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19430 Surnames | 123743 Records | 2647 Reunions
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Photo #55480
Last Name: Gladwell
First Name : Earl
Middle Name: Eugene
Subject's City: Hamilton
Subject's State: Ontario
Subject's County: Wentworth
Subject's Country: Canada
Date: 1901-1920
Photographer's City: Possibly England
Photographer's State:
Type** : WED
Comments: Earl Eugene Gladwell (Nee: ) | Hamilton Ontario Canada | 1901-1920 | Comments: Earle Eugene Gladwell 1896-1956 son of Fred Gladwell and Nellie Shaw.
Annie Florence Harris 1892-1922
Wed in London, England, Hackney District in 1918.
Digital Image Only? NO
Everyone Listed In This Photo:
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Created by a certified running coach, the magnetically attached Buddy Pouch™ family is the #1 Running Pouch. Available in a variety of sizes, this armband and running belt replacement is light & large enough to accommodate the new larger cell phones.
The Buddy Pouch fits the iPhone 4, 5, 6 &6S, 6+ & 6+S, 7, 7+, Galaxy S and Galaxy Note.
✮ Customer Reviews: Using the Pouch While Running, Cycling and at the Gym
The belt-free pouch is designed with a water-resistant inner pocket and made with soft, dri-fit material keeping all your running essentials including inhalers and EpiPens sweat-free and dry. Revolutionary, magnetic technology keep the Buddy Pouch in place no matter how intense your pace or workout. Click for Discount Active Bundles
The all new Running Buddy "Mini" was designed as a smaller magnetic pouch for those who are looking to carry the basics while on the go.
Buddy Pouch H20 provides you with the ultimate hands-free/belt-free experience, but still allows you to carry several ounces of fluid with you for that quick run. The smooth contoured bottle is very comfortable tucked behind your waist or at the side of your hip. Strong magnets keep the H20 secure on any waistband.
No more pins or holes! Simply snap & lock!
Compact, user-friend pepper sprays and personal alarms give you peace of mind without slowing you down.
The Buddy LED Safety Light is great for runners and walkers. Simple to attach and detach, without any damage to shoes. A convenient and sporty solution to stay illuminated!
Listen to your tunes and the road with our left/right and size adjustable single & double earbuds.
The HB Hand Band is designed for all versions of the iPhone and all other smart phones. Simply slide your device into the pouch, strap it on your hand and you will have complete control of the touchscreen in the palm of your hand!
Buddy Blockers help keep your personal information safe and offers the security of RFID-shielding to protect your cards from being scanned by identity thieves.

subset: commoncrawl


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Grapevine Wreath- 8 inch
Product ID : GPV8
Grapevine wreaths provide a beautiful natural, cottage look when used bare. They also provide a perfect base for dried and artificial flowers, seasonal greenery, lights, ornaments and much more. Perfect for creating your own unique home décor. 1 piece per package.
Product Type : Types
Our Price: $0.97
1 per package.


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Newegg.com - A great place to buy computers, computer parts, electronics, software, accessories, and DVDs online. With great prices, fast shipping, and top-rated customer service - once you know, you Newegg.
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Showing Results: Most Recent
This review is from: Acer P243WAid Black-Silver 24" 2ms(GTG) HDMI Widescreen LCD Monitor with HDCP Support 400 cd/m2 3000:1 ACM
Pros: I purchased this in 2008 and it's still functioning perfectly in 8/2014 and I use it quite often. I wrote this review as a little nod to Acer quality for this monitor.
Cons: NoneREAD FULL REVIEW
This review is from: KWorld VS-L883D X-Pert DVD Maker PCI Card
Pros: This capture card works fine and is very inexpensive , you can't beat that
Cons: noneREAD FULL REVIEW
This review is from: Thermaltake VOLCANO 10+ 60mm 2 Ball Cooling Fan/Heatsink
Pros: This is a great simple all copper fan to replace the oem fan. It cools really well and isn't bulky or expensive like higher models
Cons: noneREAD FULL REVIEW


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Looking for a vacation rental in Kingscliff? Tripping.com has 532 rental homes available in Kingscliff. Potential renters will find studios, 281 1-bedroom, 142 2-bedroom and 109 rentals with 3 or more bedrooms ranging from $25 a night to $902 a night. Many of these properties are instantly bookable.
The average price of a nightly rental in Kingscliff is $156 while the average price of a weekly rental is $1381. For those looking for more than a short term rental such as corporate rentals, extended stays or long term rentals, the average monthly price of a Kingscliff home is $899.
Wondering if you can bring your favorite dog or cat with you? We have 6 pet friendly vacation rentals available for you. In addition to accommodating pets, Kingscliff rentals offer a variety of amenities. In particular, 76 rentals have internet and wifi, 88 have TV and/or cable and 80 have a pool or hot tub.Search properties from 7 rental sites in Kingscliff where the top providers in that area are Booking.com, Agoda, and TripAdvisor. There are 424 Booking.com rentals, 57 Agoda short term rentals and 39 TripAdvisor vacation rentals.


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The Nike Pro Combat Branded Skull Cap is as sleek as headwear gets, but gets the job done. It will give you just enough warmth to get going, but won't make you overheat as it is well ventilated.
Thank you for visiting Nike Pro Combat Banded Skull Cap 2.0 - SP15 Running Headwear on oakleysbikes.co.uk
We bring the products from many websites to one website for you to search, save and compare.


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Please note: This item is kerbside delivery This requires an adult to sign upon delivery
Constructed using tongue and groove panels, this rectangular planter from Rowlinson is raised from the ground to save your patio or grass, and aid drainage of the plant. The planter is made from pressure-treated wood for longevity and protection against rot. It comes in a natural timber finish and can be painted or stained if desired.
Includes 15-year manufacturer's guarantee against rot. H50xW100xD50cm.


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There are 12 people in a dinner gathering. In how many ways can the host (one of the 12) arrange his guests around a dining table if a. they can sit on any of the chairs? b. 3 people insist on sitting beside each other? c. 2 people refuse to sit beside each other?1
by mamanaleyann


