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245 Tasting Notes
I have read so many good things about this blend, and was really eager to get my hands on a sample of this. Many thanks goes out to Short Sorceress for providing me with one.
This is delicious. OMG this is REALLY GOOD!! I didn't know what to expect, but I didn't expect this to taste as good as it does. I didn't add sugar, this blend does not need it. It imparts a sweet creamy lemon dessert flavour.
I have a lemon chiffon from another company, which I have yet to try…I wonder if it will be as good as this one.
This is one of four tea's that I have sipped down! I wonder if I should reward myself by buying more??!! Thank you JustJames for a sample of this one.
The dry leaf definitely smells of sweet strawberry. The tea brews to a lovely rose colour. I thought this was a good sign.
The first couple of sips I didn't get very much strawberry flavour. As the tea cooled, it imparted a light tartness that I guess was supposed to be strawberry. I always found strawberries to be a little sour, so I don't why I would think strawberry flavoured tea would be any different.
The tea was ok. I don't think it's one that I would purchase, but am glad that I got to try.
This is not a tasty tea. It says it is pomegranate green – but I'm not tasting any pomegranate. I taste a little bitterness and some strange background taste.
Not a fan, but I consider this a tasting adventure…thank you Short Sorceress!
Thanks goes out to Dexter3657 for the sample of this tea!!
When I opened the package I felt like such a newbie…how do I deal with this pressed sample? I left it for a while, even though I was dying to slurp it up. I finally overcame my fear and broke off a piece (I know that this was not the correct way to get a piece).
The dry leaf aroma is fresh and woodsy. It reminds of walking through the ravine.
The brewed taste is light and nutty. I have not yet had any issues with pu'erhs -for me this one is on the lighter side, but still an excellent tea that was sweet, flavourful and mild. For those who are unsure or wary of pu'erhs this would be the one to start with. No fishiness or strange tastes.
It is still ridiculously humid in TO, so I have still have not had any hot tea beverages, and I have so many to try!!!
Yesterday I went to 'Toys R Us' to buy a kiddie pool. Not for me…but for the furry beast (shep/lab cross) that I live with, to help him get through the heat. He still prefers the stink water creek a few minutes away…but will jump in the kiddie pool if encouraged.
Back to the tea….on opening the bag I smell over-ripe pears. This told me that this would be fairly sweet on it's own, without any sugar additives.
Because of the heat, my first tasting of this one is as a cold brew, and I have to say…I am surprised that I like this as much as I do. When I first saw the fennel seeds as part of the mix I was like "What??!!!" This will not be good. WRONG!! This is my second fave cold brew…a real surprise to self.
Cold brew tastes like sweet pear. No tartness. Just sweet pear. Sugar is not needed here…it is sweet on it's own. YUM!!!! I will be sad when it's gone (2-3 cold brews left). But, maybe I will find something as good. The tea journey continues…. :)
It is stinking hot in Toronto, and I have not been drinking any hot tea's since yesterday morning. My tea related beverages that I have been drinking recently have all been cold brewed, including this one. I have a few others on the go, since this stifling heat won't be letting up in the next couple of days.
It is tart. I am so over tart tea's. I don't like it, and find myself reaching for sugar or some other type of sweetener. I had purchased from IKEA Lingonberry syrup and added it to this cold brew, so it no longer tastes like strawberry parfait – it tastes like the Lingonberry syrup. Strawberry parfait is drinkable to me this way, but it is no longer strawberry nor parfait. But it's cold and refreshing on this stinking humid day.
oh no! it's muggy here, but nice and cool in the evenings. i find the longer i wait for my butiki and verdant orders the more mundane everything else tastes.
I saw your recent comment, I think it was about this tea…but is true, I am getting particular about what I drink! I am very surprised!! The more tea one drinks, the more sophisticated one's palette becomes. I still like dessert tea's, but I like sweet…not sour. Have never enjoyed fruit desserts, so I should have reasoned that I would not like tart fruit teas.
i used to really love this tea.. when it first came out.. but the tartness of it is offputting to me this time around.. i think my tastes have changed, and its just not a favorite anymore
For some reason, I had this idea in my head that this would taste like a strawberry sundae. I don't know why I thought this. I will finish it, with added sugar or syrup, but it will not be a re-purchase.
This is a backlog from yesterday. I had this in lieu of my morning coffee, as I had slept in, and The Man left me no coffee, and I was too lazy to brew any.
This is earthy and mushroom-y. Maybe a little too mushroom-y for my tastes. I drank it bare. I was able o finish the cup, but I think for the rest of this sample I will be adding cream (maybe sugar). Maybe I will like a little better then. I didn't get too much roasted flavour, but I was still not quite awake when drinking this. Next brew hopefully will work out better!
Thanks to Dexter3657 for a sample of this !!
No rating until I try it again with different parameters.
I like this one. I get how the lemon flavouring is in your face, but this one I enjoy. This is a tea that I bring to work, because I don't have to worry about steeping times and being all finicky about water temp and keeping an eye on how long it's steeped for. Because I don't bring extra special tea's to work, this one does just fine.
I cold brewed this one, and I really like as an unsweetened iced.
It's funny that I got this particular tisane because it is made with something called 'Lapacho' (I've never hear of this), and I was looking at a Toronto based tea company that also uses this herb (??) in a couple of blends, and I was thinking about buying them to see. Now that I've actually tried it, I know I will at least request a sample of it.
In dry leaf form it smelled like chocolate orange – not a favourite combination of mine (until I tried Terry's chocolate orange). It also looked like it would taste like chocolate orange – large pieces of orange, and a dust that I thought was cocoa powder.
Once it was brewed, it didn't have an overwhelming citrusy taste, nor chocolate. I got a hint of both, plus something else that I am not able to identify – probably due to lack of experience with this herb (?). Not bad.
Thank you JustJames for introducing me to a new tasting experience!!
Delicious coconut…one of my favourite flavours in a tea.
The dry leaf smell reminds me of coconut rum with a bit woody scent from the rooibos. Generous shreds of coconut are mixed in with the rooibos.
I had a couple of sips of this without and creamer or sugar, and it was quite tasty and sweet on its own. I decided to add almond milk to bump up the creaminess and now I'm in creamy coconut, nutty almond heaven. Yeah, this is good. No added sugar. Not required here.
Thanks goes out to Dexter3657 for this yummy surprise sample!


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Lots of Suit-less Justin Timberlakes Take Over 'SNL' in New Promos (Video)
The singer/actor/box-wearer marks his return to the late-night show with a run of short clips that find him under-dressed and a wee bit nervous.
Justin Timberlake will host and serve as musical guest on Saturday Night Live this weekend, returning to the show that helped make him as well known for putting his member in a gift wrapped box as his smash-hit singing career.
STORY: Justin Timberlake, Marcus Mumford Collaborating on 'Inside Llewyn Davis' Soundtrack
If his promos are to be believed (just go with it), Timberlake will not only be acting and crooning this weekend, but operating the boom mic and cameras, as well. Elsewhere in the quick videos made alongside cast regular Keenan Thompson, JT signs his life away and finds himself unable to remember his lines without his co-star's moral support.
Oh, and he throws a fit over forgetting his now-famed suit and tie.
After his turn on SNL, Timberlake will spend a week as a guest on Late Night With Jimmy Fallon to celebrate his new album, The 20/20 Experience.
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Gaming Posts - Page 99
Crytek and DeNA announce the launch of The Collectables
Crytek is a company that gamers know and are fond of. It is the developer behind the cool PC and console game Crysis. Crytek teamed up with DeNA to make a mobile game called The Collectables that was announced an awhile back.
That game has now landed for mobile gamers on the iPhone or iPad. The game will be coming later for Android gamers. Players are able to assemble a squad of mercenaries in the game and control the third-person tactical combat action through 3D environments.
The game is available for free on the App Store. There is no mention of it on the PR, but I would assume the game is supported by in-game purchases. Players will be able to upgrade their squad as the game progresses using a collecting card system unlocking new weapons and ways to kill the enemy.
