1 recycling between the plasma membrane and endosomal compartments in dissociated hippocampal neurons (Ehlers 2000).
The enhancement of mEPSC frequency by 194 ± 25%, observed under the whole cell configuration, was contributed by presynaptic mechanisms. One general class of such a mechanism, an upregulation of the probability of spontaneous transmitter release, might result from accelerated vesicle cycling at the readily releasable pool of neurotransmitter. Such an effect of PKA activation has been demonstrated in cerebellar granule cells (Chavis et al. 1998). Another class of presynaptic mechanism, the induction of new or silent release sites, was reported for hippocampal cultured neurons (Kohara et al. 2001; Ma et al. 1999). This effect of forskolin, however, was demonstrated in young (<14 days) or pharmacologically silenced neurons and was absent in more mature nonsilenced neurons. Thus induction of new or silent release sites could not contribute to the enhancement of mEPSC frequency in our experiments.
The conclusion regarding the presynaptic component of the forskolin-induced upregulation of mEPSC frequency should be distinguished from our results with paired recordings, which reveal little or no presynaptic contribution. We note that increased spontaneous, calcium-independent transmitter release, observed during mEPSC recording, does not necessarily contribute to stimulus-induced, calcium-dependent transmitter release, which determines eEPSC amplitudes. For example, enhancement of mEPSC frequency might result from accelerated vesicle cycling and a subsequent increased availability of synaptic vesicles at the readily releasable pool of neurotransmitter. However, only if vesicle availability is a limiting factor in evoked transmitter release will such a mechanism change eEPSC amplitudes. Vesicle availability may not be limiting in our experiments using a moderate rate of stimulation (once every 30 s). The limiting factors for the induced transmitter release during paired recordings might rather be the amplitude of the stimulus-induced calcium transients at the presynaptic release sites, or the effectiveness of these calcium transients to trigger synaptic vesicle exocytosis (reviewed by Atwood and Karunanithi 2002).
Having established the role of postsynaptic events, we attempted to address whether modulation of glutamate receptors was one of those postsynaptic events contributing to the forskolin-induced upregulation of eEPSCs and mEPSCs. PKA is targeted to AMPA receptors by means of the A kinase anchoring protein and other scaffolding proteins of the postsynaptic density (Colledge et al. 2000). Phosphorylation by PKA of the GluR1 AMPA receptor subunit at Ser845 increased the peak open probability of the receptor channel opening, a change expected to increase macroscopic current through these receptors (Banke et al. 2000).
Despite ample evidence that AMPA receptors are targeted by PKA, activation of this enzyme did not increase mEPSC amplitudes in CA1 pyramidal neurons treated with forskolin (Carroll et al. 1998). We also found no detectable increase of mEPSC amplitudes in experiments in high-density cultures (data not shown). We hypothesize that the failure to detect potentiation of mEPSC amplitudes in mature neurons could arise from two factors: low signal-to-noise ratio of mEPSC recordings and dendritic filtering. This hypothesis is supported by the report that, in very young hippocampal cultures (2–7 days in vitro), in which the dendritic filtering is minimal, forskolin did potentiate mEPSC amplitudes (Greengard et al. 1991). To avoid the problems of low signal-to-noise ratio and dendritic filtering, we studied the effect of forskolin on mEPSCs in low-density cultures. Neurons in such cultures possess only few, relatively short processes, providing mEPSC records devoid of most dendritic filtering. We have now demonstrated that, in such low-density cultures, PKA activation with forskolin enhanced mEPSC amplitudes. As expected, this effect of forskolin was disrupted by the whole cell configuration (Fig. 4B).
The differential effect of forskolin on mEPSC amplitudes in high- and low-density cultures might arise from developmental differences in these cultures, as well as from the electrophysiological considerations discussed above. For example, GluR1 AMPA receptors may be constitutively and maximally phosphorylated by PKA in a mature hippocampus (Carroll et al. 1998; Mammen et al. 1997). If this also applied to the high-density cultures, it could explain the absence of the forskolin effect on mEPSC amplitudes. However, our Western blot immunostaining argues against constitutive and maximal level of phosphorylation of GluR1 AMPA receptors. As revealed with the anti-P-GluR1Ser845 antibody, forskolin treatment of high-density cultures resulted in an extra band in the Western blot of electrophoretically separated whole cell lysates, indicating increased phosphorylation of the GluR1 subunit (Fig. 4A). An increase of the anti-P-GluR1Ser845 signal after forskolin treatment was also demonstrated in mature rat hippocampal slices (Lee et al. 1998).
It is widely acknowledged that activation of the calcium- and calmodulin-dependent protein kinase II (CaMKII), mediated by NMDA receptors, is necessary for activity-induced LTP in the hippocampus (Bliss and Collingridge 1993; Silva et al. 1992). We tested whether forskolin-induced LP of eEPSCs is NMDA receptor dependent. NMDA-receptor activation might be triggered by an increase in spontaneous firing that accompanies forskolin treatment of cultures during paired recordings. Such a mechanism for the forskolin-induced late phase of potentiation in the CA1 area of the hippocampus was recently reported by Otmakhov and colleagues (2004).
Enhanced spontaneous activity, which manifests itself as an increase of the frequency and amplitudes of spontaneous EPSCs, was also observed in our experiments. We argue, however, that, because during paired recordings the neurons were voltage clamped at −60 mV, activation of these receptors was hardly possible because of a block of NMDA receptors by magnesium ions. In addition, increase of spontaneous EPSCs was not associated with spontaneous action potential generation. In fact, under no conditions during the experiments in the low-density culture did neurons spontaneously fire action potentials (some regions of cultured cells typically escape voltage clamp, and such unclamped action potentials are revealed as transient inward currents). Nevertheless, we could not completely exclude the possibility of NMDA-receptor activation during enhanced spontaneous activity because, at a concentration of external Mg2+ used in our experiments (0.5 mM), a small fraction NMDA receptors might remain unblocked even at high negative membrane potentials (Hestrin et al. 1990).
Our data do not confirm a role of NMDA receptors in the forskolin-induced LP in hippocampal cultures. The NMDA-receptor blocker AP-5 (50 μM) did not alter forskolin-induced LP (Fig. 4C). The observed potentiation of eEPSCs was also stimulus independent. In fact, LP was even more pronounced if the presynaptic neuron was not stimulated during, and 40 min after, forskolin treatment (Fig. 4C).
In summary, results presented here challenge the conclusion of previous electrophysiological studies that the forskolin-induced potentiation of synaptic transmission is mediated by presynaptic mechanisms (Carroll et al. 1998; Chavez-Noriega and Stevens 1994). We have demonstrated that postsynaptic mechanisms are essential for the forskolin-induced potentiation of eEPSCs in hippocampal embryonic neuronal cultures. Phosphorylation of the GluR1 subunit of AMPA receptors, subsequent upregulation of their function, and/or their membrane targeting are among the possible mechanisms contributing to the effect of forskolin on synaptic currents in hippocampal-cultured neurons.
This research was supported by National Institute of Mental Health Grant MH-49176.
We are thankful to S. McKinney for neuronal cultures, A. Kleschevnikov for discussion and valuable suggestions, and K. Diba for help in preparing the manuscript.
↵✠ Deceased 14 February 2002.
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 © 2006 by the American Physiological Society