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Between the economic downturn and the looming concern over climate change, small businesses are finding more reasons than ever to emphasize sustainability. Here we explore the companies and the strategies that are performing best.
In Denmark, Brewery's Departure Offers a Chance to Go Green
Carlsberg's decision to vacate its brewery gave Copenhagen an opportunity to regenerate a neighborhood, and meet a target to become a carbon-neutral city.
February 22, 2017, Wednesday
MORE ON SUSTAINABILITY AND: Real Estate and Housing (Residential) , Greenhouse Gas Emissions , Energy Efficiency , Carlsberg Group , Copenhagen (Denmark)
Conservatives' Plan for a Carbon Tax
Readers suggest that the plan is workable, or not.
February 21, 2017, Tuesday
MORE ON SUSTAINABILITY AND: Greenhouse Gas Emissions , Taxation
How an Interoffice Spat Erupted Into a Climate-Change Furor
A retired scientist became a hero to some climate-change deniers after he criticized his former boss at NOAA over data handling.
February 20, 2017, Monday
MORE ON SUSTAINABILITY AND: Bates, John (Scientist) , Global Warming , National Oceanic and Atmospheric Administration , United Nations Framework Convention on Climate Change , Research , Workplace Environment
Protests in Iranian City Where 'Everything Is Covered in Brown Dust'
Residents of Ahvaz, a center of oil production, protested for days over dust storms, power failures and government mismanagement.
February 19, 2017, Sunday
MORE ON SUSTAINABILITY AND: Iran , Demonstrations, Protests and Riots , Dust and Sand Storms , Air Pollution , Ahvaz (Iran)
A Push for Diesel Leaves London Gasping Amid Record Pollution
Britain's efforts to aid the environment have inadvertently led to a spike in an even more insidious form of air pollution than the deadly smog of 1952.
February 17, 2017, Friday
MORE ON SUSTAINABILITY AND: London (England) , Air Pollution , Great Britain , Fuel Emissions (Transportation) , Diesel Power
Eco-Friendly, Nontoxic and Vegan: It's a Condom
The market for natural alternatives to household staples like diapers, hand soaps and paper towels has another niche business: condoms.
February 17, 2017, Friday
MORE ON SUSTAINABILITY AND: Condoms , Sustain Natural , Hollender, Meika , Seventh Generation , Hollender, Jeffrey , Sustainable Living , Organic Foods and Products , Hazardous and Toxic Substances
Environmental Doomsday
A reader urges Democrats to protest the administration's assault on the environment.
February 16, 2017, Thursday
MORE ON SUSTAINABILITY AND: Democratic Party , Environment , Global Warming , Republican Party , United States Politics and Government
Disappearing Seagrass Protects Against Pathogens, Even Climate Change, Scientists Find
An ecological cornerstone is vanishing rapidly from the oceans, and scientists fear the consequences for climate change, fisheries and disease.
February 16, 2017, Thursday
MORE ON SUSTAINABILITY AND: Carbon Capture and Sequestration , Coral , Fish and Other Marine Life , Fishing, Commercial , Global Warming , Flowers and Plants , Chesapeake Bay , Indonesia
A Bee Mogul Confronts the Crisis in His Field
Beekeeping on an industrial scale is central to American agriculture, and "colony collapse" has proved to be a severe test.
February 16, 2017, Thursday
MORE ON SUSTAINABILITY AND: Bees , Adee Honey Farms , Adee, Bret , Environmental Protection Agency , California , Agriculture and Farming , Environment
Greenpeace Links Beijing's Air Pollution Surge to Steel Factories
A report shows how state-owned enterprises and local officials have acted to keep steel companies operating out of self-interest despite an overcapacity problem.
February 16, 2017, Thursday
MORE ON SUSTAINABILITY AND: Greenhouse Gas Emissions , Economic Conditions and Trends , Greenpeace , Beijing (China) , China , Steel and Iron , vis-multimedia
Cleaning Up Our E-Waste Mess
Traci Phillips of Tulsa, Okla., is in the business of recycling old cellphones, discarded laptops and other electronic waste, a fast-growing environmental problem.
Pedal-Powered Delivery
A Portland company moves freight using six electric-assisted tricycles and strong riders in lieu of trucks.
Tom Szaky, the founder and C.E.O. of TerraCycle, explains how he saved his company despite mounting manufacturing costs.
Preserving the Music
Guitar makers are rethinking the notion that there is an inexhaustible supply of wood to make their instruments.
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Carlos Pl, London, W1K 2AL
020 3404 3387
If you are looking for a highly qualified remodeling or repair service, you've come to the right place.If you have additional questions, or if you are ready to hire Handyman Mayfair, please call or provide the details of your job via the contact form on our website and we will get back with you as quickly as possible. Thanks for letting us be your personal handyman!
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A Japanese radiation expert has claimed that life just one kilometre outside the restricted area surrounding the stricken nuclear plant leaking radiation is "as safe as London".
In the latest proclamation by officials trying to reassure people that they face minimal danger from the nuclear crisis a government adviser said the public had been misled by inaccurate information.
But it came amid more sinister news as it emerged that the plant had contained far more spent fuel rods than it was designed to store, while its technicians failed to carry out the necessary safety checks, according to documents from the reactor's operator.
Despite hopes of progress in the world's most major nuclear crisis in a quarter of a century, plant operator Tokyo Electric Power Co (TEPCO) said it needed more time before it could say the reactors were stabilised. The documents also showed that the the company repeatedly missed safety checks over a 10-year period up to two weeks before the 11 March disaster, and allowed uranium fuel rods to pile up inside the 40-year-old facility.
Yesterday smoke and steam were seen rising from two of the most threatening reactors, two and three, whilst several blasts of steam are thought to probably have released a small amount of radioactive particles.
The news is likely to aggrieve the public, especially in the wake of claims earlier in the day from a Japanese radiation expert that life just 1km outside the restricted area surrounding the stricken nuclear plant leaking radiation is "as safe as London".
"The people we should worry about are those working at the site of the Fukushima Daiichi nuclear power plant. Otherwise we do not have to worry about exposure," said Professor Shunichi Yamashita.
Professor Yamashita, from Nagasaki University, said radiation in Tokyo was minuscule and being hyped needlessly by the media: "We're talking about microsieverts [SI unit of radiation] a year not millisieverts," he said. "The dose is crucial. It's nonsense to worry about microsieverts."
Concerns about radiation in Tokyo have prompted thousands of foreigners and Japanese citizens to leave. Many businesses have relocated to the west of Japan or Hong Kong. The latest radiation reading over 24 hours was 0.34 microsieverts though the figure is increasing slightly. Yesterday the professor said exposure to 11.4 microsieverts per hour was the safety benchmark.
Officials from the International Atomic Energy Agency found radiation 1,600 times higher than normal around the town of Namie, near Fukushima. But Professor Yamashita said at a briefing in Tokyo: "People who evacuated from the 20km zone are completely safe." Japan's Prime Minister last week advised 140,000 people living within between 20km and 30km of the complex to leave. Many have criticised the perimeter as arbitrary. The US government says 80km is the safe limit.
Professor Yamashita said most radiation-linked illnesses in the 1986 Chernobyl disaster, including thyroid cancer, came not from direct exposure but from consuming food, especially contaminated milk. "Compared to Chernobyl the Fukushima radiation release is still very, very low," he said. "I want the media to make this clear."
Chernobyl, the world's worst nuclear disaster, has been linked to thousands of thyroid cancer cases since the plant in Ukraine went into meltdown.
The Japanese government has imposed a ban on food products from Fukushima and surrounding areas. Small quantities of radiation have been detected in spinach and milk. The government has also begun studying contamination of fish food after discovering radioactive materials 128 times higher than the legal limit in the seas around the plant.It was reported yesterday that minuscule amounts of radioactive particles believed to have come from the Fukushima power plant had been detected as far away as Iceland.
For those people who have been evacuated from the danger zone near the nuclear plant, these are days of anxiety. Around 1,400 people are living in the town's gymnasium which has been turned into an evacuation centre, and yesterday there were long queues for bowls of hot noodle soup. As many as 2.4 million people are still without water and nearly 250,000 households have no power.
Profiting from the doom
The huge earthquake and tsunami that ripped through north-eastern Japan 11 days ago has led to lots of stories of stoicism and pulling together. But not in every case.
Police said the disaster crippled a bank's security mechanisms and left a safe wide open. That allowed someone to walk off with ¥40m, about £300,000.
The tsunami washed over the Shinkin Bank, like much else in Kesennuma in Japan's north-east region, and police said that with the combination of the flooding and the ensuing power outages the vault came open.
"The bank was flooded and things were thrown all over. It was a total mess. Somebody stole the money in the midst of the chaos," a police official in the Miyagi region said.
The bank notified police yesterday when the theft was discovered. AP
- More about:
- Nuclear Power
- Tokyo Stock Market
- Tsunami
- Ukraine


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Steaming is always better than boiling. If you submerge corn on the cob into a big pot of boiling water, you'll have water that tastes like corn and corn that tastes like water. Rather than direct contact with the hot liquid where your fresh corn on the cob can lose flavor, add only a small amount of liquid to your stock pot.
Serving size Per servingComplete recipe
Calories 38 Calories from Fat 4
% Daily Value*
Total Fat 0.52 g0.79%
Saturated Fat 0.08 g0.4%
Trans Fat 0 g
Cholesterol 0 mg
Sodium 6.6 mg0.3%
Total Carbohydrates 8 g2.8%
Dietary Fiber 1.2 g4.7%
Sugars 1.4 g
Protein 1 g2.8%
Vitamin A 1.6%
Vitamin C 5%
Calcium 0.09%
Iron 1.3%
*Based on a 2000 Calorie diet


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Weddings, Corporate and Private Events, Restaurants and Wineries in Southern California: Orange, San Diego, Los Angeles Counties, and Inland Empire
*Brian performs regularly at Ponte Winery in Temecula.
* Brian's professional goal is to combine the highest quality musicianship with a personable, detail-oriented approach resulting in a memorable wedding celebration that is both seamless and worry-free. Ask about the upgrade possibilities to make your day just perfect!
Professional Live Music
* Brian Stodart and Mike Gross offer quality live music that will complement, not overwhelm, your wedding celebration with their versatile duo (piano, vocals, percussion and guitar). Brian's WEDDING MUSIC PACKAGE provides music for your ceremony and reception. In addition to choosing from their well-rounded playlist of over 100 songs, you may request up to two special selections and they will add them to their repertoire specifically for your wedding day. Ask about Brian's Portable "Baby Grand" Piano!!
Personal Consultation
* Brian's goal is to enable you to be completely focused on the significance of this once-in-a-lifetime event. During your in person consultation, you will have the chance to get better acquainted as you walk through all of the important details of your wedding day. Mike and Brian pride themselves on their ability to set your mind at ease combining attention to detail with a friendly, relaxed atmosphere.
Customized "DJ" Service
* This dynamic duo offers tailor-made playlists to create an atmosphere that is perfectly suited to the tastes of you and your guests.
Multiple PA Systems
* Mike and Brian provide all necessary amplification, including up to three independent systems (for separate ceremony, cocktail hour and reception locations) and wireless microphones for all public speaking applications.
* As your Emcee for the day, Brian will ensure that your wedding day celebration flows effortlessly, on schedule, just the way you envisioned it!
Up to 6 Hours of Service
* Service begins as the first guests arrive at your ceremony (usually 30 minutes prior to your official ceremony time).
Copyright © 2001-2017 OneWed Inc.
All Rights Reserved.
Copyright © 2001-2017 OneWed Inc. All Rights Reserved.