Continue reading 'Crytek and DeNA announce the launch of The Collectables' (full post)
Thrustmaster Ferrari themed racing wheel for Xbox One unveiled
There are a few companies out there that make racing wheels for game consoles and PCs. Some of them are very expensive and others are affordable. Thrustmaster has announced a new racing wheel for the Xbox One that falls into the affordable category.
The new racing wheel is called the Ferrari 458 Spider Racing Wheel and it is an officially licensed product from Ferrari. The racing wheel has a red rubberized grip and dual paddle shifters for changing gears.
The racing wheel is a nine-button device that includes a view and menu button. The engine start button is actually a d-pad. The racing wheel comes with a pedal set with adjustable travel and linear feedback. The wheel itself has a diameter of 11-inches.
Continue reading 'Thrustmaster Ferrari themed racing wheel for Xbox One unveiled' (full post)
Respawn to update Titanfall, will support 4K and other NVIDIA tech
Titanfall will soon be updated, where we will see the first-person shooter include the ability to be played at 4K. Not only that, but Titanfall will finally support multi-GPU setups with NVIDIA GeForce GPUs.
NVIDIA's Manager of Developer Content and Technology, Ashu Rege, said: "Respawn will also release updates to support high-definition 4K display, and our SLI multi-GPU technology, for a fantastic high-end PC experience." Respawn and NVIDIA are also working together to see NVIDIA's GameWorks technologies built into the mech shooter.
This will see Titanfall include PhysX for clothing and destruction simulation, and VisualFX for hair, fire and liquid simulation. Not only that, but we'll also see Temporal anti-aliasing (TXAA), which will reduce screen flickering, and Horizon Based Ambient Occlusion+ (HBAO+) which will provide Titanfall with "more lifelike shadows."
Galactic Strongholds expansion comes to Star Wars: The Old Republic
Gamers that have spent some time with Star Wars: The Old Republic MMO will be glad to hear that a new free to play expansion is coming soon. This expansion brings a new social feature for the game that allows players to design their own strongholds.
The stronghold is really just an apartment or dwelling for the player to invite their friends to for hangouts and chats. You can choose your own custom decorations and place them where you want in the stronghold.
As you fix up your stronghold, you will earn Prestige Score points and the chance to compete against other players in the Stronghold Directory. The update also brings with it Guild Flagships that can't participate in battles, but can be meeting places. Strongholds can be acquired in the game by earning credits or with Cartel Coins in the Cartel market.
Continue reading 'Galactic Strongholds expansion comes to Star Wars: The Old Republic' (full post)
Crytek offers CryEngine as an 'Engine-as-a-Service', is royalty free
GDC 2014 - Epic Games announced that it is now offering its Unreal Engine 4 on a subscription-based model, which will open the graphics engine up to many more developers, but now Crytek has entered the same market - announcing the "Engine-as-a-Service" (EaaS) program.
This will allow developers to use CryEngine and its associated development tools for a flat monthly fee. This all kicks off in May, starting with the first tier of $9.90 per month, which gives developers access to the engine, without a royalty fee attached to sold games. There's not much more known on this at the moment, but Crytek will unveil more details on the service in the coming we eks.
AMD partners with Crytek, will see Mantle supported in CryEngine
GDC 2014 - It looks like AMD's Mantle API is gaining some traction, with the chipmaker announcing a new deal with Crytek, the developer behind CryEngine, and the popular Crysis games, Forbes reports.
The news is coming directly from Matt Skynner, who is the Corporate VP and GM of AMD's Graphics Business Unit, who said the company has had productive couple of months. We've seen the chipmaker team up with Roberts Space Industries - the developer behind the upcoming Star Citizen, which is built on CryEngine, as well as the very popular Battlefield 4, which runs on Frostbite 3 from EA and DICE.
With over 35 studios working with CryEngine and over 50 games powered by the engine, this is quite the coup for AMD. This new announcement of a partnership between AMD and Crytek is an interesting one, as it seems AMD's API is picking up some steam. We still have to see how DirectX 12 fares, and then we have SteamOS and Steam Machines coming out later in the year.
Snowdrop engine detailed in an amazing new video of The Division
GDC 2014 - Massive Entertainment, the developer behind The Division, has shown off a new trailer for the game's engine, Snowdrop, which you can check out below.
It looks absolutely beautiful, one of the first games that truly looks next-gen. The team talks about how versatile Snowdrop is, and how great it is to develop with. The video shows off some bits where you can see it is quite like connecting Lego together, in terms of ease. The last time we saw The Division we thought it looked great, but this video is a big step up on that.
Remember, the team has said that The Division won't just be a port on the PC, but it will "stand out" on PC. Hopefully Massive keep to its word, and we don't see the game being scaled down in terms of graphics and features when it gets closer to release.
Continue reading 'Snowdrop engine detailed in an amazing new video of The Division' (full post)
Titanfall 2 rumored to not be exclusive to Microsoft platforms
According to a GameSpot article and Polygon's sources, EA has just landed the deal for the sequel to Titanfall 2 from Respawn Entertainment. Polygon's sources have said that Titanfall 2 will this time around, not be exclusive to Microsoft platforms.
Titanfall, which was only released a couple of weeks ago, was exclusive to Microsoft platforms: Xbox 360, Xbox One and PC. An EA representative told Polygon that it wouldn't talk about the rumor, saying: "We do not comment on rumor or speculation. Right now all our focus is on supporting all the fans worldwide enjoying Titanfall."
Rewinding back to October, Respawn co-founder Vince Zampella tweeted that his company would eventually make games for the PS4, implying that Titanfall's exclusivity to Microsoft platforms was limited to just the first Titanfall. Hopefully we see this materialize into Respawn making Titanfall 2 for the PS4.
NeoCore Games opens up pre-orders and closed beta for Van Helsing II
The Incredible Adventures of Van Helsing was one of those games that gained massive popularity not only for its epic gameplay, but for its pricing point as well. When the game first released, NeoCore Games priced it at just $15 which was unheard of for a game of its caliber, and the company has done it again with the games sequel.
The Incredible Adventures of Van Helsing II is based on the same RPG game play type as the first, but features more character classes, multiplayer modes, and an overall more complex story line. With more than 250 unique weapon models, and over 1000 item types to collect, The Incredible Adventures of Van Helsing II is a no brainer purchase at just $15 on Steam. I played through the closed beta some today and the game is quite fun and I lost track of time and spent about an hour in-game.
Continue reading 'NeoCore Games opens up pre-orders and closed beta for Van Helsing II' (full post)
DICE has released the official trailer for Battlefield 4 Naval Strike
DICE has just released the official trailer for Battlefield 4's upcoming Naval Strike Expansion, and WOW was it worth the wait. Naval Strike brings in four new maps, a new game mode, and a new vehicle plus a treasure trove of new weapons, assignments, and achievements to Battlefield 4. Perhaps the biggest addition is the new Carrier Assault game mode which is reminiscent of the old Titan Mode found in Battlefield 2142.
The trailer above illustrates the new maps well, and we can even see that some ancient cannons are able to be fired from some of the map's fortresses. Only in battlefield are you able to take down a modern fighter jet with a historic cannon! Naval Strike is expected to drop next week for Battlefield 4 Premium subscribers, and two weeks later, arrive to the rest of Battlefield 4 players.
Continue reading 'DICE has released the official trailer for Battlefield 4 Naval Strike' (full post)


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Zama Community Spouses' Association (ZaCSA) offers two scholarship opportunities: 1) Graduating high school seniors in Camp Zama and NAF Atsugi, 2) Camp Zama Family Members. Find about more information and to download a application form: ZaCSA Scholarships.
Application Deadline is March 31, 2015.
ZaCSA Membership is now only $10 from now until the end of the fiscal year (June 30). We still have many great events for you to get involved with — we hope to see you soon!