text:
This photo titled Royal Baby Shower Invitations combined with unique baby shower invitation background images dl43 with the ID 1426 was published at this website on May, 7 2017 by our team. Is it useful for you ? If you need to save this cool ideas, just right click on the picture and save them to your device. The original dimension is 1470×1132 pixels. And the downloable size is arround 0.7 MB only.
There are so many inspiration that you can find on this site. You can get all of them for free since we collect them from search engine like Google and Bing Image. But please make sure that you make use of them for personal use only. We are not responsible for any claims relating to this content.


text:
When Georges St-Pierre steps back inside the Octagon, Firas Zahabi prefers it's against a high-profile fighter.
Ultimate Fighting Championship (UFC) President Dana White claims St-Pierre's middleweight title fight against Michael Bisping is now off the table. When St-Pierre comes back, he's supposedly now competing for the welterweight title. Presumably, it'll be against the winner of a potential Tyron Woodley vs. Demian Maia title bout.
St-Pierre's longtime coach Zahabi would still like to see "Rush" fight Bisping. At the very least, he wants his fighter in a marquee match-up (via MMAJunkie.com):
"I think I want Georges to fight a high-profile fight. He doesn't need any more to go and try to be champion of the welterweight division and try to defend the title every three-four months. I think he's done that, and he's past that. I don't want to speak for him. I don't know if it's a lifestyle he wants to go back to."
If Bisping is no longer an option, a bout with Conor McGregor would certainly draw interest from St-Pierre's team.
"I think he should fight Conor McGregor or Michael Bisping, and I'll tell you why: Michael Bisping will give him a two-title championship, and if he fights McGregor, it'll be the greatest fight in UFC history."