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The Navarre Beach Area Chamber of Commerce is a vibrant membership organization with more than 500 business, civic and individual members. The Chamber serves as a collective voice for the Navarre area - representing their interests. Additionally, the Chamber organizes a host of community-centered activities and events.
Navarre is located in Northwest Florida in Santa Rosa County. The community is well-known for its beautiful Gulf of Mexico beaches and Santa Rosa Sound. However, residents and visitors quickly discover the Navarre area is much more. Top-rated schools, quality healthcare and a variety of housing options make Navarre a great location to not only visit, but also call home.
On our website you will find useful information about our members and the community. If you should have any questions, please do not hesitate to contact us at email@example.com
8543 Navarre Parkway Navarre, FL 32566
(850) 939-3267
*Prices in US dollars (USD). Prices reflect lowest average nightly rates. Wemake every attempt to get accurate prices, however prices are not guaranteed. N/A = Not Available
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Wego.com recently found 3 hotels in Oxelosund, Sweden. Book last minute hotel or find great hotel deals such as Stjarnholmsslott Hotel for US$147 per night. Save more on cheap hotels with rates from as low as US$147 per night.
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Flights to Sweden Flights from Sweden Sweden Airports Sweden Airlines


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We offer the full range of flavors and here's a piece of flavor trivia :
For many years, Zesty Ranch was advertised on the back of the packages but that flavor was never officially produced. To many Andy Capp snack lovers, this flavor is considered the "holy grail"
Andy Capp Cheddar Fries Andy Capp Cheddar Fries are a perennial favorite and have been since they were first offered in 1971 by Goodmark Foods. In 1998, this classic snack favorite was acquired by Con Agra Foods. The name comes from the fact that an Andy Capp comic strip was fea
- Price: $28.97


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40 User Opinions
"Battery good Camera OK OK"
"Battery an issue . As soon as the one year warranty period got over, the performance of the battery started to dip."
"The battery backup is even worse. It doesnt even last for half a day."
"Charges faster, The camera does far better than its predecessor."
"Battery back up is very poor . Battery back up is total waste and sometime iphone temperature is high , Apart from battery its a awesome phone."
"Good phone but low battery backup and extra small scree. ."
"This may led to shortage of memory, battery draining etc."
"Apart from the heating issue ( a little while charging or using Wifi for longer time) it works nicely. You can really enjoy the brand."
"Battery life is low. ."
"This phone is awesome to use battery backup"


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Annie Hobnail Glass Floral Vase 5.5" Wide x 5.25" Tall
DetailsLooking for wedding containers to complete your floral centerpieces? Check out this gorgeous Annie hobnail glass floral vase in clear. This cute glass container would make a simple yet beautiful arrangement for your vintage glam wedding!
- 5.5" Wide x 5.25" Tall
Shop All Glass Vases
Shipping & Returns
- Economy Shipping: 5-10 business days
- Ground Shipping: 1-5 business days
- Expedited, International, and SmartPost options available at checkout.
- More Shipping Information »
Return this item within 30 days for a full product refund. Return Policy »


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Features: Software Reviews
The Top Portfolio Management Software
October 15, 2016 by Andrew Plis
The four best programs for managing your portfolio allow you to view breakdowns of assets, track your transactions and calculate performance.
Manage Your Investment Portfolio With Fund Manager
January 16, 2016 by Mark Beiley
Beiley Software's owner describes how to track the performance of your investments and your portfolios with his Fund Manager program.
Manage Your Portfolios With Investment Account Manager 2
May 16, 2015 by Hareesh N Jayanthi
An in-depth look at software that helps you assess the holdings in your portfolios and gauge their performance.
A Comparison of Tax Preparation Services
March 21, 2015 by Hareesh N Jayanthi
A review of three popular websites for tax preparation show many similarities but also some clear differences.
Quicken 2015 (Premier) for Windows
December 20, 2014 by Hareesh N Jayanthi
After a few years with minimal changes to its popular personal finance software, Intuit has made some valuable upgrades this time around.
Track Your Finances on a Mac With iBank
First Quarter 2012 by Joe Lan
A look at a personal finance program designed for the Mac and capable of syncing with mobile devices.


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Photo Collections
Looking ahead
Blog Entry by David Howard and viewed 183 times. Updated 35 months ago.
Looking for the Devil's Study
Geo-trip by Anne Burgess, 114 images. Updated 44 months ago.This had been on my list for some time, because I had read the description in... (more)
Looking for the Gunpowder mills
Blog Entry by Bill Nicholls and viewed 263 times. Updated 54 months ago.
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Geo-trip by Bill Nicholls, 92 images. Updated 55 months ago.A short walk from Garliston to trace an old railway that ran there, taking in a... (more)
Query '@title looking' retrieved 4 of 4 matches in 0.001 sec.


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Web Button Design
This project was successfully completed by schmeisserV for $60 USD in 3 days.Get free quotes for a project like this
Project Budget$30 - $250 USD
Completed In3 days
Total Bids29
Project Description
I need 3 web buttons designed for a website. The buttons are light up switches, see example. I will need an at rest button, and a hover state for each button. The hover state should look like the switch is illuminated. The final size of the buttons will be 390 X 50 pixels. I will need a RED, ORANGE and YELLOW button with editable text. I want the graphic designed in adobe Illustrator.
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Icecat: syndicator of product information via global open catalog with more than 3845116 data-sheets 14559 brands – register (free)
Add to compare Don't compare Add to compare
You can only compare products that are fully described in the Icecat standard format, and belong to the same category.
The general trademark of a manufacturer by which the consumer knows its products. A manufacturer can have multiple brand names. Some manufacturers license their brand names to other producers.HP
Product name:
Product name is a brand's identification of a product, often a model name, but not totally unique as it can include some product variants. Product name is a key part of the Icecat product title on a product data-sheet.1 GB Secure Digital Memory Card
Product code :
The brand's unique identifier for a product. Multiple product codes can be mapped to one mother product data-sheet if the specifications are identical. We map away wrong codes or sometimes logistic variants.FA283A
Category :
Pocket-sized card with embedded integrated circuits. Smart cards can provide identification, authentication, data storage and application processing. Smart cards may provide strong security authentication for single sign-on (SSO) within large organizations.smart cards
Data-sheet quality: created by HP
The quality of the product data-sheet can be on several levels:
only logistic data imported: we have only basic data imported from a supplier, a data-sheet is not yet created by an editor.
created by HP: a data-sheet is imported from an official source from a manufacturer. But the data-sheet is not yet standardized by an Icecat editor.
created/standardized by Icecat: the data-sheet is created or standardized by an Icecat editor.
only logistic data imported: we have only basic data imported from a supplier, a data-sheet is not yet created by an editor.
created by HP: a data-sheet is imported from an official source from a manufacturer. But the data-sheet is not yet standardized by an Icecat editor.
created/standardized by Icecat: the data-sheet is created or standardized by an Icecat editor.
Included since: 15 Jul 2005
The date Icecat noticed for the first time that this product was included in the price list of a channel partner
Info modified on: 06 Feb 2017 11:22:23
The date of the most recent change of this product data-sheet in Icecat.
Product views: 32528
This statistic is based on the 66464 using ecommerce sites (eshops, distributors, comparison sites, ecommerce ASPs, purchase systems, etc) downloading this Icecat data-sheet since 15-07-05 Only sponsoring brands are included in the free Open Icecat content distribution as used by 64733 free Open Icecat users .
HP iPAQ hx2000 Pocket PC series (0.5MB)
Here, we only show product PDFs of sponsoring brands that joined Open Icecat. As Full Icecat channel partner login to see all product data or request a Full Icecat subscription.
Warranty: Your Option Limitedis a one (1) year (HP Option LimitedPeriod) parts replacementon any HP-branded or Compaq-branded options (HP Options). If your HP Option is installed in an HP Hardware Product, HP may provideservice either for the HP Option LimitedPeriod or the remaining Limited Warranty Period of the HP Hardware Product in which the HP Option is being installed, whichever period is the longer but not to exceed three (3) years from the date you purchased the HP Option.
Long product name HP 1 GB Secure Digital Memory Card:
The short editorial description of HP 1 GB Secure Digital Memory CardNeed more storage? This high-performance 1 GB Mini SD memory card is perfect for backing up a large amount of files and data. You can use it to create music cards, photo/video libraries or for storing maps if you have GPS navigation capability.
Marketing text HP 1 GB Secure Digital Memory Card:
The official marketing text of HP 1 GB Secure Digital Memory Card as supplied by the manufacturerSD Memory Cards let you store more programs and files than ever before. Create music cards to use during your daily commute or to take with you on long trips. Use SD cards to back up sensitive files that are stored on your iPAQ Pocket PC. Store special programs on an SD card to free up memory on your iPAQ Pocket PC for daily operations. Also great for sharing pictures from an SD digital camera or music with an MP3 Player.
Short summary description HP 1 GB Secure Digital Memory Card:
This short summary of the HP 1 GB Secure Digital Memory Card data-sheet is auto-generated and uses the product title and the first six key specs.HP 1 GB Secure Digital Memory Card
Long summary description HP 1 GB Secure Digital Memory Card:
This is an auto-generated long summary of HP 1 GB Secure Digital Memory Card based on the first three specs of the first five spec groups.HP 1 GB Secure Digital Memory Card
This is a demo of a seamless insert of an Icecat LIVE product data-sheet in your website. Imagine that this responsive data-sheet is included in the product page of your webshop. How to integrate Icecat LIVE JavaScript.
1 No information available on Specs