East Meets West Gift Shop
We need volunteers every week to keep our store open (Store hours: Tues, Thurs 11-6, and first Sat 10-2). If you would like to help, but have questions or find shifts listed on this page do not work with your availability, you can also coordinate directly with Gloria Workman, East Meets West Gift Shop Manager, at email@example.com.
ZaCSA's accepts Community Grant applications each month.
The deadline this month is February 18, 2015.
Find more information, an application, and other application deadlines: Community Grants
ZaCSA's accepts Community Grant applications each month.


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EUR 260 М of Loans for Bulgarian SMEs under JEREMIE
Bulgarian small and medium-sized enterprises (SMEs) have a new chance to benefit from loans with low interest rates and reduced collateral under the Joint European Resources for Micro to Medium Enterprises (JEREMIE) initiative.
The European Investment Fund has signed agreements with four banks (Allianz Bank, First Investment Bank, ProCredit Bank and Société Générale Expressbank) under the JEREMIE initiative to benefit SMEs in Bulgaria.
With the JEREMIE funding, the four banks will be able to extend loans with reduced interest rates of at least 50% to over 3,300 SMEs across Bulgaria looking to strengthen or expand their businesses.
Information on how to access loans financed under the JEREMIE initiative will soon be available from the selected banks.
The JEREMIE Initiative is implemented in Bulgaria within the framework of the OP "Increase of the competitiveness of the Bulgarian economy 2007- 2013″ co-financed by the European Regional Development Fund and the State Budget.
The four banks will double the initial budgetary allocation of EUR 130 М and channel an amount equal to approximately EUR 260 М as low interest rate loans to Bulgarian SMEs.
- » Profit of Bulgarian Banks Grew by BGN 47 M Y/Y in Feb 2015
- » Bulgaria Registers BGN 250 M Budget Surplus for First Quarter of 2015
- » Bulgaria Objects to 'Discriminatory' Change to Greece's Income Tax Code
- » Bulgaria's Finance Minister Expects New Governor of Bulgarian National Bank by end-April
- » Bulgaria's Gross External Debt at EUR 39 B in end-January
- » Bulgaria Repays EUR 1.5 B Bridge Loan


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by Chez
(St. Paul, MN)
We are purchasing a 55' Kingscraft houseboat, all aluminum with plans for year around living. We are looking for ideas or tips on cold climate houseboat living. Any ideas on how to insulate perimeter walls and heating. Any experience or suggestions?
Thanks Chez, St Paul, MN
Reply - Answer
Well Chez, congratulations on your purchase of an excellent all aluminum Kingscraft houseboat. They have always been one of my favorite models, and the all aluminum construction make for a sturdy, long lasting houseboat.
As to tips and ideas for year round living in a cold climate, you may want to read some of our pages with helpful information:
Houseboat Living Tips - Winter or Cold Climates
Houseboat Living - Winter Heating Tips
As to tips and ideas for insulating perimeter walls, condensation is an issue when the inside is heated and the outside is cold. This all depends on what type of changes that you would like to do?
Do your refurbishing plans involve more than just changing the curtains and painting, or are your looking at removing the interior paneling and adding an "R factor" insulation.
It would be wise to add some insulation between the interior & exterior walls. This would call for some more extensive refurbishing, but well worth your time and effort. It may also allow you to update any electrical and plumbing.
You may also want to add some clear exterior heat shrinkable plastic warp to the windows, this helps in cutting down on cold drafts. (Home Depot's have it)
Lastly, hopefully some of our readers will share and post comments about their winter houseboat living or insulation experiences.
IAN - from www.all-about-houseboats.com
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What to say when pet owners can't pay for veterinary care
Most pet owners want the best care for their pets. Erase the embarrassment factor for clients on tight budgets with these five tips to talk about cost.
Jan 01, 2014
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Quartz countertops and support
I know the first answer to this question will be ask the fabricator and I will, but I would like a little knowledge first.
We have the fabricator coming next Friday for LG quartz. We have a small island / peninsula that will have seating. The end of the peninsula is an open top bookcase. The countertop will overhang the bookcase edge about 12", then about 12" on the other side for seating, with the rail at the back edge of the bookcase and the 3/8 inch back panel being the only support. Not sure if I explained that well, here's the picure. Do I need support anywhere?
I am a fabricator.
Rule of thumb is max. 12" unsupported cantilever so theoretically you're ok.
However, I've seen 2 instances in 13 years where a chunk of overhang was broken off; once by a teenager hopping up to sit on the counter and the second when two 24 packs of beer were dropped onto a counter.
so - I always recommend modest corbels (like 6 " horizontal) or buried rodding.
Thanks oldryder. So the bookcase area should be fine, but consider supports for the 12" overhang? I found the lg quartz fabricator guide online, and it says their quartz can go to 14" without support. I'm not opposed to some hidden supports though.


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Robin's parents, Stephanie & Joel, stayed at our apartment ---- it was their 2nd visit and all went incredibly smooth. Great guests!
Robin's parents stayed in my place for a week and were perfect guests. They left everything so neat and tidy and were very respectful of the property. I would gladly host them anytime.
Robin booked our place for her parents...Stephanie & Joel. They are a really nice couple and it was great to host them I would welcome them back anytime.
Robin and her parents were fantastic. Booking with Robin was very easy and her parents Stephanie and Joel were so friendly, fun, and neat that i would love to have them back often. I hope to host them again when they return to the SF area.


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A question about Farscape ep "Bad Timing"
View Single Post
September 9 2009, 04:53 AM
Gary Mitchell
Location: ¯\_(°·O)_/¯ Florida
A question about Farscape ep "Bad Timing"
I just recently rewatched all of
except for "Peacekeeper Wars". Something that I hadn't noticed before was part of a scene on the Command Carrier with Braca, Scorpius and Sikozu:
Scorpius to Braca:
"Next time we encounter Crichton, see to it that the older cook is brought aboard - unharmed."
I know that it was the last episode and they had been planning stuff for the fifth season that they should've had, but does anyone have a clue why Scorpy Sue would want Noranti?
If no one comes from the future to stop you from doing it, then how bad of a decision can it really be?
Gary Mitchell
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The Tamil Nadu Hindu Aalaya Paadhukaapu Kuzhu – a forum to protect temples in Tamil Nadu – has strongly condemned the proposal to hike marriage and darshan fees at Meenakshi Sundareswarar Temple.
The meeting, which was presided, by forum's State general secretary P. Sundara Vadivel was attended by members from various Hindu outfits including the Hindu Munnani, BJP and ABVP.
According to a press release issued here on Sunday, the members objected to the fee hike for conducting marriages in the Meenakshi temple. A resolution was passed asking the temple administration to withdraw the proposal forthwith and respect the sentiments of the devotees.
Similarly, the proposal to hike special darshan fee too should be withdrawn in the larger public interest.
The administration should take steps to recover arrears due to the temple from various sources and not tax the common man.
A delegation would submit a memorandum to the temple administration on October 6 in this connection, the release said.
Keywords: Madurai, Meenakshi Temple, Entry fee hike, Protest


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Emergency Physician
6 years of experience
Accepting new patients
6 years of experience
Accepting new patients
1934 Day Island Blvd W
University Place, WA 98466 Phone number & directions
University Place, WA 98466 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Joshua Bigler:
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Dr. Joshua Bigler, MD is board certified in Emergency Medicine.
Education & Awards
Dr. Bigler has 5 hospital affiliations:
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- Swedish Medical Center - Cherry Hill
Home > Find an Emergency Physicians > WA > University Place Emergency Physicians > Dr. Joshua Bigler, MD
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- Dr. Joshua B Bigler MD is a male Emergency Medicine Specialist, has 6 years of experience and practices in Emergency Medicine.
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Dr. Joshua Bigler practices Emergency Medicine near University Place, WA.
See University Place Emergency Physicians.