text:
Looks like Heritage Park - 3 Bedroom Home is no longer available on Rent.com which means this information is likely out of date.
1 1/2 year old concrete block home on pond in lovely new community.
Lovely new home, open floor plan, spacious master suite & Kitchen, breakfast nook, formal dining room, large screened in lanai overlooking pond, 2 car garage, beautifully landscaped front & back yard, friendly neighbors, clubhouse, pool, tennis & basketball courts, baseball & soccer fields, picnic, playgrounds and walking trails. Located between US-1 and I-95 off State Road 16. Rent includes, housekeeper, inside pest control, lawn treatments and mowing. Appprox. 5 miles to dowtown St. Augustine.
Cats Accepted. Call for service animal policy.
Don't have a Rent.com account? Create one.
Creating an account allows you to acccess your saved and contacted properties on any device. Already have an account?
Log In
We are experiencing technical difficulties at the moment. Please try again later.


text:
An ex-servicemen (ESM) rally will be conducted at Chilkuri Laxmy Gardens, Mavala, in Adilabad town on Sunday, 14th September, 2014. The rally is being organised by Artillery Centre, Hyderabad under the aegis of Headquarters Andhra Tamil Nadu Karnataka and Kerala (ATNK&K) Area in coordination with Department of Sainik Welfare (Adilabad district). Ex-servicemen belonging to Indian Army, Navy and Air Force, veer naris, widows and next to the kin residing in Adilabad, Nizamabad and Karimnagar districts will attend the rally. The opening ceremony of the ESM Rally will be graced by General Officer Commanding (GOC), Andhra Tamil Nadu Karnataka and Kerala Area apart from prominent civilian dignitaries are also invited to the event.
A large number of facilities are being planned to help the ex-servicemen and war widows, sort out their problems relating to medical and health, pension, rehabilitation and re-employment. This is first time that an ex-servicemen rally is being organised at a far flung area like Adilabad.


text:
1983 South Prairie View Road
Directions Chippewa Falls, WI 54729
Let Our Family Take Care of Your Family, For Over 50 years, Chilson Automotive
Learn More About Buying a $vehicle.year $vehicle.make $vehicle.model in Chippewa Falls
Visit Chilson Chrysler Dodge Jeep Ram FIAT for a great deal on a new $vehicle.year $vehicle.make $vehicle.model. Our sales team is ready to show you all of the features that you will find in the $vehicle.make $vehicle.model and take you for a test drive in the Chippewa Falls Area. At our $vehicle.make dealership you will find competitive prices, a stocked inventory of $vehicle.year $vehicle.make $vehicle.model cars and a helpful sales team. Stop by our car dealership today and take a $vehicle.year $vehicle.make for a spin.
Interested in a different Jeep? Click a link below and find the Jeep that fits your lifestyle.
* The advertised price does not include sales tax, vehicle registration fees, other fees required by law, finance charges and any documentation charges.
* Images, prices, and options shown, including vehicle color, trim, options, pricing and other specifications are subject to availability, incentive offerings, current pricing and credit worthiness.


text: Updated from 10:17 a.m. EDTBristol-Myers Squibb ( BMY) and AstraZeneca ( AZN) announced Thursday that the U.S. Food and Drug Administration had extended its timeline for review of their diabetes drug ONGLYZA. The FDA extended its deadline until July 30, 2009, for review of ONGLYZA, also known as saxagliptin. The original deadline was April 30. The drug is intended to be used to treat type 2 diabetes in conjunction with diet and exercise and a combination of three other drugs, and in initial combination therapy with metformin, as an adjunct to diet and exercise. It's part of the DPP-4 inhibitor drug family, which includes Merck's ( MRK) Januvia.


text:
Hey guys
this is my first post so if I am posting this in the wrong place I appologize. I was wondering if anybody had the time, if they could make me something with cadillac, whether it be a theme, a slider, battery icon, dialer, or whatever, I would greatly appreciate it. I've attached (or something like that )an image that you could incorporate, unless you have something better. I have a DTS if you were gonna take that into consideration too..thanks alot and I appreciate it!


text:
Titles ▾
This movie is one of the best Korean movies I have ever watched, I cried, laughed and smiled because of it. The story is absolutely heartbreaking, some scenes made me cry like a baby, I couldn't stop thinking about how could a human being could do that to a child but besides that the movie is incredible. The story is not just heartbreaking but it's an inspirational . If you are a sensible person this movie is not for you but if you can endure violence scenes than you will endure it just fine. The cast is completely amazing and they fit the characters perfectly well. I couldn't find anything bad about this movie, everything is very well done.