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With Instructables you can share what you make with the world, and tap into an ever-growing community of creative experts.
So simple and fastUnlock all your System PreferencesThis i found on a google search, not my idea
Check you System Preferences (SP from not on), usually locked at school or work, make sure that yours are locked, if not then your done.Do a Spot light ...
Rename SPto something you want I am going to name it SP2, so you know what is the unlocked version
Right (control click) on the SP2go to Show Package Contentsnow navigate to /Contents/Resourcesnow delete the file NSPrefPaneGroups.plistnow close package Contents and run SP2
Now run it, dont worry all the easy to use buttons are gone but all you have to do is go to viewyou may have to click view ...
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Housing excellently located next to the River Turia garden. House with elevator located on A fifth with 3 bedrooms and a bathroom. The kitchen is near the living room and is equipped and reformed. Then we access the spacious living room that has a window and a balcony from where you can see the Turia gardens. The whole house has gas heating radiators.
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It is estimated that more than 3% of the world's population carry mutations in the 2-globin gene locus that cause more or less severe cases of anemia. One of the best known and characterized of these mutations is an amino acid change in the adult 2-globin chain that causes sickle cell disease (SCD). Other mutations lead to thalassemias, which are characterized by reduction in globin gene synthesis. Currently, there is no satisfactory treatment for hemoglobinopathies that will benefit all patients. The 2-globin gene locus consists of five genes that are competitively regulated by a locus control region (LCR) during development. The LCR is composed of five DNase I hypersensitive (HS) sites that are spread over more than 10 kbp of DNA and which act together to mediate high-level globin gene expression. Despite advances in understanding globin gene regulation over the last three decades, how the LCR operates to mediate high level globin gene expression is not understood. A detailed knowledge of the molecular mechanisms leading to stage specific expression of the globin genes will aid the development of novel and more broadly applicable therapies for hemoglobinopathies. This is best illustrated by the fact that expression of the fetal 3-globin in adults homozygous for the sickle cell mutation ameliorates the severe form of the disease. The development of strategies that silence the mutant 2-globin gene and at the same time activate expression of the 3-globin genes would offer novel therapies for a variety of hemoglobinopathies. Clearly, a detailed understanding of how the genes are competitively regulated by the LCR is crucial for the development of therapies that target or take advantage of LCR mediated mechanisms of globin gene regulation. Approach: The goal of this application is to decipher the mechanism(s) by which the LCR regulates 2-globin gene expression. It is proposed that the LCR serves as the primary attachment and assembly site for elongation active transcription complexes in the 2-globin gene locus. The elongation active transcription complexes are transferred to globin gene promoters in a developmental stage specific manner. This hypothesis will be addressed in transgenic mice by examining if the LCR is sufficient to form transcription foci in erythroid cells and how transcription factor upstream stimulatory factor (USF) in conjunction with erythroid specific transcription factors regulates this process (Specific Aim 1). Furthermore, to analyze the mechanism of transcription complex recruitment and assembly, components of transcription complexes recruited to the LCR and to the adult 2-globin gene promoter will be characterized using a novel in vitro assembly assay as well as using chromatin immunoprecipitation (ChIP) in intact cells (Specific Aim 2). We will map accessibility in the LCR and the rest of the 2-globin gene locus during differentiation using a novel approach, called MAPit, designed to assay protein DNA interactions at high resolution on single molecules (Specific Aim 3). Finally, to identify cis-regulatory DNA elements that mediate interactions between the LCR and the globin genes we will express zinc finger DNA binding domains that neutralize transcription factor binding sites and examine the effect of these proteins on globin gene expression and LCR globin gene interactions (Specific Aim 4).
The goal of this study is to increase our knowledge of how the 2-globin genes are regulated by the locus control region and to develop new tools that could aid in finding better cures for hemoglobinopathies. The major focus of this application is to determine the role of transcription factors and cis-regulatory elements in the high-level expression of the 2-globin gene. Interfering with activities that mediate high-level expression of the adult 2-globin gene could lead to increased expression of the therapeutic 3-globin genes in patients with sickle cell disease.
|Barrow, Joeva J; Li, Ying; Hossain, Mir et al. (2014) Dissecting the function of the adult ?-globin downstream promoter region using an artificial zinc finger DNA-binding domain. Nucleic Acids Res 42:4363-74
|Deng, Changwang; Li, Ying; Liang, Shermi et al. (2013) USF1 and hSET1A mediated epigenetic modifications regulate lineage differentiation and HoxB4 transcription. PLoS Genet 9:e1003524
|Rosenberg, Michael; Fan, Alex Xiucheng; Lin, I-Ju et al. (2013) Cell-cycle specific association of transcription factors and RNA polymerase ii with the human *-globin gene locus. J Cell Biochem 114:1997-2006
|Anantharaman, Archana; Lin, I-Ju; Barrow, Joeva et al. (2011) Role of helix-loop-helix proteins during differentiation of erythroid cells. Mol Cell Biol 31:1332-43
|Litt, Michael; Qiu, Yi; Huang, Suming (2009) Histone arginine methylations: their roles in chromatin dynamics and transcriptional regulation. Biosci Rep 29:131-41
|Luo, Yi; Jian, Wei; Stavreva, Diana et al. (2009) Trans-regulation of histone deacetylase activities through acetylation. J Biol Chem 284:34901-10
|Thurisch, Boris; Liang, Shermi Y; Sarioglu, Nanette et al. (2009) Transgenic mice expressing small interfering RNA against Gata4 point to a crucial role of Gata4 in the heart and gonads. J Mol Endocrinol 43:157-69
|Liang, Shermi; Moghimi, Babak; Yang, Thomas P et al. (2008) Locus control region mediated regulation of adult beta-globin gene expression. J Cell Biochem 105:9-16