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Page 1
Attenuated sensitivity to neuroactive steroids in
?-aminobutyrate type A receptor delta subunit
knockout mice
Robert M. Mihaleka, Pradeep K. Banerjeeb, Esa R. Korpic, Joseph J. Quinlana, Leonard L. Firestonea, Zhi-Ping Mid, Carl Lagenaurd,
Verena Trettere, Werner Siegharte, Stephan G. Anagnostarasf, Jennifer R. Sagef, Michael S. Fanselowf, Alessandro Guidottig,
Igor Spigelmanh, Zhiwei Lib,h, Timothy M. DeLoreyj, Richard W. Olsenb,k, and Gregg E. Homanicsa,i,l
Departments ofaAnesthesiology?Critical Care Medicine,iPharmacology, anddNeurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA
15261;jMolecular Research Institute, 2495 Old Middlefield Way, Mountain View, CA 94043;cDepartment of Pharmacology and Clinical Pharmacology,
University of Turku, FIN-20520 Turku, Finland;eUniversity Clinic for Psychiatry, Section of Biochemical Psychiatry, Wa ¨hringer Gu ¨rtel 18–20, A-1090 Vienna,
Austria;fDepartment of Psychology, University of California, Los Angeles, CA 90095-1563;gPsychiatric Institute, Department of Psychiatry, University of
Illinois, Chicago, IL 60612;hDivision of Oral Biology and Medicine, University of California School of Dentistry and Departments ofbMolecular and Medical
Pharmacology andkAnesthesiology, University of California School of Medicine, Los Angeles, CA 90095
Edited by Erminio Costa, University of Illinois, Chicago, IL, and approved September 8, 1999 (received for review July 21, 1999)
?-Aminobutyric acid (GABA) type A receptors mediate fast inhib-
itory synaptic transmission and have been implicated in responses
to sedative?hypnotic agents (including neuroactive steroids), anx-
iety, and learning and memory. Using gene targeting technology,
we generated a strain of mice deficient in the ? subunit of the
GABA type A receptors. In vivo testing of various behavioral
responses revealed a strikingly selective attenuation of responses
to neuroactive steroids, but not to other modulatory drugs. Elec-
trophysiological recordings from hippocampal slices revealed a
significantly faster miniature inhibitory postsynaptic current decay
time in null mice, with no change in miniature inhibitory postsyn-
aptic current amplitude or frequency. Learning and memory as-
sessed with fear conditioning were normal. These results begin to
illuminate the novel contributions of the ? subunit to GABA
pharmacology and sedative?hypnotic responses and behavior and
provide insights into the physiology of neurosteroids.
acid type A receptors (GABAA-Rs). These ligand-gated recep-
tors are believed to be pentamers, with subunits selected from
(at least) 15 possible variants (?1–6, ?1–4, ?1–3, ?, and ?) (1).
GABAA-Rs are modulated by many drugs, including ethanol,
benzodiazepines, various anesthetics, and neuroactive steroids
(2–5). Specific roles for various GABAA-R subunits in mediating
anesthetic responses are beginning to be elucidated (6–9).
Additionally, GABAA-Rs have been shown to be involved in
epilepsy (4, 10, 11), various behavioral states such as depression
and anxiety (12), and learning and memory (11, 13).
Several studies have shown that the ? subunit participates in
GABAA-Rs that exhibit a unique pharmacology. Such receptor
isoforms are benzodiazepine-insensitive (14), neuroactive ste-
roid-insensitive (15), and Zn2?-sensitive (16). Nearly 30% of
cerebellar GABAA-Rs appear to contain the ? subunit; levels of
? mRNA are highest in the cerebellar granule cells, secondarily
in hippocampus and thalamus (17, 18).
Neuroactive steroids are naturally occurring metabolites of
endogenous steroid hormones, or synthetic analogs, which exert
very rapid, nongenomic effects on membrane-bound
GABAA-Rs (19, 20). Those synthesized in the brain, termed
neurosteroids, are believed to regulate anxiety, stress, and
neuronal excitability by modulating GABAA-R function in vivo
(5, 21). Examples include metabolites of progesterone and
corticosterone that enhance GABA inhibition and preg-
nenolone (3?-hydroxy-5?-pregnan-20-one) sulfate that inhibits
GABA action (13, 19–21). The synthetic analog alphaxalone
(3?-hydroxy-5?-pregnan-11,20-dione) enhances GABAA-R
function and has been used clinically as an i.v. anesthetic. The
chemical analog ganaxalone (3?-hydroxy-3?-methyl-5?-
nhibitory ion currents in the vertebrate central nervous system
pregnan-20-one) was developed for improved bioavailability and
potential anxiolytic and anticonvulsant activity (22).
To investigate the contribution of ?-containing GABAA-R
isoforms to behavior and various drug responses, we used gene
targeting in embryonic stem (ES) cells to create a strain of mice
lacking a functional ?-subunit gene. Here we report the char-
acterization of the ? knockout mice.
Materials and Methods
Generation of Mutant Mice. A 9.5-kb PstI restriction fragment was
isolated from a strain 129?SvJ P1 phage library (Genome
Systems, St. Louis) with ?-specific primers from exon 2 (5?-
CAGGGCAATGAATGACATTG-3?) and exon 3 (5?-
CAAGCGCCACATTCACAG-3?). A replacement-type DNA
targeting vector was constructed in which the MC1Neo gene
(Stratagene) was blunt-end ligated into a blunted HindIII site in
exon 4 (Fig. 1A). The targeting vector was linearized with ClaI
before electroporation into R1 ES cells (23). G418-resistant ES
cell clones were screened for targeting by Southern blot analysis
of BamHI-digested genomic DNA hybridized with a 3? probe
(probe D) (Fig. 1A).
Three correctly targeted ES cell lines were microinjected into
C57BL?6J blastocysts, two of which produced chimeric mice.
Highly chimeric males were mated to C57BL?6J females (The
Jackson Laboratory). Agouti offspring that were heterozygous
were wild type (??/?), ??/?, and homozygous null (??/?). The
mice used for the studies reported here were derived primarily
from the ?#767 ES cell line. A more limited analysis of the
?#773 ES cell line yielded similar results. The genetic back-
ground of all mice was C57BL?6J X strain 129Sv?SvJ, F2-F5
Northern Blot Analysis. Total RNA was isolated from adult mouse
brain by using Trizol reagent (Life Technologies, Grand Island,
NY). Approximately 10 ?g of total RNA was electrophoresed in
a 1.9% formaldehyde?1% agarose gel, blotted to Hybond-N
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: GABA, ?-aminobutyric acid; GABAA-R, GABA type A receptor; mIPSC, min-
ES, embryonic stem.
lTo whom reprint requests should be addressed at: University of Pittsburgh School of
Medicine, Departments of Anesthesiology?Critical Care Medicine and Pharmacology,
W1356 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: firstname.lastname@example.com.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
October 26, 1999 ?
vol. 96 ?
no. 22 ?
Page 2
(Amersham Pharmacia) and hybridized as described (24). After
the membrane was hydridized with probe D, it was rehybridized
with a human ?-actin cDNA probe (CLONTECH) as a control
for RNA loading in each lane.
Western Blot Analysis. Antibody production, gel electrophoresis,
and blotting were performed as in ref. 25. Briefly, the ?(1–44)R5
polyclonal antibody (26) was prepared by immunizing rabbits
with a maltose binding protein-?(1–44)-7His fusion protein and
the ? subunit and does not precipitate ?1?3?2 receptors (26).
Equal amounts of cerebellar membrane proteins were subject to
SDS?PAGE and immunoblotted onto poly(vinylidene difluo-
ride) membranes. Membranes were incubated with digoxigenin-
labeled antibodies and treated with anti-digoxigenin-alkaline
phosphatase Fab fragments (Boehringer Mannheim). Proteins
Bedford, MA). Blots were exposed to Kodak X-Omat S film and
recorded with a DocuGel 2000i gel system using RFLPSCAN
software (MWG Biotec, Ebersberg, Germany).