text:
< Back to Leisure and Licensing
Betting and Gaming
We have many years of experience of applying for betting office licences and permits and have made applications on behalf of some of the biggest names in the business as well as individual owner operators.
We are therefore able to advise in relation to all aspects of betting, gaming and lotteries in Norwich, Norfolk and on a nationwide basis.
Related Pages
Bruce Faulkner is one of Norwich's few specialist licensing lawyers"


text:
- This event has passed.
Alex Weitz Group (Wynwood Live)
May 4 @ 7:30 pm - 10:30 pm
Event Navigation
Wynwood Live is a new concert series at Wynwood Cafe & Lounge featuring some of Miami's finest musical and visual artists. Come enjoy great music, artwork, food, drinks, and cocktails in the heart of the Miami art district.
Before the show, from 5-7pm enjoy our Happy Hour featuring 50% off on cocktails, beer, and wine.
Student Special: Quesadilla and Corona for $10
"what (Alex Weitz Group) projects together is something you (really, REALLY) don't often find… total comfort with each other, and an extreme dedication to pleasing the listener who wants more than "just another tune"… totally unique phrasing and a most graceful style"
-Contemporary Fusion Reviews
" It is impossible not to be knocked out by the sheer creative firepower of the artist."
-Raul Da Gama, The Jazz Word
Saxophonist and composer Alex Weitz began his career in Arizona as a member of the award-winning Tucson Jazz Institute Ellington Band, versing himself in the music of its namesake and the classic repertoire of Count Basie. This foundation in swing distinguished him early on as an artist who knows that, to architect a sustainable future, one must mine raw materials from the past. Tapping the roots of tradition in the Southwest gave him more than enough minerals to sustain his relocation to the Southeast, where he studied at the prestigious University of Miami Frost School of Music. While there, he completed his undergraduate studies in Jazz Saxophone Performance and graduate studies in Studio Jazz Writing, gaining invaluable mentorship and experience as a member of the Henry Mancini Institute Jazz Septet under famed trumpeter Terence Blanchard. With Blanchard's encouragement, Weitz strengthened his personal voice, and then some, emerging from the Miami program with an in-depth knowledge of composition that imbues his originals with robust, memorable melodies and structural integrity. Since graduating, Alex's primary musical focus has been on performing, composing, and producing music with his ensemble, the Alex Weitz Group.


text:
Gold Class Transfers will not be responsible for any delays incurred due to traffic congestion or vehicle breakdown circumstances beyond our control.
Gold Class Transfers is not responsible to compensate any funds that travellers incur due to breakdowns or any other problems.
Gold Class Transfers is not responsible for the loss or damage of luggage, personal and/or valuable items.
All transfer services are NON-SMOKING. Due to government law, smoking or the consumption of alcohol is illegal on vehicles by Queensland law.


text:
Today's Featured Artist: Michael Godard
Transform your photos into one-of-a-kind, hand painted masterpieces!
Boundary: Bleed area may not be visible.
Click and drag to re-position the image, if desired.
The more you buy... the more you save.
Image Size
The image is near the edges of the product but doesn't cover the entire product. Some of the background color may appear around the outside edges of the image.
Background Color
Our greeting cards are 5" x 7" in size and are produced on digital offset printers using 100 lb. paper stock. Each card is coated with a UV protectant on the outside surface which produces a semi-gloss finish. The inside of each card has a matte white finish and can be customized with your own message up to 500 characters in length. Each card comes with a white envelope for mailing or gift giving.
Ships Within
1 - 2 business days
Ships From
Charlotte, NC
Front View
Back View
Inside Message
Inside View
There are no comments for Love-fish. Click here to post the first comment.
This is a serie of Pisces which has been created for an Exhibition with the Title: 'Pisces from the Galaxy'. A totally of 38 different fishes have been painted so far. Many of he Originals are available on request, the others are acquirable only as Postcards or Prints
Our return policy is very simple:
If you're not happy with a purchase that you made on FineArtAmerica.com, for any reason, you can return it to us within 30 days of the order date. As soon as it arrives, we'll issue a full refund for the entire purchase price. Please note - Fine Art America does not reimburse the outgoing or return shipping charges unless the return is due to a defect in quality.
Fine Art America sells thousands of pieces of artwork each month - all with a 100% money-back guarantee. We take great pride in the fact that hundreds of thousands of artists have chosen Fine Art America to fulfill their orders, and we look forward to helping you select your next piece!
Customer Service: 877-807-5901
(Call Any Time - 24 Hours Per Day)
All greeting cards ship from our production facility within 1 - 2 business days of your order.