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Recent studies have focused attention on mechanisms of spontaneous large-scale wavelike activity during early development of the neocortex. In this study, we describe and characterize synchronous neuronal activity that occurs in cultured cortical networks naturally without pharmacological intervention. The synchronous activity that can be detected by means of Fluo-3 fluorescence imaging starts to develop at the beginning of the second week in culture and eventually includes the entire neuronal population about 1 wk later. A synchronous increase of [Ca2+]i in the neuronal population is associated with a burst of action potentials riding on a long-lasting depolarization recorded in a single cell. It is suggested that this depolarization results directly from synaptic current, which was comprised of at least three different components mediated by AMPA,N-methyl-d-aspartate (NMDA), and GABAA receptors. We never observed a gradually depolarizing pacemaker potential and found no evidence for a change of excitability during inter-burst periods. However, we found evidence for a period of synaptic depression after bursts. Network excitability recovers gradually over seconds from this depression that can explain the episodic nature of spontaneous network activity. Using pharmacological manipulation to investigate the propagation of activity in the network, we show that synchronous network activity depends on both glutamatergic and GABAAergic neurotransmission during a brief period. Reversal potential of GABAA receptor-mediated current was found to be significantly more positive than resting membrane potential both at 1 and 2 wk in culture, suggesting depolarizing action of GABA. However, in cultures older than 2 wk, inhibition of GABAAreceptors does not result in block of synchronous network activity but in modulation of burst width and frequency.
Development of highly organized structures and connections in the CNS depends on both activity-independent and activity-dependent mechanisms. In recent years, the occurrence of spontaneous synchronous neuronal activity during defined periods of early CNS development has been described in a wide range of structures and species (for review see Ben-Ari 2001; Feller 1999; O'Donovan 1999). This rather primitive form of network activity is suggested to be an essential step in the formation of functional networks. It coincides with the depolarizing action of GABA and glycine (Ben-Ari 2001), which act as excitatory transmitters via chloride permeable receptor channels in young neurons (Cherubini et al. 1991; Leinekugel et al. 1995;Owens et al. 1996), although the activity may be carried by different neurotransmitters and modulators varying with structure and age. In the retina, excitation is initially provided by GABA and acetylcholine and later switches to glutamate (Wong 1999). Similarly, in the spinal cord, GABA and acetylcholine start out as the dominating transmitters in the generation of patterned spontaneous activity (Milner and Landmesser 1999) but are relieved later by glutamate (O'Donovan 1999). Thus in most structures, GABA or glycine contribute initially to the generation of spontaneous synchronous neuronal activity in developing networks.
Very recently, large-scale spontaneous correlated neuronal activity has been described in the immature rat neocortex (Garaschuk et al. 2000; Peinado 2000, 2001), which differs from oscillations in local cortical domains (Yuste et al. 1992) in the requirement of action potential firing. Although it is still a matter of debate if this activity is carried by glutamatergic transmission (Garaschuk et al. 2000) or dendro-dendritic gap junctions (Peinado 2001), there is no compelling evidence for the need of GABAAreceptor activation in the neonatal cortex.
As in the neonatal cerebral cortex, synchronized rhythmic activity also develops spontaneously in neuronal networks formed by embryonic neurons in cell culture. It can be observed as slowly rhythmic synchronous bursting of larger numbers of neurons accompanied by Ca2+ transients (Maeda et al. 1995; Murphy et al. 1992; Robinson et al. 1993), thus resembling hallmark features of spontaneous correlated activity recorded in cortical slices. Interestingly, failure of neurons to participate in the spontaneously generated activity of a network resulted in their elimination from the network (Voigt et al. 1997). In most cases, however, spontaneous activity of cultured cortical neurons was described under conditions facilitating glutamatergic synaptic transmission, for instance by use of Mg2+-free extracellular solution, which prevents observations on the role of GABA. In a recent study, we found evidence that the development of synchronous calcium oscillations in neocortical cultures depends on the presence of a certain type of GABAergic neuron originating in the cortical preplate (Voigt et al. 2001). This suggests the necessity of GABAergic transmission for the expression of early synchronous network activity also in networks of neocortical neurons. Here we describe in detail the development of synchronized activity in unstimulated cultures of rat cerebral cortex in conditions as similar as possible to the standard culturing conditions. This activity appears comparable to large scale activity recorded from acute brain slice preparations in that it involves action potential firing and correlated increase of intracellular Ca2+ concentration ([Ca2+]i). We provide evidence for synaptic origin of synchronous network activity and find a period of synaptic depression after bursts. Furthermore, we show that GABAergic transmission is necessary for synchronous activity in young cultures but rather modulating oscillatory network activity of older ones although GABA still acts depolarizing.
Cell culture
Cortical cells were cultivated on poly-d-lysine coated coverslips in serum free N2 medium in the presence of a glial feeding layer (for detailed description of the method see de Lima and Voigt 1999). Neocortical cells from rats at embryonic day 16 were prepared by trypsin treatment followed by mechanical dissociation and were plated with a density of 200 cells/mm2. Excessive proliferation of glial cells was prevented by adding cytosine arabinoside (Ara-C) to the cultures at a final concentration of 5 μM at the 4th day in vitro (DIV). One-third of the medium volume was then changed after 24 h and no further medium change was performed for the rest of the cultivation period.
Fluo-3 videomicroscopy
Fifty micrograms of Fluo-3 pentacetoxy-methylester (Molecular Probes, purchased from MoBiTec, Goettingen, Germany) were dissolved in 45 μl DMSO. Of this stock, 15 μl were added to 3 ml N2 culture medium to load the cells with dye (final concentration: 4.9 μM Fluo-3, 0.5% DMSO). For recordings in N2 culture medium, one-half of the medium was saved for later use before Fluo-3 was added. After 1 h of dye loading, cultures were washed twice with DMEM, and the saved portion of the original medium was restored. Dishes were tightly sealed to prevent degassing during recording that would lead to major pH changes. In most cases, however, fluorescence recording was performed in HEPES-buffered artificial cerebrospinal fluid (aCSF). The ionic composition of this aCSF was (in mM) 140 NaCl, 5 KCl, 1.5 CaCl2, 0.75 MgCl2, 1.25 NaH2PO4, 20d-glucose, and 15 HEPES/NaOH (pH 7.4) and resembled the main components of the N2 culture medium with the exception of the pH buffering system (HEPES vs. bicarbonate). After ≥30 min to allow deesterification of the dye, culture dishes were transferred to an inverted microscope (Axiovert S100 TV; Zeiss) equipped with a cooled charge coupled device camera (Princeton Instruments). Fields for recording were chosen randomly and sometimes marked with a diamond tool when repeated imaging or later identification was required. We used 20× or 40× objectives (Plan-Neofluar; Zeiss), resulting in images covering an area of 380 × 380 μm2 or 190 × 190 μm2, respectively. Frames were recorded at different intervals with sample time adjusted to cell loading (typically 200–400 ms). Excitation wavelength was 470 ± 20 nm (Chroma Technology, Brattleboro, VT). A differential interference contrast (DIC) image of each field was also acquired for later cell identification. Frames were stored on computer and processed off-line using MetaMorph software (version 3.5; Universal Imaging, West Chester, PA). To recognize changes in the fluorescence, we subtracted from each frame the one preceding it, and shifted zero (i.e., no change) to a value representing a medium gray level (=2046 for a 12-bit image). This procedure yields images as shown in Fig.1, where white depicts increased and black represents decreased fluorescence. These differential images enabled us to identify regions of interest (we used circles of 7.5 μm diam placed on neuronal somata) that corresponded to active neurons as confirmed with the help of DIC images. Ca2+signals from astrocytes, when present, appeared as slowly propagating waves along processes. They could be clearly distinguished from signals of neuronal origin and were excluded from analysis. Average gray values of regions of interest were calculated, and a change in [Ca2+]i was considered significant when the absolute difference of gray values exceeded five times the SD of background noise measured in cell-free areas. Neuronal density was calculated using DIC images of the recorded fields and usually expressed as number of neurons per square millimeter. Neurons could be clearly identified and distinguished from nonneuronal cells in these images.
Drugs and drug application
All drugs were dissolved to 100×–1000× stocks, stored at −20°C, and diluted to final concentration just before application. We purchased tetrodotoxin (TTx) from Alomone Labs (ICS Clinical Service, Munich, Germany), 5-aminomethyl-3-hydroxyisoxazole hydrobromide (muscimol) and (−)-bicuculline methiodide (BMI) from RBI (RBI/Sigma, Deisenhofen, Germany), andd-2-amino-5-phosphonopentanoic acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) from Tocris Cookson (Biotrend, Cologne, Germany). Drugs were applied either directly from the stocks in case of static bath recordings (some of the Fluo-3 fluorescence imaging experiments) or via a gravity-fed perfusion system. For stimulation of a smaller part of the network or a single cell, we used multichannel device that was controlled by magnetic valves (ALA Scientific Instruments, New York, NY) and had an inner tip diameter of 250 μm.
Electrophysiological recordings
The feeder glia surrounding the neuronal culture was wiped off, and an acrylic ring was fixed to the culture dish with silicon grease, resulting in a chamber with a volume of 1–1.5 ml. This chamber was mounted to the stage of the inverted microscope and continuously superfused with aCSF at 1–2 ml/min. In some experiments (illustrated in Fig. 1), we used another aCSF to examine possible effects of HEPES versus bicarbonate buffering. This aCSF contained the following (in mM): 123 NaCl, 5 KCl, 1.5 CaCl2, 0.75 MgCl2, 1.25 NaH2PO4, 20d-glucose, and 23 NaHCO3 (pH 7.35 after gassing with 95% O2-5% CO2). All electrophysiological recordings were performed at room temperature. Whole cell current-clamp and voltage-clamp recording was carried out with an Axoclamp-2B (Axon Instruments, Foster City, CA) or EPC-7 (HEKA Electronics, Darmstadt, Germany) amplifier. For most recordings of membrane potential or current, the patch pipette (tip resistance 3–5 MΩ) contained the following (in mM): 135 potassium gluconate (C6H11O7K), 15 KCl, 2 MgCl2, 10 HEPES, 0.2 EGTA, 2.5 Mg ATP, and 0.25 Na GTP (pH 7.2). For some voltage-clamp recording (Fig.4 C), another pipette filling solution was used (in mM): 120 cesium methanesulfonate (CH3O3SCs), 10 CsCl, 1 MgCl2, 10 HEPES, 1 CaCl2, 11 EGTA, 2.5 Mg ATP, and 0.25 Na GTP (pH 7.2). To monitor membrane potential in parallel to changes of [Ca2+]i, cultures were loaded with Fluo-3 AM as described above. The recording electrode was filled with the following solution (in mM): 135 potassium gluconate, 15 KCl, 2 MgCl2, 10 HEPES, 2.5 Mg ATP, and 0.25 Na GTP (pH 7.2), supplemented with 10 μM Fluo-3 pentapotassium salt. While recording membrane potential continuously, sequences of images (20–60 frames at 1 Hz, exposure time 200–400 ms) were captured to disk, processed off-line as described above, and correlated with the electrophysiological signals. To determine the reversal potential of GABAA receptor activated current (EGABA), we employed the perforated patch method with gramicidin as the pore-forming agent because this prevents disturbance of intracellular [Cl−] (Kyrozis and Reichling 1995). Recording electrodes were filled with the following solution (in mM): 120 KCl, 1 CaCl2, 11 EGTA, and 10 HEPES, (pH 7.2), supplemented with 50 μg/ml gramicidin (Sigma). Although we did not use gramicidin-free solution in the electrode tip, no problems with gigaseal formation were encountered. After stabilization of series resistance (typically 30–50 MΩ within 10–20 min) we locally applied 30 μM muscimol. A voltage-ramp protocol (20 to −100 mV within 800 s) was started when the current had reached steady state. After leak subtraction, I-V plots were constructed and reversal potential was computed by linear regression. Throughout this paper, means are ± SD.
To test if neocortical neuronal networks in vitro are capable to develop synchronous neuronal activity spontaneously without pharmacological intervention and under conditions present in the incubator, we recorded [Ca2+]i in the original N2 culture medium. Because of the interference of phenol red in the N2 medium with Fluo-3 fluorescence measurements and the instability of the bicarbonate-based buffer system, however, we developed an aCSF that closely matched the ionic composition of the N2 medium used for culturing (see methods). Recordings were performed in 8-day-old cultures using both media subsequently in the same dish and yielded virtually the same results (Fig. 1), but the images in aCSF were crisper with no background bleaching, making the aCSF a suitable choice for the recordings described in this paper. In N2 medium, fluorescence image sequences revealed synchronous increase of [Ca2+]i in 51 ± 10 (n = 6) neurons per 380 × 380-μm2 field (Fig.1 A1 ). In aCSF, 55 ± 32 (n = 6) neurons per field showed synchronously elevated [Ca2+]i (Fig.1 A2 ). Synchronous activity occurred at a fairly low frequency and was observed only once in each 1-min recording sequence (Fig. 1, B and C).
Rhythmic network activity could also be monitored with electrophysiological recording, which allowed observations over longer time periods at high time resolution. We employed this method to examine possible effects of the HEPES-buffered aCSF compared with bicarbonate buffering that is used in the culture media. At 13–16 DIV, neuronal networks showed robust rhythmic activity (Fig. 1 D). On a switch from HEPES- to bicarbonate-buffered aCSF, network activity slowed down substantially and bursts appeared less coherent (Fig.1 D, a and b). A small (3–6 mV) transient hyperpolarization was found in most neurons (7 of 9). Input resistance did not change significantly (580 ± 188 vs. 555 ± 122 MΩ in HEPES- and bicarbonate-buffered aCSF, respectively). However, network activity recovered to frequency and burst appearance observed in HEPES-buffered aCSF within 5–15 min (Fig. 1, D andE).