GABAA-R Ligand Binding. [3H]Ro15–4513 and [3H]muscimol bind-
ing to whole brain homogenates was determined exactly as
described (7). To visualize brain regional distribution of GABA
and benzodiazepine binding sites, 14-?m horizontal sections
were cut from ??/?and ??/?brains by using a Microm cryostat,
thaw-mounted onto gelatin-coated object glasses, and used for
[3H]Ro 15–4513 (a benzodiazepine site ligand) and [3H]musci-
mol (a GABA site ligand) autoradiography as described in detail
by Ma ¨kela ¨ et al. (27).
Electrophysiology. Transverse slices of dorsal hippocampus were
obtained by using standard techniques (28). Pharmacologically
isolated miniature inhibitory postsynaptic currents (mIPSCs)
were recorded from cells located in the upper blade of the
dentate gyrus at 34.5°C during perfusion with artificial cerebro-
spinal fluid (ACSF) composed of 125 mM NaCl, 2.5 mM KCl,
2 mM CaCl2, 2 mM MgCl2, 26 mM NaHCO3, and 10 mM
dextrose. The ACSF was continuously bubbled with a 95%?5%
mix of O2?CO2to ensure adequate oxygenation of slices and a
pH of 7.4. Patch pipettes contained 135 mM Cs gluconate, 2 mM
and 0.2 mM Na2GTP, pH to 7.5 with CsOH. mIPSCs were
recorded in the presence of 0.5 mM tetrodotoxin, 40 mM
D(-)-2-amino-5-phosphonopentanoate, 10 mM 6-cyano-7-
nitroquinoxaline-2,3-dione, and 1 mM CGP 54,626. Signals were
recorded with an amplifier (Axoclamp 2A, Axon Instruments,
Foster City, CA). Data were acquired with PCLAMP 7 (Axon
Instruments) software, digitized at 20 kHz (Digidata 1200B,
Axon Instruments), and analyzed by using the Mini Analysis
Program (version 4.1.1, Jaejin Software, Leonia, NJ).
Behavioral Characterization. All animal experiments were ap-
proved by the Institutional Animal Care and Use Committee.
Mice were grouped together by age, weight, and generation such
that they were all between 8 and 16 weeks old and 15–35 g. Mice
of both sexes were used for all studies.
Sleep Time Assay. Alphaxalone (8 mg?kg and 16 mg?kg), preg-
nanolone (16 mg?kg), midazolam (25 mg?kg), and propofol (50
mg?kg) were administered i.v. Etomidate (20 mg?kg), pento-
barbital (45 mg?kg), and ketamine (150 mg?kg) were given by
i.p. injection. Injection volumes were 5 ?l?g body weight for i.v.
and 20 ?l?g for i.p. Neuroactive steroids [alphaxalone (Research
Biochemicals, Natick, MA) and pregnanolone (Sigma)] were
dissolved in a 22.5% (wt?vol) solution of 2-hydroxypropyl-?-
cyclodextrin (Research Biochemicals). Pentobarbital (Abbott)
and ketamine (Sigma) were dissolved in sterile saline. Midazo-
lam HCl (Roche Clinical Laboratories, Burlington, NC) was
dissolved in 0.8% NaCl, 0.01% EDTA, and 1% benzyl alcohol.
Sleep times were determined as follows: after drug injection
and loss of the righting reflex, mice were placed on their backs
in a V-shaped trough and a timer was started. The sleep time
period ended when the animal was able to flip over six times in
45 sec (neuroactive steroids) or three times in 30 sec (all other
drugs). Normothermia was maintained with the aid of a heat
lamp. All assays were performed by an investigator who was
unaware of the genotypes of the individual mice being tested.
Effect of genotype was compared by Student's t test.
Loss of Righting Reflex (LORR) and Tail-Clamp?Withdrawal Assay.
LORR and tail-clamp?withdrawal using volatile anesthetics
were performed as described (7). Briefly, mice were placed in
small, cylindrical wire-mesh cages attached to a carousel housed
in a sealed Plexiglas box. Anesthetics diluted with oxygen were
delivered from an anesthetic-specific vaporizer. After 15-min
equilibration at each concentration, the carousel was rotated five
times while the mice were observed. Scoring was quantal; mice
that passively rolled over twice were scored as positive for
LORR. For tail-clamp, the carousel was removed and a 10-
compartment divider was installed. After a 15-min exposure to
Fig. 1.
locus. Numbered black boxes represent exons in genomic DNA. Probe D is
830-bp PCR product used for genotyping. The neo gene was flanked on the 5?
side by 8.3 kb of genomic DNA and 1.2 kb of 3? genomic DNA. Probe D
hybridizes to an ?7.7-kb BamHI restriction fragment at a correctly targeted
Southern blot analysis of BamHI digested DNA illustrating mice of all three
genotypes. (C) Northern blot analysis using adult whole brain total RNA
hybridized with probe D or human ?-actin probe for loading control. (D)
Western blot analysis of total cerebellar protein from ??/?and ??/?with
?(1–44) polyclonal antibody. The 54-kDa ? protein present in wild-type lanes
is completely absent from ??/?lanes. Higher molecular mass bands of non-
specific binding show equal loading of samples in all lanes.
Gene targeting and molecular characterization. (A) Structure of ?
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If any motor activity occurred as a result of the stimulus, the
concentration was scored as one that permitted a positive
Elevated Plus-Maze Test. The anxiolytic effect of the synthetic
neuroactive steroid ganaxolone (CoCensys, Irvine, CA) was
tested by using the elevated plus-maze. The plus-maze was
constructed as described (29). A stock solution of ganaxolone
was made in DMSO and diluted with saline (pH 7.1) before
systemic administration. The final concentration of DMSO was
0.1%, a concentration known not to interfere with GABAA-R
function (30).
Ganaxolone (10 mg?kg, i.p.) or an equal volume of saline was
injected 10 min before testing (n ? 9?group). To start the 5-min
test session, a mouse was placed on the central platform of the
maze, facing an open arm. Open-arm and closed-arm entries and
the cumulative time spent on the open and closed arms was
recorded. A mouse was considered to be on the central platform
when all four paws were within its perimeter. The ratio of
open?total arm entries (% open-arm entries) was calculated and
expressed as mean % open-arm entries ? SEM. Data were
analyzed by using one-way ANOVA, and ganaxolone-treated
group means were compared with their respective saline-treated
controls by using Bonferroni's post-hoc test.
Low-Dose Pentylenetetrazol (PTZ) Seizures. Pretreatment of ??/?
and ??/?mice with ganaxolone (10 mg?kg, i.p.) or saline
occurred 10 min before PTZ (Sigma) (20 mg?kg, i.p.) injection.
The behavior induced by saline?PTZ versus ganaxolone?PTZ
was observed and recorded for 2–3 h post-PTZ treatment. Data
are expressed as mean duration of hypoactivity ? SEM (n ?
9?group). One-way ANOVA followed by Bonferroni's post-hoc
test was used to compare multiple group means.
Pavlovian Fear Conditioning. Associative memory was examined
with context and tone-dependent fear conditioning (31, 32).
Briefly, a tone conditional stimulus was paired with an aversive
unconditional foot shock stimulus in a novel context. Mice
trained in this manner develop a fear of both tone and context,
which was measured as freezing, an adaptive defense reaction.
One day after conditioning, mice were returned to the training
context for an 8-min context test. One day after the context test,
mice were placed in a novel context for an 8-min tone fear test.
After a 2-min baseline period, the training tone was played for
both tests. To assess exploratory activity, crossovers were scored
during the 3-min period before tone-shock pairing on the
conditioning day. To assess pain sensitivity, velocity was com-
puter-tracked for 2 sec immediately before and for 2 sec during
the first foot shock (33). Consolidation and retention of contex-
tual fear was examined by repeating the context test 50 days after
Production of Mice. Of 307 ES cell clones analyzed for gene
targeting, three displayed the predicted BamHI restriction frag-
ment length polymorphism indicative of correct gene targeting
at the ? locus. Two cell lines (?#767 and ?#773) yielded
germ-line competent chimeric males. As indicated in Fig. 1B,
probe D hybridized to a 6.6-kb BamHI fragment from the
allele. The ? locus was examined with 12 restriction enzymes,
three unique genomic probes, and a neo probe. All results (data
not shown) were consistent with the targeting event depicted in
Fig. 1A.