text:
The idea of making isn't just reserved for handmade bikes, artisan pickles, and Arduino helicopters. The future of making is a product of our human needs and the possibilities we create through technology. This is about a larger shift towards making and the unexpected movements that might occur. It's about how everyone from you to your grandma might design, make and consume products or experiences in the next 10 to 15 years. In this session Joi Ito, Director of the MIT Media Lab, and Tim Brown, CEO of IDEO, will host a conversation that considers how we might fashion new tools for the future and then how those tools might influence our lives.
Check out all the Circuit Playground Episodes! Our new kid's show and subscribe!
Have an amazing project to share? Join the SHOW-AND-TELL every Wednesday night at 7:30pm ET on Google+ Hangouts.
Join us every Wednesday night at 8pm ET for Ask an Engineer!
Learn resistor values with Mho's Resistance or get the best electronics calculator for engineers "Circuit Playground" – Adafruit's Apps!
Maker Business — "ORANGE PI: MEETING WITH STEVEN ZHAO IN SHENZHEN"
Wearables — Putty in your hands
Electronics — Multimeter Bandwidth – AC Signal
Biohacking — Google Searching for "My Eyes Hurt" Peaked After Yesterday's Eclipse
No Comments
No comments yet.
Sorry, the comment form is closed at this time.


text: Cost2 Kairu Defense Zone 1None Defense Zone 2None Defense Zone 3NoneAttack Power350 Red - Damages Top Defense ZoneAbility:METACHARGE: If this hits, your other Attacks have +300 power this turn.HealsNoneRarityTBA Anime Add a photo to this gallery Retrieved from "http://redapedia.wikia.com/wiki/Bone_Crusher?oldid=13120" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Attack Cards Game Metacharged Cards


text: True, I need more sleep! Apologies and thanks, my mind is on autopilot. s/your/the/ Sam On 1/17/06, Holger Krull <holger.krull@...> wrote: > Sam Moffatt schrieb: > > I'm not sure, might want to try it and see if this resolves your > > issue. Sounds like the right command though. > > > > I've got issues? Well, they belong to Giacomo. > > > ------------------------------------------------------- > This SF.net email is sponsored by: Splunk Inc. Do you grep through log fi= les > for problems? Stop! Download the new AJAX search engine that makes > searching your log files as easy as surfing the web. DOWNLOAD SPLUNK! > http://sel.as-us.falkag.net/sel?cmd=3Dlnk&kid=3D103432&bid=3D230486&dat= =3D121642 > _______________________________________________ > coLinux-users mailing list > coLinux-users@... > https://lists.sourceforge.net/lists/listinfo/colinux-users >


text: 4Chan, I.. Adjust content blocking dethmajick next random Comment Top NextComment This was what 4chan was like before all the summerfags came in and started trolling everything. next Tags: fucking | Cars | Flying | killing | Shooting | violence | Porn | Awesome | shit | tits do or C! File: KB, 1536x2048, . jpg) "*5 Hello there, I' m posting to see if any of you are local to Richmond, Virginia. I' m looking for a one night stand, and i kid you not, this is srs laws. I am no trap, and i' m not fucking around. This is legit. I' m just looking for a guy under old thats got the balls to fuck me tonight. I' m not a dirty whore with the herps either, ijust haven' t gotten laid in a few months and want something that wont lead to a relationship, so post your picture and if you tickle my fancy i' ll ask for your email then you can send me your number. And here' s a timestamp for proof. Lets get to it, the sooner i find a guy worthy, the better. File: -( 524 KB, 1536x2048, ) I posting a few other pictures of me. Anonymous no Immense} 07/ 22/ 12( Sun) 23: 23: 09 No. File: -( 206 KB, 1224x2048, ) lpg) and another. Anonymous no ) 07/ 22/ 12( Sun) 23: 23: 17 OH CKD- ... +8 Views: 1950 Submitted: 07/23/2012 Facebook Twitter Google+ Tumblr Pinterest