To explore the development of synchronous activity in our culture system, we recorded spontaneous [Ca2+]i changes in cultures of the same preparation at 6, 9, 12, and 15 days after plating for a total of four different preparations. In each dish we recorded fluorescence images at a frequency of 1 Hz from four to five fields using a 20× objective, resulting in a field size of 380 × 380 μm2. For each field, the total number of neurons as well as the number of active neurons was determined. Analysis of these experiments is summarized in Fig.2. At six DIV (median, 28%; range, 14–42%) of all neurons were spontaneously active and the same portion of neurons remained active in the presence of 10 μM CNQX and 20 μM BMI (median, 27%; range, 17–41%). During this spontaneous intrinsic activity we found the maximal fraction of simultaneously active neurons to be 7.5%. Thus for further analysis of the developmental time course of synchronous activity, a significant rise of [Ca2+]i in more than 10% of the neurons in the field was considered a synchronous event. Such synchronous activity was first detected at 9 DIV in 10 of 20 examined fields. In these fields, 41% (median; range, 10–82%) of all neurons participated in synchronous events. At 12 DIV we observed synchronous increases of [Ca2+]i also in only about one-half of the examined fields (11 of 20). However, the fraction of neurons that were involved in the synchronous events now averaged 91% (median; range, 37–100%) of all neurons in the fields. Fifteen days after plating, synchronous activity could be recorded in almost all examined fields (15 of 16), and in these fields, 94% (median; range, 81–100%) of the neurons took part in those events. Over the 9-day observation period, the neuronal density declined steadily from 1804 ± 59 neurons/mm2 at 6 DIV to 626 ± 31 neurons/mm2 at 15 DIV (n = 16 fields), a phenomenon usually observed in cortical cultures over this time period (Voigt et al. 1997). At the same time, however, the number of active neurons as assessed by Fluo-3 fluorescence imaging remained relatively stable with 512 ± 24 neurons/mm2 at 6 DIV and 568 ± 33 neurons/mm2 at 15 DIV (n = 16 fields). Thus similar to cultures examined in Mg2+-free solution (Voigt et al. 1997), after 2 wk in culture, the steady decrease of neuronal density and the increasing number of neurons participating in synchronous activity lead to a network with virtually all members being synchronously active.
To correlate the changes in [Ca2+]i with the actual changes in membrane potential patch-clamp recordings were combined with Fluo-3 fluorescence imaging in a 12-day-old culture. These recordings showed that a rise of [Ca2+]i coincided with a pronounced depolarization and firing of action potentials (Fig.3 A) in all neurons that were measured in this set of experiments (n = 7). Importantly, when [Ca2+]iincreased synchronously in a larger number of neurons in the field under observation, the electrophysiological recording displayed a burst of action potentials riding on top of a long-lasting depolarization. The electrophysiological response roughly correlated with the number of adjacent cells that displayed a significant rise of [Ca2+]i. When only a small number of neurons were active (i.e., showed a [Ca2+]i increase) at a time, single or compound excitatory postsynaptic potentials (EPSPs) were observed that did not reach action potential threshold. When a larger number of cells were synchronously active, the neuron under investigation fired a burst of action potentials (Fig. 3 A). Thus although only one neuron at a time could be observed, electrophysiological recordings allowed registration of synchronous network activity, which could be investigated for longer time periods than with Fluo-3 fluorescence imaging. The inter-burst interval and hence the time between two synchronous network events was relatively stable over long time periods. In the example shown in Fig.3 B, the inter-burst intervals lasted 10.9 ± 2.2 s (n = 43) with no significant trend to slow down or accelerate during the 40 min of measurement (Fig. 3 C) and followed a Gaussian distribution (Fig. 3 D). However, the interval between the synchronous events was variable among different cultures and was found to be in the range of 9–87 s (22 cultures from 4 different platings analyzed). There was no apparent correlation between age of the network and frequency of synchronous activity (R 2 < 0.1).
A detailed characterization of the synchronous burst activity was carried out in neurons after about 2 wk of culture (12–16 DIV). When the recording mode was switched from current clamp to voltage clamp near resting membrane potential (RMP), large inward currents were observed at the same phase and frequency as the bursts (Fig.4 A). These currents appeared to be barrages of synaptic currents (Fig. 4 B) of which three different components could be isolated. At a holding potential of −70 mV (which is close to the estimated Cl− reversal potential), the inward current could be partially inhibited by theN-methyl-d-aspartate (NMDA) receptor antagonist d-APV (50 μM), leaving a component most probably driven by AMPA receptors (Fig. 4 C). The effect ofd-APV was especially pronounced with regard to the kinetics. A time constant of the current decay was derived by a single exponential fit but was found to vary substantially between different preparations. However, it was relatively constant for bursts recorded from the same cell, making quantitative comparisons of current decay before and after d-APV application feasible. For the neuron illustrated in Fig. 4 C1 , for example, τdecay of the burst-associated currents was calculated to be 649 ± 268 ms under control conditions, but only 356 ± 166 ms after application of 50 μM d-APV (n = 7 bursts). In another five cells, τdecay of currents recorded in the presence ofd-APV was found to be one-half to one-tenth of the control values. At a holding potential of 0 mV, large outward currents were observed that could be totally blocked by the GABAA receptor antagonist BMI (20 μM) and were assumed to represent GABAergic inputs during burst activity (Fig.4 C2 ).
The membrane potential during the bursts recorded in current-clamp measurements and the correspondent inward currents observed in voltage clamp shared approximately the same time course, suggesting the maintained synaptic current as the source of the long-lasting depolarization. To exclude that a voltage-dependent sodium or calcium "plateau" current is also activated, we applied short strongly hyperpolarizing current pulses during burst depolarizations, which should switch off a plateau current if present (Fig.5 A). This protocol did not affect the time course of the decay (n = 5 cells), suggesting that the depolarization results directly from synaptic current. A further indication that this suggestion is correct was that direct stimulation of the neuron by short (30–50 ms) depolarizing current pulses was able to trigger single action potentials but never induced bursts nor influenced the rhythm of the intrinsic bursts (n = 10 cells).
A common mechanism of rhythmic bursting is the appearance of pacemaker currents that lead to gradual depolarization of the neuron between discharges. However, in all neurons that were recorded in current-clamp mode during this study (n = 36 cells), the membrane potential remained relatively stable between bursts, and we never observed a pacemaker potential. Another mechanism of rhythmicity could employ a gradual increase of excitability after the bursts so that the neuron becomes increasingly prone for participation in a network activity. To explore this possibility we applied short (30–50 ms) depolarizing current pulses in regular intervals to bursting neurons (Fig. 5 B), estimated the threshold potential to fire an action potential for every inter-burst current injection, and examined for correlation with time left until the next burst. The rationale of this protocol was to detect a decreased action potential threshold if excitability rises. However, in none of the five neurons studied with this protocol did we found evidence for a change of the threshold potential between two bursts (Fig. 5 C). No strong correlation between temporal proximity to a burst and action potential threshold could be observed (R 2 = 0.037).
Another mechanism that has been suggested to render spontaneous activity an episodic nature is activity-dependent depression of network excitability. To test for a period after a burst during which the network is not able to trigger the next burst we recorded the membrane potential of a neuron and stimulated part of the network (about 2–5.5 mm away from the recording site in the direction of the aCSF flow to avoid direct effect on the recorded neuron) by brief pulses of aCSF containing elevated [K+] (1 s, 15 mM KCl) at random times after each fifth spontaneous burst. Such a stimulus was able to induce neuronal activity that spread synaptically (tested by blocking with 10 μM CNQX, data not shown) through a large part of the network and could be recorded even >5 mm from the stimulation site as long-lasting depolarization with a burst of action potentials (Fig.6 A). Plotting the width of the evoked burst against the interval between stimulus and preceding spontaneous burst revealed a period during which the network could not be activated (Fig. 6 B). The duration of this period of network depression appeared variable between different cells (3.7–9.3 s, n = 4 cells) and could not be determined accurately because after a number of stimuli (>10) the network did not respond reliably anymore.
To gain more insight into the involvement of activity-dependent depression in synchronous network activity, we analyzed the statistical relationship between burst width and inter-burst interval. If the network is depressed always to the same level after a burst and recovery is gradual, then the duration of the next burst should depend on the time of recovery. On the other hand, if the level of depression depends on the burst width, then there should be a correlation between burst duration and the following inter-burst interval. For this analysis we selected networks with slow and more irregular burst frequency (inter-burst intervals ≤1 min) for a larger range of values. Burst duration and inter-burst intervals were calculated from long-term membrane potential recordings of 6 neurons containing 27–52 bursts. Plotting the burst width against the interval preceding the burst revealed that the duration of a spontaneous burst became larger with longer recovery from the last synchronous network activity (Fig.6 C). On the contrary, there was no apparent dependence of the following inter-burst interval on the burst duration (Fig.6 D). Statistical analysis yielded a good correlation of the burst width with the preceding but not the following interval (Fig.6 E).
Voltage-clamp experiments suggested the presence of both glutamatergic and GABAergic neurotransmission during rhythmic burst activity (Fig.4 C). We investigated the influence of the two transmitter systems on synchronous network activity at various time points during the cultivation period. Fluo-3 fluorescence was recorded in fields of 190 × 190 μm2 in a recording chamber perfused with aCSF for 10 min. During this time, transmitter receptor antagonists were applied for 2 min. Images were taken only every 3 s to prevent bleaching over the long recording period; however, this low sampling rate still allowed save detection of synchronous events that usually exceeded 5 s. When synchronous activity was first detected by Fluo-3 fluorescence imaging at the beginning of the second week of cultivation, it could be totally blocked by 10 μM CNQX, 50 μM d-APV, or by 20 μM BMI (Fig.7 A, left). Also, the Na+ channel blocker tetrodotoxin (1 μM) inhibited synchronous network activity (data not shown). It is noteworthy, however, that neither CNQX nor BMI were able to decrease the number of neurons in which a spontaneous increase of [Ca2+]i could be detected between synchronous events (see Fig. 2 B: difference between the total number of active neurons and the number of synchronously active ones). This suggests that substantial spontaneous neuronal activity (as recorded by Fluo-3 imaging) does not depend on synaptic transmission at this early age. One week later, in cultures at the beginning of the third week of cultivation, the effects of the same three transmitter receptor antagonists differed from each other. While CNQX (10 μM) still blocked synchronous activity completely,d-APV (50 μM) significantly reduced but did not completely abolish the recorded Ca2+ transients (Fig. 7 A, right). Blockade of GABAA receptors with 20 μM BMI did not prevent synchronous network activity. In fact an increase of the Ca2+ transients was observed in 11 of 12 fields (380 × 380 μm2, 90 ± 30 neurons/field participated in synchronous events) when recorded at higher time resolution (1 Hz, Fig. 7 B), whereas the frequency of events was reduced. For quantification the baseline-subtracted area under the Ca2+transients was calculated and found to be increased 2.6-fold in BMI compared with control conditions (range: 1.3- to 4-fold). It should be noted that the above described effects of CNQX, d-APV, and BMI were stable for ≥30 min when examined with longer-lasting drug application (data not shown). Also, in electrophysiological recordings, we did not find notable effects on RMP of the transmitter receptor antagonists.
We employed electrophysiological recordings to seek possible causes of the larger Ca2+ transients in the presence of BMI. A first assumption was a possible developmental transition of GABA to become an inhibitory transmitter. We had demonstrated that EGABA in neurons of 8- to 12-day-old cultures is significantly more positive than the RMP (EGABA: −44.6 ± 1.4 mV, RMP: −54.8 ± 1.1 mV, n = 13; Voigt et al. 2001). Now we determined EGABA in neurons of 14- to 16-day-old cultures but found it still significantly more positive than the RMP measured in the same neurons (EGABA: −38.1 ± 4.7 mV, RMP: −55.7 ± 6 mV, n = 9). In current-clamp recordings from neurons in 15- to 19 day-old cultures (Fig.7 C), we observed that application of 20 μM BMI tended to decrease the number of spikes per burst from which one would rather predict a decrease of Ca2+ transients. However, another very robust effect of BMI on rhythmic bursts appeared to be a substantially prolonged depolarization. This finding could be verified in voltage-clamp experiments (Fig. 7 D). Application of 20 μM BMI significantly prolonged the burst-associated inward current recorded at a holding potential of V H= −70 mV but clearly reduced its amplitude (241 ± 64 vs. 145 ± 18 pA, n = 5). Interestingly, calculation of charge transfer during burst activity revealed no significant difference between control condition and BMI treatment (535 ± 81 vs. 564 ± 236 nA s, n = 5). Assuming that in these older cultures virtually all neurons participate in synchronous network activity (Fig. 2), the number of inputs per burst should be relatively constant and should not increase by BMI application. A possible explanation for the prolonged depolarization is that the activity of neurons during rhythmic bursting becomes less coherent when GABAAergic transmission is blocked. This view is supported by voltage-clamp measurements atV H = −70 mV in 50 μMd-APV (Fig. 7 D), a condition when AMPA receptors are the major contributors to the recorded currents. Burst-associated currents appear very brief compared with recordings in the absence ofd-APV. However, after application of 20 μM BMI, the currents became substantially prolonged with smaller amplitude (342 ± 35 vs. 114 ± 16 pA, n = 5) but equal charge transfer (58 ± 10 vs. 54 ± 6 nA s, n= 5; Fig. 7 D).