Northern and Western blots were used to examine ? gene
expression. Hybridization of adult cerebellar total RNA with
probe D showed an abundant 1.9-kb band in the ??/?mice. In
contrast, samples from ??/?mice revealed the absence of the
wild-type 1.9-kb mRNA (Fig. 1C). In addition, in mice bearing
a targeted allele, a novel 2.3-kb band was observed. Rehybrid-
ization of the same blot with a neo probe revealed that only the
2.3-kb message from ??/?and ??/?mice contained neo-
homologous sequences (data not shown). These results suggest
that the targeting event resulted in a ? allele that was transcribed
as a chimeric ??neo mRNA.
with the highest level of ? expression) of three different ??/?and
three different ??/?mice were probed with the rabbit polyclonal
antibody ?(1–44) (26). The ? subunit exhibits an apparent
molecular mass of 54 kDa (Fig. 1D) and is readily apparent in the
??/?lanes. In contrast, samples from the ??/?mouse cerebel-
lums were completely devoid of the ? protein. Together, these
results demonstrated that the gene targeting event disrupted the
? locus and prevented production of ? protein, i.e., a true null
The size, histological appearance, and folding of the folia in
the cerebellum all appeared normal and indistinguishable be-
tween ??/?and ??/?mice, as did the staining patterns of
toluidine blue, RT97, and antisynaptophysin (data not shown).
Of 1,030 F2and F5pups genotyped at weaning from hetero-
zygote mating pairs, only 211 (20.5%) were null. This percent of
null pups is significantly below the expected Mendelian fre-
quency distribution of 25% (P ? 0.005). Thus it appears that
? knockout had any effect on fecundity, true breeding F3lines
were established. These breeding experiments revealed that ??/?
breeding pairs produced statistically fewer pups per litter than
the ??/?breeding pairs [??/?matings: 7.6 ? 0.4 pups?litter (n ?
47 litters) vs. ??/?matings: 6.3 ? 0.3 pups?litter (n ? 50 litters)]
Pharmacological Characterization. Ligand binding in whole brain
homogenates from ??/?and ??/?mice was studied with
[3H]Ro15–4513 and [3H]muscimol. The slopes of the binding
of the Kdvalues. Maximal receptor number and binding affinity
for [3H]muscimol in homogenates from ??/?brains were similar
to previous reports (7, 34). However, the maximal binding for
muscimol was markedly decreased in ??/?homogenates (Table
??/?was similar to previous reports (7). The maximal receptor
number and affinity constants for [3H]Ro15–4513 were not
different between ??/?and ??/?brain homogenates (Table 1).
Table 1. Binding of [3H]Ro15-4513 and [3H]muscimol to whole
brain homogenates
Wild type Null
[3H]Ro15-4513 binding*
Apparent Kd(nM)
Maximal binding (pmol?mg)
[3H]Muscimol binding†
Apparent Kd(nM)
Maximal binding (pmol?mg)
8.9 ? 1.2
1.4 ? 0.1
1.2 ? 0.2
9.1 ? 1.0
1.2 ? 0.1
1.3 ? 0.3
37.0 ? 9.9
4.5 ? 0.4
1.1 ? 0.2
21.0 ? 10.9
2.4 ? 0.4‡
1.1 ? 0.4
*These data were pooled from four separate binding experiments for each
for each [3H]Ro15-4513 concentration tested.
†These data were pooled from three separate binding experiments for each
genotype. Each experiment consisted of five specific binding determinations
at each [3H]muscimol concentration tested.
Mihalek et al.PNAS ?
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vol. 96 ?
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Using in situ autoradiography to examine the abundance and
distribution of GABA and benzodiazepine sites, it was observed
that [3H]muscimol binding was drastically reduced in most
regions of ??/?brains (Fig. 2). In contrast, the [3H]Ro 15–4513
binding was slightly elevated in several regions of the ??/?brain,
including the thalamus, striatum, and the cerebellar granule cell
Delta Knockout Mice Exhibit Altered Synaptic GABAA Currents.
mIPSCs were recorded in hippocampal slices from adult ??/?
and ??/?mice to directly investigate the possibility of altered
GABAA-R current kinetics in mutant mice. Table 2 illustrates
that the average amplitude, rise time, and frequency of mIPSCs
recorded at 0 mV did not differ between genotypes. However,
the decay time (?) of mIPSCs from ??/?mice was significantly
faster than that of ??/?controls.
Delta Knockout Mice Are Resistant to Neuroactive Steroids: Reduced
Sleep Time. Using a sleep time assay, significant differences were
found in response to i.v. injections of alphaxalone and preg-
in sleep time duration compared with ??/?at the two concen-
trations of alphaxalone used (8 mg?kg and 16 mg?kg), respec-
tively. Comparison of ??/?and ??/?after an 8 mg?kg injection
of pregnanolone revealed a 42% reduction in sleep time in ??/?
mice. Intraperitoneal injection of alphaxalone (75 mg?kg) also
revealed a similar difference (data not shown). Similar effects
were observed in ??/?mice derived from the ?#773 cell line
(data not shown). Wild-type and ??/?mice responded similarly
to etomidate, pentobarbital, midazolam, propofol, and ketamine
(an NMDA receptor antagonist) (Table 3).
A trivial explanation for the change in neuroactive steroid
response could be an unexpected pharmacokinetic effect. To
investigate this possibility, pregnanolone concentration in brain
tissue was determined (35) in a limited number of mice at waking
from a 16 mg?kg i.v. injection. After an average sleep time of
45.3 ? 7.4 min, ??/?brains had 5.5 ? 0.8 (n ? 2) ?g?g
pregnanolone, and after an average sleep time of 33.2 ? 4.9 min,
??/?brains had 7.3 ? 1.2 (n ? 3) ?g?g pregnanolone. The level
of endogenous allopregnanolone also was measured in the same
mice: ??/?, 514 ? 48 pg?g (n ? 2) and ??/?, 394 ? 118 pg?g (n ?
3). These preliminary results indicate no gross differences
between genotypes in neuroactive steroid pharmacokinetics or
endogenous levels.
A second explanation for the difference in sleep times is that
they result from the influence of genetic background. Therefore,
the two strains used to make the ??/?mice, C57BL?6J and strain
129?SvJ mice, were tested with alphaxalone (16 mg?kg) and
pregnanolone (16 mg?kg). Sleep times were not statistically
different between strains for either compound: alphaxalone,
C57BL?6J (8.1 ? 1.3 min., n ? 10) and strain 129?SvJ (9.1 ? 2.7
min., n ? 10) (P ? 0.34) and pregnanolone, C57BL?6J (38.4 ?
Fig. 2.
binding sites in horizontal brain sections of GABAA-R ??/?and ??/?mice.
Representative images illustrate the reduction of [3H]muscimol binding and
region-specific elevation of [3H]Ro 15–4513 binding. Gr, cerebellar granule
cell layer; Th, thalamus; Str, striatum.
Autoradiographic distribution of [3H]muscimol and [3H]Ro 15–4513
Table 2. Hippocampal electrophysiology
GenotypeWild typeNull
Rise time (msec)
Amplitude (mV)
Frequency (Hz)
Decay, ? (msec)
# Cells?mice
1.53 ? 0.11
11.30 ? 1.05
3.04 ? 0.42
7.05 ? 0.39
1.49 ? 0.10
9.92 ? 0.65
2.85 ? 0.26
5.74 ? 0.18*
? is the time from 90% to 37% of decay phase of mIPSC.