text:
Teenage Mutant Ninja Turtles (2014)
April O'Neil
Vernon Fenwick
Eric Sacks
(written by)
(written by) and
list by I.T.log
list by schwarzerabt
list by JPHOLT
list by johanlefourbe
list by Tumppi
Teenage Mutant Ninja Turtles Videos
112 Views, 3 votes
Added 3 years ago
63 Views
Added 3 years ago
53 Views
Added 3 years ago
Cover art, photos and screenshots
April O'Neil: What are you?
Leonardo: We're ninjas.
Raphael: We're mutants!
Donatello: Technically, we're turtles.
Michelangelo: And we're teenagers. But we can have adult conversations.
April O'Neil: So, you're... Ninja Mutant Turtle Teenagers?
Donatello: When you put it like that, it sounds ridiculous!
Alright so despite ... read more
Teenage Mutant Ninja Turtles review
Teenage Mutant Ninja Turtles was probably the first fandom i joined as a kid. I absolutely loved them. I watched the cartoon and the first two movies over and over again. Even as a kid i knew the third one sucked. I also had them on my clothes, shoes, wrist watch, lunch boxes, and everything else they could plast... read more
Update feed
"Katsottu: 29.1.2017, rewatch, TV (Nelonen) En kyllä muistanut että tämä oli näin paska. Tarina tuntuu väkisinväännetyltä ja leffan huumori aiheuttaa enimmäkseen myötähäpeää. Eilen tuli aloiteltua tämän jatko-osaa, mutta nukahdin kesken. Ei taida tulla katsottua loppuun. "
"28.1. TV Älytön leffa, mutta ehkä just sen takia tykkäsin. Rakastin turtleseita muksuna eikä tää leffa millään tasolla pilannut mun lapsuusmuistoja, joten sikäli tää oli ihan onnistunut tekele. Huonoja vitsejä ja ihan kivannäkösiä toimintakohtauksia. Ois sitä voinu lauantai-illan huonomminkin viettää, kuten esimerkiksi ihmisten seurassa."
"Didn't offer anything new, what the trailers or reviews has already showed. Story was uninteresting and villains non threatening dummies. Though the action was pretty enjoyable. Didn't like the turtles personality a much, but liked a lot their design. I think i was one of the fews who liked their look when the first trailer came out. And ou my god the amount of those adds throughout the movie. Level of Transformers. Shared With Friends! Users: Cuomi & hsallinen and his brother"
"Bernadette Thompson"
"Re-Watched 06/04/2016 Rating 6.0/10 Format: Blu Ray Still a pretty enjoyable flick i think. "
"Ei vain lähde,äh."
"for "Teenage Mutant Ninja Turtles: Out of the Shadows" Director: Jonathan Liebesman Starring: Megan Fox, Will Arnett, William Fichtner"
"This was just 100 mins of stupid shit, boredom and ads. The jokes were shitty and the story didn't have any effect on me at all. The turtles themselves were okay, I guess, but there wasn't anything else in this."


text:
Thursday's Picks
March 17, 2005
Home Team in CAPS
Anthony's Picks:
Last Night: (3-2), Week: (6-4)
Be wary- Ant will probably be sloshed by the time he puts his picks up.
Paul's Picks:
Last Night: (3-0), Week: (7-2)
- GOLDEN STATE [17-21-2] -2 over Sacramento [17-17-2]
- West Virginia -1 over Creighton
- Nevada +1.5 over Texas
- LSU -3.5 over UAB
- Utah St. +5.5 over Arizona
- Winthrop +14 over Gonzaga
Sorry if you don't like the green font- just thought I'd do something lame to commemerate St. Patty's Day since I can't celebrate the approriate way (I'd kill for a pint of Guinness right now). That GS/Sac line is a bit misleading-- GS had been on the road for awhile up until last game in which they played a red-hot Houston team very tough. Linesmakers are banking that betters will forget this and assume the team is struggling. I'd go with GS on this one.
I'm also going to try my luck with some tourney games. West Virginia if you'll remember got off to a hot start this season after feasting on mid-majors, then stumbled during the first half of the Big East season due to their lack of talent, and pulled together late in the season thanks to some sharpshooting and a very underrated coach John Beilen. I think they pull this one out over the Blue Jays of Creighton. Texas is without 2 starters and has a freshman point guard-- I can't remember the last time a team who's best player was a frosh PG did anything in the tourney-- take tourney-tested Nevada. LSU is a team to keep an eye on-- they played great in the second half of the season, have a star in Brandon Bass, and aren't getting as many points as they should because a lot of people are taking UAB based on last season's run. I can see them making a deep run--possibly as far as the Elite 8. That Utah St./'Zona line is shocking-- Utah St. only got in because they won their conference tourney and Zona is the kind of team that can drop 90 against anyone. The line suggests that the Aggies will control the tempo and given that everyone is taking Arizona, I'm going to stick with Vegas and take Utah St. Hell, I have them winning anyways. And Winthrop is a very underrated team who supposedly is athletic enough to run with Gonzaga in what should be an entertaining game. Once again everyone is banking on the Zags, so I'm siding with Vegas once again. Good luck tonight...