In this paper, we describe and characterize synchronous neuronal activity that occurs in cultured cortical networks naturally without pharmacological intervention. The synchronous activity that can be detected by means of Fluo-3 fluorescence imaging developed at the beginning of the second week in culture and included virtually the entire neuronal population about 1 wk later. Ca2+signals arising from astrocytes could be observed only sporadically and in no temporal relation to the neuronal synchronous activity. It has been reported that astrocytes are required for oscillatory activity in cultured hippocampal neurons (Verderio et al. 1999). Although we cannot rule out that astrocytes influence synchronous neuronal activity in our cortical cultures, there are indications against their role as a permissive element. We found synchronous oscillatory activity in cultures that were treated with Ara-C at the second DIV. Such early termination of mitotic activity yields cortical networks that are virtually free of astroglia. Furthermore, preliminary experiments with a broad-spectrum glutamate transporter antagonist showed no profound influence on synchronous activity as reported byVerderio et al. (1999) for hippocampal cultures.
Rhythmic synchronized activity appears to be a prerequisite of developing neuronal networks (for review see Ben-Ari 2001; Feller 1999; O'Donovan 1999). It has been described in a number of different CNS regions of different species, and therefore, does not seem to depend on a special network architecture. Consequently synchronous activity has been observed even in cultures of dissociated neurons; however, in most cases after pharmacological intervention like prolonged inhibition of glutamatergic neurotransmission (Furshpan and Potter 1989), enhancement of synaptic transmission by means of Mg2+ removal from the aCSF (Higashi et al. 1999; Robinson et al. 1993; Streit et al. 2001), or block of inhibition (Misgeld et al. 1998; Murphy et al. 1992; Streit et al. 2001), but also in ventral horn neurons without pharmacological alterations (Latham et al. 2000b). In the present study, we found that synchronous activity in cultures older than 1 wk in vitro can be detected in aCSF closely resembling the ionic composition of the culture medium or even in N2 culturing medium itself. The most prominent difference between incubator conditions and recording conditions was the temperature, since the recordings were made at room temperature instead of 36°C. Because synaptic transmission is temperature dependent (Hardingham and Larkman 1998;Weight and Erulkar 1976), this could have resulted in an underestimation of network activity. However, experiments addressing this issue indicated that the frequency of synchronous events but not the number of participating neurons changes when temperature is raised from 20°C to 35°C (Opitz and Voigt, unpublished observation). Another difference to incubator conditions is the use of HEPES for pH buffering of the aCSF instead of bicarbonate. It has been reported that a switch from bicarbonate- to HEPES-buffered solutions or vice versa affects a number of functional aspects in neurons. A consistent finding is a change of RMP to more positive values when cells are bathed in HEPES-buffered solution (Church 1992; Cowan and Martin 1995; Gu et al. 2000). In agreement with this, we observed a small but transient hyperpolarization when the aCSF was switched from a HEPES- to a bicarbonate-buffered one. Changes of the neuron's input resistance was reported by some authors (Church 1992; Gu et al. 2000) but was not always observed (Cowan and Martin 1995; see also our data in here). The different pH buffers also affect Na+ channel kinetics (Gu et al. 2000) and possibly some Cl− and K+ conductances (Cowan and Martin 1996; Stea and Nurse 1991). Taken together it appears that neurons show enhanced excitability on a switch from bicarbonate- to HEPES-buffered solutions that may also be of relevance for synchronous network activity described in this paper. However, our experiments showed only a transient effect of a pH buffer change on burst frequency. We hypothesize that the degree of recurrent excitation in cultured neocortical networks is high enough to provide strong inputs that override a possible drop of excitability. Alternatively, this drop may be only transient.
The correlation of synchronous increase of [Ca2+]i with firing bursts of action potentials superimposed on long-lasting depolarizations is in agreement with other reports on rhythmic activity in cortical cell cultures (Murphy et al. 1992;Robinson et al. 1993). Interestingly, there appear to be no major phenomenological differences between recordings with (this study; Murphy et al. 1992) or without Mg2+ (Robinson et al. 1993) in the extracellular solution: rhythmic burst activity was found to be mediated by synaptic excitation without the appearance of a gradually depolarizing pacemaker potential. If NMDA receptor channels are not a major source of Ca2+ influx during bursts (Robinson et al. 1993) and the generation and propagation of synchronous activity depend on the level of spontaneous presynaptic firing and the degree of connectivity of the network (Maeda et al. 1995), one would deduce that Mg2+-free medium would simply facilitate burst generation and possibly prolong depolarization. Indeed we found an increase in burst frequency and number of action potentials per burst when the extracellular [Mg2+] was changed from 2 to 0 mM (Opitz and Voigt, unpublished observation).
In the immature neocortex, several kinds of coordinated network activity have been described. A locally, rather restricted one, appears to be a biochemically mediated coactivation that involves gap junctional coupling and has been termed "neuronal domains" (Kandler and Katz 1998; Yuste et al. 1992). Only recently, waves of activity that involve large neuronal populations and can span long distances have been described in the neonatal cortex (Garaschuk et al. 2000;Peinado 2000, 2001). Peinado (2001)reported two types of wave-like neuronal activity: one that initiates in infragranular layers and spreads toward upper cortical layers requires glutamatergic transmission, and another type propagating along the longitudinal axis of the cortex that is not blocked by antagonists of glutamate receptors but relies on the presence of dendrodendritic gap junctional connections. In contrast, Garaschuk et al. (2000) found oscillatory Ca2+ waves traveling along the longitudinal axis of the cortex that are totally blocked by the AMPA receptor antagonist CNQX, suggesting spread of activity by glutamatergic neurotransmission. This pharmacology is in agreement with our findings in cultures of dissociated cortical neurons. It has been noted, however, that glutamate could be only involved in neuronal excitation but not in the mechanism of wave propagation per se (Peinado 2001). Indeed, compared with the situation in neuronal domains where activity is transmitted biochemically (Kandler and Katz 1998), long-range waves supposed to involve gap junctions require strong depolarization and action potential firing. Thus the fact that synchronous activity in cortical cultures can be blocked effectively by CNQX or TTx would still leave the possibility for gap junctional contribution. On the other hand, in whole cell voltage-clamp recordings, we found barrages of glutamatergic synaptic currents associated with rhythmic bursts but never attenuated unclamped spikes from supposedly coupled cells. This suggests that the synchronous activity in the network of cultured cortical neurons spreads via synaptic transmission.
Modeling studies have shown that it is sufficient for the expression of synchronous oscillatory network activity if there are recurrent, functionally excitatory connections rendering hyper-excitability to the network, and activity-dependent depression of network excitability (Tabak et al. 2000). Our cortical cultures appear to fulfill these two requirements. We found that the two major transmitters glutamate and GABA are both causing excitation. Furthermore, we demonstrated a period of network depression lasting seconds after a synchronous event (Fig. 6). Synchronous bursting has been characterized by a positive correlation between burst duration and the preceding interval (Staley et al. 1998;Streit et al. 2001; Tabak et al. 2001), suggesting that burst duration is controlled by parameters recovering from depression after the previous burst. The same kind of correlation could be observed in neocortical cultures (Fig. 6). Thus it is possible that the synchronous oscillatory activity is a feature of the network without the need for a specific pacemaker. Indeed we never observed a gradually depolarizing pacemaker potential and found no evidence for a change of excitability during inter-burst periods (Fig. 5). Instead bursts could be initiated by spontaneous activity. Very recently it was demonstrated that burst generation induced by disinhibition in spinal cord cultures is controlled by intrinsic spiking of some neurons (Darbon et al. 2002). Theoretical considerations showed that the firing patterns in a recurrent network are controlled largely by the fraction of endogenously active cells (Latham et al. 2000a). It was predicted and experimentally shown that networks with a larger fraction of spontaneously active neurons fire at low rates, whereas lowering the number of endogenously active cells led to bursting (Latham et al. 2000b). In line with this we registered a much higher number of neurons with spontaneous Ca2+ transients in young cultures (6–9 DIV) than in older ones (12–16 DIV) under conditions of blocked synaptic transmission.
In this study, GABAergic transmission was found to be involved in generation of synchronous activity in cultured neocortical neurons. During the first 3–4 days after their emergence, synchronous events could be blocked totally by application of the GABAA receptor antagonist BMI, suggesting that GABA acts as an excitatory neurotransmitter in these young networks. Perforated-patch recording that avoid disturbance of intracellular chloride concentration and [Ca2+]i imaging during specific activation of GABAA receptors showed that this is indeed feasible (Voigt et al. 2001). Depolarizing action of GABA and even excitation have been described in immature CNS structures and are based on a high intracellular [Cl−] which results in a shift of EGABA to more positive values (Cherubini et al. 1991; Leinekugel et al. 1995;Owens et al. 1996). Accordingly GABAA receptor antagonists block rhythmic synchronous activity in neonatal hippocampus (Ben-Ari et al. 1989; Garaschuk et al. 1998), but they fail to do so in immature cerebral cortex (Garaschuk et al. 2000). There are, however, striking similarities in the action of BMI between cortical slices of newborn rats and our culture system. In cortical cultures older than 2 wk, synchronous neuronal activity persisted despite the presence of BMI, but we found that inhibition of GABAAergic transmission decreases burst frequency, prolongs depolarization, and increases burst-associated Ca2+ transients (Fig. 7). A robust decrease of burst frequency and an increases of Ca2+transients (but only in 5 of 9 cases) has been also described in immature neocortical slices. In consideration of the difference to the situation in neonatal hippocampus, it was suggested that maturation of excitatory glutamatergic transmission in the cortex occurs earlier than in hippocampus (Garaschuk et al. 2000). Our results in culture suggest that there is a period in cortical development when GABAergic neurotransmission is necessary for synchronous network activity (Fig. 7) and a specific type of preplate neuron was shown to be sufficient for the initiation of synchronous oscillatory network activity in culture (Voigt et al. 2001). Neurons of the preplate, however, mature far earlier than those of the cortical plate. Thus with ongoing development and synaptogenesis (de Lima et al. 1997), GABAergic cells will lose their decisive role to the now well-developed glutamatergic neurons. In the rat, this might happen before birth; a blockade of synchronous activity by GABAA receptor antagonists could not be observed in neonatal slices (Garaschuk et al. 2000). Our findings regarding the prolonged depolarization and increased burst-associated Ca2+ transients during inhibition of GABAAergic transmission in older cortical cultures (Fig. 7) are also consistent with those of Murphy et al. (1992), who observed larger Ca2+transients after picrotoxin-treatment in neocortical cultures older than 3 wk. However, the increase of Ca2+transients in picrotoxin was explained with the block of inhibitory GABAergic transmission. Thus one would assume a larger excitatory input in BMI or picrotoxin. Voltage-clamp experiments revealed a significant longer inward current but unchanged charge transfer in the presence of BMI, suggesting a change of excitatory input in time but not size. Furthermore, we found EGABA more positive than the RMP suggesting that GABA still acts depolarizing in 14- to 16-day-old cultures. We propose that in early cortical development GABAergic neurotransmission is able to "bundle" the activity of glutamatergic neurons in time possibly by providing fast additional depolarization.
It was reported that the major source of [Ca2+]i increase during rhythmic bursting of cultured cortical neurons in Mg2+-free external solution are voltage gated Ca2+ channels (VGCC) (Robinson et al. 1993). Likewise, synaptically induced [Ca2+]i increase in hippocampal CA1 neurons was reported to be due to VGCC activation (Miyakawa et al. 1992). In line with these findings, we observed no Ca2+ transients in neurons when they were voltage clamped at −70 mV or when they received only subthreshold inputs (compare voltage and fluorescence traces of cell 1 in Fig. 3). However, in the presence of BMI, burst-associated depolarizations appeared not only longer lasting but also more shallow. Consequently they tended to generate less action potentials that in turn would reduce VGCC activation. This seems to be in contradiction with the increase of Ca2+ transients, and we suggest to consider additional Ca2+ sources. The voltage-clamp and Ca2+ imaging experiments illustrated in Fig. 6 provide evidence that NMDA receptors contribute to the rhythmic synchronous network activity. This contribution may be twofold. First, NMDA receptors render glutamatergic postsynaptic currents longer lasting because of the slow decay kinetics (Forsythe and Westbrook 1988; Stern et al. 1992), which can explain the pronounced shortening of burst-associated currents by d-APV (Figs. 4 C and6 D). Second, the NMDA receptor channel is also permeable for Ca2+ (MacDermott et al. 1986;Mayer and Westbrook 1987). The prolonged depolarization in BMI could keep the voltage-dependent Mg2+block (Nowak et al. 1984) effectively removed, thus increasing the time period of possible NMDA receptor activation. Ca2+ entry through NMDA receptor channels, however, has been reported to contribute insignificantly to synaptically evoked increase of [Ca2+]i in the neuronal soma (Alford et al. 1993; Miyakawa et al. 1992). Even so, in the dendrite, Ca2+influx through NMDA receptors is able to induce substantial Ca2+ release from intracellular stores (Alford et al. 1993). In addition Ca2+ release could be triggered by activation of metabotropic glutamate receptors during the bursts. A synaptically evoked increase of [Ca2+]i in dendrites may therefore be responsible for the increased Ca2+transients that we observed in the presence of BMI by triggering regenerative Ca2+ waves toward the soma (Jaffe and Brown 1994).
The authors thank B. Adam for excellent technical assistance.
This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grants VO 519/4–1 and VO 519/6–1.
Address for reprint requests: T. Opitz, Otto-von-Guericke University, Institute for Physiology, Leipziger Str. 44, D-39120 Magdeburg, Germany (E-mail: opitz{at}medizin.uni-magdeburg.de).
- Copyright © 2002 The American Physiological Society