Table 3. Sleep time assays
Drug (dose)
Sleep time, min
(mean ? SEM)
Alphaxalone (8 mg?kg) 18
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
Wild type
3.5 ? 0.5
1.6 ? 0.4*
8.6 ? 0.8
5.3 ? 0.7†
24.1 ? 1.8
14.0 ? 2.2‡
56.4 ? 3.2
52.2 ? 3.6
14.1 ? 1.8
13.7 ? 2.5
14.4 ? 4.0
13.2 ? 4.8
55.3 ? 5.0
42.0 ? 4.5
40.1 ? 3.8
37.5 ? 2.6
Alphaxalone (16 mg?kg)
Pregnanolone (8 mg?kg)
Pentobarbital (45 mg?kg)
Propofol (50 mg?kg)
Midazolam (25 mg?kg)
Etomidate (20 mg?kg)
Ketamine (150 mg?kg)
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www.pnas.orgMihalek et al.
Page 5
3.5 min., n ? 9) and strain 129?SvJ (43.8 ? 1.6 min., n ? 10) (P ?
0.16). Thus, the phenotype observed in the ??/?mice is not likely
caused by genetic background effects from the parental strains.
Reduced Anxiolysis. Using an elevated plus-maze assay, the ten-
dency of mice to enter or remain on the open arm of the maze
is increased by anxiolytic drugs (36). Basal levels of anxiety (i.e.,
anxiety levels in drug-naive mice) were not statistically different
between genotypes. With percent open-arm entries as a measure
of anxiolysis, a 10 mg?kg i.p. dose of ganaxolone in ??/?mice
0.01). No significant increase in open-arm entries occurred in
??/?mice. Thus, the well-characterized anxiolytic effect of
neuroactive steroids was absent in ??/?mice.
Reduced Pro-Absence Seizure Effect. Absence seizures are exacer-
bated by GABAA-R agonists, including neuroactive steroids
(37). Using a low-dose PTZ absence seizure model, ganaxolone
(10 mg?kg, i.p.) failed to prolong PTZ-induced absence-like
freezing in ??/?mice, whereas it increased absence-like freezing
74% in ??/?mice (Fig. 3B;**, P ? 0.001). PTZ-induced
hypoactivity was not statistically different between genotypes.
Normal Behavioral Responses to Volatile Anesthetics. Wild-type and
??/?mice were compared for their sensitivity toward the ob-
tunding (LORR) and pain suppression (tail-clamp?withdrawal)
effects of two halogenated volatile anesthetics. There was no
difference statistically in the tail-clamp response between ??/?
and ??/?mice in the EC50 value for either anesthetic or for
LORR with halothane (Table 4). Similar results were obtained
in our analyses of the ?#773 mice (data not shown).
Normal Fear Conditioning, Exploratory Activity, and Pain Sensitivity.
Fig. 4A depicts freezing during the 8-min context test. There
were no significant differences in context conditioning. Fig. 4B
depicts cued conditioning, during the 2-min baseline period
before and during the 6-min tone: there were no significant
differences. Mutant mice showed comparable levels of activity to
??/?during the baseline period on the conditioning day and
showed robust foot shock reactivity equivalent to controls (data
not shown). Finally, ??/?mice showed robust context freezing
during the 50-day posttraining memory test, not significantly
different than ??/?(data not shown).
The absence of the ? subunit of the GABAA-R resulted in a
duration of alphaxalone?pregnanolone anesthesia and the anxi-
olytic effect and pro-absence seizure effect of ganaxolone all
were reduced?ablated. The reduced response of ??/?mice to
neuroactive steroids reveals a potential role of ?-containing
GABAA-Rs in modulating behavioral responses to endogenous
neuroactive steroids. This dramatic reduction in whole animal
neuroactive steroid sensitivity was specific, as deletion of ? had
no effect on response to several other sedative?hypnotic agents
in the sleep time assay or on the LORR or tail clamp?withdrawal
response after exposure to volatile anesthetics. The observed
difference does not appear to be confounded by pharmacoki-
netic or genetic background influences. These results show an
animal model that demonstrates a selective alteration in behav-
ioral responses to neuroactive steroids. If responses to endoge-
nous neuroactive steroids are similarly attenuated, these mice
Fig. 3.
??/?mice. (A) Mice were injected with ganaxolone (10 mg?kg, i.p.) 10 min
before testing on the elevated plus-maze assay (n ? 9?group). Ganaxolone
in ??/?. (B) Mice were treated with ganaxolone (10 mg?kg, i.p.) or saline (n ?
Behavior was observed for 2–3 h posttreatment. Ganaxolone significantly
prolonged absence-like behavior in ??/?(**, P ? 0.001, Bonferroni's post-hoc
test), but not in ??/?mice (mean ? SEM).
Anxiolytic (A) and pro-absence (B) effects of ganaxolone in ??/?and
Fig. 4.
ings in a distinctive context. (A) Context fear. One day after training, mice
were returned to conditioning chambers and contextual freezing (% time,
mean ? SEM) was assessed for 8 min. Mutant mice exhibited normal levels of
contextual fear. (B) Tone fear. One day after the context fear test, the mice
were brought to a novel context, and after a 2-min baseline (BL) period, the
conditioning tone was played for 6 min. Freezing (%time, mean ? SEM) was
assessed for both periods.
Pavlovian fear conditioning. Mice were given three tone-shock pair-
Table 4. Behavioral responses to volatile anesthetics
(atm % ? SEM)
(mean ? SEM)
Tail clamp
Wild type
Wild type
LORR assay
Wild type
1.55 ? 0.03
1.50 ? 0.04
2.43 ? 0.05
2.32 ? 0.05
10.71 ? 1.66
11.11 ? 1.72
11.11 ? 2.03
11.86 ? 2.09
0.72 ? 0.04
0.71 ? 0.04
8.83 ? 2.74
10.65 ? 1.47
Mihalek et al.PNAS ?
October 26, 1999 ?
vol. 96 ?
no. 22 ?
Page 6
will be invaluable for elucidating the physiological mechanisms
of these enigmatic compounds.
These results support the findings that neuroactive steroids
have unique, noninteracting binding requirements on
GABAA-Rs distinct from those of benzodiazepines and barbi-
turates (19, 21). Additionally, the differential behavioral re-
sponse to sedative?hypnotic agents indicates that different drugs
have different molecular targets as suggested by Eger et al. (38)
and illustrated by the GABAA-R ?3 knockout (9). It remains to
be determined whether ? subunit-containing GABAA-Rs rep-
resent actual targets of steroid action or whether the behavioral
sensitivity is indirectly altered in the ??/?mice.
Although these results (i.e., reduced behavioral response to
neuroactive steroids in ??/?mice) may appear at odds to those
with Zhu et al. (15) (i.e., enhanced sensitivity to neuroactive
steroids by GABAA-Rs lacking ?), one must keep in mind that
in vivo behavioral changes may not necessarily be directly related
to in vitro findings using recombinant receptors, because of the
complexity of the nervous system, the circuits involved, pleio-
tropic changes in a chronic model (lifetime null mutation), etc.
Detailed in vitro studies will be needed to analyze the possible
mechanism(s) responsible for the altered neuroactive steroid
sensitivity. One possibility would be that the GABAA-Rs re-
maining in ??/?mice have reduced sensitivity to neuroactive
The dentate gyrus granule cell electrophysiology (chosen
because it is one of three major cell types that express ?) suggests
that there is no drastic reduction in synaptic inhibition in these
cells because of the lack of decrease in mIPSC amplitude and
frequency. Rather, there may be a change in the molecular
composition of the GABAA-Rs involved. The observed faster
decay of mIPSCs is consistent with such a possibility. The
electrophysiological data also suggest ?-subunit involvement in
normal synaptic transmission in the dentate gyrus.