text:
When authors first publish their book, the fresh release date makes it easier to mention the book in day-to-day conversations and more exciting to seek out new promotion opportunities, but what do you do after your book has been out for a few years? Hard core authors say that the best way to sell a book is to write another one, and book marketing professionals say to promote it to a new audience or even repurpose the book content into something new. No matter which approach you choose, APSS may be just the community you need to help you reach your goal of maintaining long-term book sales.
What I appreciate the most about APSS is that they offer a free membership option that includes notification about free educational webinars and conference calls. If you get hooked and you are interested in a paid membership option, you will get a discount on the already affordable membership that offers more for less than comparable organizations for publishers. It also helps that the new head of the organization is book marketing guru Brian Jud who has consulted for traditional publishers, self-publishers and hybrid authors to get bulk and long-term sales. If it has something to do with selling books-especially with selling books in bulk-he has experience with it.
I don't get any benefit by recommending the organization. I've had a free membership for over a year since Jud took over, and I love getting information that I can pass along to authors. I was skeptical about how helpful and practical their webinars would be, but Jud has a long track record of success and I can tell by the way he does business that he's in the business of putting people before profit.
So whether you have a contract with a traditional publishing company or you are self-published, I highly recommend participating in a couple free webinars to see if the kind of information they offer is the kind of thing you need to keep making sales over time. You'll have to explore it for yourself to find out whether it can help you with long-term sales like it has for so many others. Learn more on their website: http://community.bookapss.org/.


text:
"You don't want love. You want a love experience."
– Knight of Cups
In the Tech Age, the "human experience" has become harder to define. Before, the "human experience" meant all of the things that a person experiences as a mortal on planet Earth: joy, grief, enlightenment, fear, anger and lust. These are experiences and emotions that occur naturally through our interaction with the world, with each other, through our five senses. Nowadays, experiencing the joy of listening to a beautiful piece of music for the first time, or seeing magnificent landscapes thousands of miles away from your home, can be achieved digitally.
Through synthetic realities–such as virtual reality simulations or simply photos online–we can feel and think we know the backstreets of Le Marais, in Paris, as if it were our hometown by navigating through it on Google Street View a hundred times.
When we are in love with someone, are we truly "in love" with them or are we actually addicted to the holistic experience of above-average infatuation (as I like to define the term "in love")?
After all, humans are, to put it simplistically, sophisticated animals that learned to respond to external stimuli through complex processes in our mind (psychological) and brain (physiological/neurological) that was developed via evolutionary adaptation. We've also created social constructs, such as codes of ethics based on religion and laws based on those codes of ethics (and perceptions of the state as an administrator of public safety and order–e.g. why it's illegal to run a red light or corporations are fined millions of dollars for improperly disposing hazardous waste).
Through these social constructs and psychological processes, we've evolved to become biological machines that are constantly thinking, analyzing and making judgments every minute or less. So how can we discern what it is love–which to this day still does not have a clear definition–and what is the love experience?
I once thought I loved a woman overseas. We wrote letters to each other, communicated online, and went through ups and downs like any other couple (although we were never officially in a relationship). We both admitted to each other that we "loved" or was "in love" with the other at certain points in our timeline. We've known each other for almost a decade. We've never met. Was love possible without physical presence, without physical intimacy, without shared memories of actually doing things together in person? Perhaps we were just in love with the experience of what we thought was love.
When I was 19, I wrote a poem about walking through the woods in the northern parts of New England. I was able to depict the scenery in vivid details, despite never having been to New England at that time, due to extensive reading and thorough visual research online. About 8 years later, I finally went to New Hampshire and when I stood on the ridge of a mountain after walking through a heavily wooded trail, I felt a sense of déjà vu.
Did the "memory" I construct through my imagination when I wrote that poem and through web photos overlap with my actual first-hand experience?
I believe that first-hand accounts are important. I believe that seeking genuine experiences are important. If we rely heavily on second-hand information–whether through travel stories from a friend or YouTube videos–we can become susceptible to misinformation, deception and mind pollution. In fact, military and political propaganda is applied using this premise.
On a personal note, I believe that seeking "genuine experiences" like the love experience is just as good as seeking love itself. After all, what is love but an experience, right?