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I will not be fearful. I will not toss aside the values that are fundamental to our democracy and exchange them for a vigilante mentality.
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Campus preachers: when they're not explaining that women were made for men or arguing with professors, they're holding up signs that say, "You Deserve...
If sitting down and rationally and peacefully talking among people of different religions is unworkable, perhaps creating interfaith families would be a way to bring disparate people together.
I caught up with Christian scholar Philip Jenkins to discuss his new book, militant Muslims, and the current scandal surrounding the pope.
Those who would manipulate the masses to their own advantage invariably draw on the power of passion. And for most of history, and never more than now, religion is the passion of choice.
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'Canoe Adventure' Bear Fest weekend
Friends of Bayou Teche National Wildlife Refuge are hosting a Canoe Adventure into Bayou Teche Refuge on Saturday, April 20 in conjunction with the Bayou Teche Bear Festival in Franklin.
Participants will have the opportunity to learn about the wildlife of the refuge in this guided, easy pace paddle suited for beginners or families.
The adventure begins at 8:30 a.m. A pre-registration fee of $20 per canoe is required and must be received by April 18 to participate.
Only two adults and one child are allowed per canoe. Children must be at least 6 years old and accompanied by an adult to participate. For an entry form or for additional information, visit online at www.bayoutechebearfest.org, email email@example.com or call Donovan at 337-923-9718. Canoes are limited.
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