Analysis of the expected Mendelian ratio of pups and the
fecundity of wild-type versus null breeding pairs indicated that
deletion of the ? subunit impacted reproduction: litters from
??/?parents had fewer than the expected number of null pups
and ??/?parents had fewer pups per litter than ??/?parents. The
reduced sensitivity to neuroactive steroids may affect multiple
aspects of reproduction and development, an intriguing possi-
bility that warrants further investigation.
In conclusion, we have shown that a global deletion of the ?
subunit of the GABAA-R resulted in a decrease in the sensitivity
of mice to the sedative?hypnotic, anxiolytic, and pro-absence
effects of neuroactive steroids, and this change was remarkably
specific. Together, these results reveal a central involvement of
?-containing GABAA-Rs in neuroactive steroid action in mul-
tiple behavioral modalities.
We thank Carolyn Ferguson, JoAnn Steinmiller, Frank Kist, Janey
Whalen, and Jodi Daggett for expert technical assistance. This work was
supported by the University Anesthesiology and Critical Care Medicine
Foundation, the National Institutes of Health (Grants AA10422 to
G.E.H., GM52035 to L.L.F., and NS28772 to R.W.O.), and the National
Science Foundation (Grant IBN 9723295 to M.S.F.).
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- March 22: Government Investigates Recall of 1 Million Nissan Vehicles
- March 6: Nissan Recalls 625,000 Altimas With Hoods That Could Fly Open
- February 11: Nissan Steering Problems Cause Recall of 17,000 Vehicles
- January 31: Nissan Recalls 639,000 Pathfinders, Rogues and Infiniti QX60 Vehicles
- January 22: Nissan Recalls 3 Models For Braking and Steering Problems
Radiator Causes Transmission Failure
2005 Nissan Xterra (Page 2 of 12)
This problem may be covered under warranty. Ask your Nissan dealer.
CarComplaints.com Notes: The 2005-2007 Xterra appears to have a widespread defect where coolant leaks into the transmission, causing transmission failure around 90,000 miles. Repair costs are typically upwards of $3,500.
The same defect exists in the Pathfinder & Frontier as well. And according to the New York Times & a class action lawsuit, the defect actually extends through 2010 model year. More info here.
9.3really awful
- Typical Repair Cost:
- $3,781.00
- Average Mileage:
- 103,281 miles
- Total Complaints:
- 226 complaints
Most Common Solutions:
- new radiator and transmission rebuild (83 reports)
- replaced radiator and transmission (37 reports)
- not sure (31 reports)
- replace transmission (27 reports)
- new radiator, transmission, and flush fluids (24 reports)
- buy new car (7 reports)
transmission problem
Helpful websites
- Nissan XTerra Problems - This site tells about all the problems we have had with our nissan xterra
- Nissan Xterra Transmission Lawsuit - Class Action regarding radiator defect that results in transmission failure.
- Nissan Cracked Radiator And Transmission Problems - A description of the problem and actions you can take.


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Oakley Square Wire Sunglasses 1 1
Great glasses GREAT Deal on TGW Got this during one of the TGW flash sales - a super bargain compared to what was out there on other sites - including eBay. Love the glasses and it really helps ease the glare on the eyes. Don't go cheap, you'll end up buying a lot of glasses to equal what you get here. July 18, 2014
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The following is a compilation of excerpts from my forthcoming book, Holistic Energy Magic: Charms and Techniques for Creating a Harmonious Life...
Fresh Brew: Wake Up and Smell the Magic
An infusion of inspiration for your magical life.
Tess Whitehurst
Every single human on earth experiences personal conflicts and challenges. Many of us on the magical path believe that we incarnated into this realm because our soul is learning about its true divine nature, and conflicts and challenges are catalysts for this process. (Not to mention, let's be honest: life would be boring without them.) But whether or not you see things in this way, as a magical practitioner – whether your problem is old or new, immediate or systemic – you have the opportunity to transmute your challenges into lessons and conflicts into blessings.
Choosing to live in this way is the way of the magical warrior. Indeed, an alive, energized, activated tension occurs when every challenge and conflict becomes grist for the mill of learning, growing, and expanding. It's a brave way to live, and the only way to truly own your personal power and not feel hopelessly buffeted about by the annoyances and heartbreaks that characterize every single life experience....
A Midsummer Night's Dream is your favorite Shakespearean play. Tinkerbell is your favorite Disney character. And you always want to wear faerie wings, even when it isn't Halloween. Could you be a faerie-person? Chances are the answer is yes.
Before we look at the signs that you are, in fact, an incarnated member of the realm of the fae, let's discuss what exactly that may mean. It's true that it's a rather mysterious classification, but let's note for a moment that for the majority of time that humans have been here on planet earth, we have been much more closely aligned to her cycles and mysteries than we are now. Instead of watching TV or sitting under a roof, we gazed at the stars and were intimately familiar with their dance. We listened to the wind and noted its intensity and direction, and knew inherently what other factors went along with those conditions. We could communicate with animals, because we were awake to their wisdom and aliveness. In short, we knew ourselves to be one with Mother Earth; and, in the same way that one tiny bit of DNA contains the blueprint to the entire organism in all its glorious wholeness, we knew that we were not just on the earth, but rather that we contained its entire essence within our every cell....
what if you have 4 of 8. and #7 while i don't know what they may be used for, i definitely attracted to certain herbs & crystals (
Oh yes, I'm aware of some other faerie qualities: http://forestdoor.wordpress.com/2014/09/18/are-you-a-faerie/ Of course, I may
Dver, oh yes indeed!
Dver: HILARIOUS! Hahahaha. OMGoddess I love it.
Yup! Nice to meetcha!! I advise use of glitter as a self-help tool in one of my books, which embodies not only your glitter com
This is an excerpt from the brand new Kindle book I just co-wrote and released with Ellen Dugan, Every Witch Way: Spells and Advice from Two Very Different Witches. In it, we explore different sides of a number of issues, including Angels and Magick. The first piece is written by, and appears courtesy of, Ellen Dugan - the second is by me!
Go Carefully My Friend (by Ellen Dugan)...
Last week I got a parking ticket in the exact same place where I got a parking ticket last time Mercury was retrograde, but for a different reason. Last time, I fed the wrong meter. This time, I failed to read the street sweeping sign. It's said that Mercury retrograde periods will bring back the same lesson until you've learned from it and received the hidden blessing within. And, as I relaxed in the bathtub yesterday it popped into my mind that it might be very fun to create an amulet to ward off parking tickets and to hang it from my rear view mirror...Could that be the hidden blessing? It's certainly a way of making something magical out of something dreary, so why not?
You may have heard that Mercury goes direct tomorrow. Also, Wednesday is Mercury's day, so this Wednesday would be an excellent time to create this amulet, but really any Wednesday when Mercury is direct will do....
Let's face it: our modern lifestyles are filled with frenetic energy. Between gadgets, appliances, wifi, sirens, car alarms, and the frantic pace that often defines the average day, many of us find that our everyday consciousness has strayed far from the silence and stillness of the sacred grove.
In the past, I did my best not to fret about the challenging effects of electronics in interior spaces (which include but are not limited to challenges to the immune system, brain wave interference, insomnia, increased anxiety, and radiation exposure), just because I felt that there was nothing to be done about them anyway, other than moving off the grid. Then, the other day, while dowsing, I discovered that I was actually able to significantly reduce the negative effects of electromagnetic stress simply by setting the intention to do so while clearing the space energetically. Afterwards, my city apartment felt notably more still, nourishing, and serene....
It's beginning to look a lot like summer (at least where I live). And when it's warm out, fresh fruit just feels right. In addition to cleansing, hydrating, and nourishing the body, it can also help align the spirit with sweetness, beauty, and the luxurious abundance of the earth. Plus, each fruit possesses its own unique magical properties. So I thought it'd be fun to explore the magical properties of various types of fruits, and to discuss some hints and tips about how we can effectively incorporate them into our edible enchantments.
So, without further ado, here are some of the fruits that seem to feel best this time of year, along with a sampling of their magical properties....


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