text: SOUND HOLIC MEETS TOHO ~東方的幽々舞踏劇~ Shangri-la Storm Pages in category "M3-19" The following 2 pages are in this category, out of 2 total. S Shangri-la StormSOUND HOLIC MEETS TOHO ~東方的幽々舞踏劇~ Retrieved from "http://touhou.wikia.com/wiki/Category:M3-19?oldid=89260" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Conventions


text:
As a boy, he dreamed of being a rock star. Now, one local musician just may get his opportunity.
"It was a chance in a lifetime," Jeremy Jackson said during a telephone interview.
Jackson, front man of local rock band Subcurrent, recently was tapped to travel to Los Angeles to audition for the lead singer position of the multi-platinum rock band Fuel.
Jackson submitted an electronic demo through the band's Web site and was one of 20 people chosen during a nationwide search to get the opportunity to perform with the band in front of several Epic Records music executives.
The winner of the auditions, which took place April 21-22 at Swinghouse Studios in West Hollywood, will be featured on the group's next album.
In addition to lesser-known artists, Fuel recently has shown an interest in former "American Idol" contestant Chris Daughtry. A post on the band's Web site May 12 says the remaining two members of Fuel "are entertaining the idea, and the offer to Chris was that we would be willing to entertain him as an option."
The posting, by guitarist Carl Bell, goes on to note that there are many contractual details on both sides to be worked out.
Daughtry hit the band's radar after performing "Hemorrhage" on "American Idol." That is the same song Jackson submitted to get an audition.
Jackson, a Gadsden native who works as a painter by day for T&D Painting, submitted an MP3 of his vocals over "Hemorrhage" along with a photo and a brief bio. Executives called him within a week.
Jackson admitted to being nervous initially, but thinks he did pretty well at the audition.
"Once the music started, it was just like being back home playing with my band," he said.
Jackson said the crowd of 10 to 15 music executives applauded when he finished performing his first song.
"Then, they said sing another song, and I did," Jackson said, "It took about 40 to 45 minutes, then I was back on the streets of West Hollywood."
Jackson said although it was only a brief exchange, he got along well with the other band members, and there was chemistry on stage.
"The band was really cool," he said.
Bell broke a string during the intro to the band's hit song "Shimmer."
After the last notes, he switched guitars and asked Jackson to sing "Hemorrhage."
"They made it real light. They couldn't have made it anymore comfortable, I don't think. They were real nice. Up until that point, I had been kind of nervous. I immediately felt comfortable. As far as getting into the music, that was definitely happening. We got to the end of it, and they said it was all really good," Jackson said.
"Until the end, walking out, it really hit me," he added. "I don't think I could have done anything different or any better without changing who I am."
Amanda, Jackson's wife, told him afterward that Bell had been staring intently at him during the entire performance.
"They heard me. That's what they got. They didn't get a carbon copy of their last guy. They got me. I wanted to make sure I stayed true to myself. I guess that's what he was trying to see, if I was really feeling the music or acting like I was feeling the music," Jackson said.
Jackson said he had not heard from Epic Records yet, but even if he doesn't, his experience was memorable.
Jackson said he and his wife spent the rest of their time "stargazing" and exploring Hollywood.
It was the couple's first time visiting the West Coast.
Since he's returned home to Gadsden, Jackson said people ask him how things went and continue to congratulate him.
He also said he was able to secure some contacts that could help the band in the future.
"What it's done for Subcurrent, it's been really good. I don't think anything but good things could have come from this. Everybody's been like family. It's really supportive.
"Every day I get somebody asking me about it," he said.
When asked what he would do if he received a call to become the lead singer of Fuel, he said, "I don't know. How do you prepare for something like that?"


text:
L:Ú L:Ú Clothing Singapore
To suit your home, most L:Ú L:Ú Clothing are manufactured with either Yellow, White and Silver. You can find popular L:Ú L:Ú Clothing, such as Dresses and Leggings. Looking for a L:Ú L:Ú Clothing? Find good ones not only from L:Ú L:Ú Clothing but also from Summer, Asos and trendsgal. Can you believe they are only S$ 30.00 - S$ 353.00 for L:Ú L:Ú Clothing on iprice? Depending on your preferences and budget, you can choose between Pants, Dresses and Cardigans.


text:
Need Appliance Repair Help? Ask a Technician for Answers ASAP
Hello from JA My name is Woody. I will be glad to assist you today. I have been servicing ALL Major Brands of Appliances for 35 Years, and have over 100,000 in home repairs and over 80,000 internet repairs under my belt.
The use and care booklet show to turn it off ,you turn the time selector knob to off.
Page #7 .
here is a link to the use and care booklet.
ok if you have the time knob set to off and it comes back on .has by chance the knob been removed and put back on in correctly?
And how old is the unit?