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Grow Together: Gardening Group for Young Children and their CaregiversSubmitted by Suzanne Ramos on Thu, 07/27/2017 - 12:50pm
Address of Samaritans of FR/NB, Inc.: 627 State Rd, Westport, MA 02790
This can be for a recent suicide or not a recent one, perhaps a special occasion that would trigger emotional pain for the survivor, ex. birthday, anniversary.
At this time, this is an adult group, and we welcome survivors to attend once or as many times as they wish. Contact for more information.

subset: commoncrawl


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Sleight of Hand: A Novel of Suspense
NOOK Book(eBook)
Private investigator Dana Cutler must take down a cunning psychopath before he can pull off the perfect crime, in Sleight of Hand, a novel of suspense from Phillip Margolin, New York Times bestselling author of Capitol Murder and Supreme Justice.
Charles Benedict – charismatic criminal defense lawyer, amateur illusionist, and professional hit man – has performed his greatest sleight of hand yet: framing a millionaire for the murder of his much younger wife.
When Horace Blair married Carrie, the prosecutor in his DUI trial, he made her sign a prenuptial agreement guaranteeing her twenty million dollars if she remained faithful for the first ten years of marriage. Just one week before their tenth anniversary, Carrie disappears, and Horace is charged with her murder. Desperate to clear his name, the millionaire hires D.C.'s most ruthless defense lawyer – Charles Benedict.
P.I. Dana Cutler is in the Pacific Northwest on the trail of a stolen relic dating from the Ottoman Empire. Hitting a dead end sends her back to Virginia perplexed and disappointed – and straight into the case of Horace and Carrie Blair.
Now Dana must conjure a few tricks of her own to expose Benedict's plot, before he can work his deadly magic on her...
Product Details
|HarperCollins Publishers
|Publication date:
|Dana Cutler Series , #4
|Sold by:
|NOOK Book
|Sales rank:
|File size:
About the Author
Phillip Margolin has written nineteen novels, many of them New York Times bestsellers, including his latest novels Woman with a Gun, Worthy Brown's Daughter, Sleight of Hand, and the Washington trilogy. Each displays a unique, compelling insider's view of criminal behavior, which comes from his long background as a criminal defense attorney who has handled thirty murder cases. Winner of the Distinguished Northwest Writer Award, he lives in Portland, Oregon.
Place of Birth:New York, New York
Education:B.A. in Government, American University, 1965; New York University School of Law, 1970
Customer Reviews
Average Review
Most Helpful Customer Reviews
It's fun to read a story in which the villain is known. The author then must weave in the details in a way that keeps the reader involved. Phillip Margolin does this. One thing that impressed me: In his acknowledgments, Margolin recognizes his copy editor by name. It's easy to see why. This is one of those rare books in which I found no glaring grammar or spelling errors. Kudos to Margolin and to his editors.
I love Phillip Margolin's books and can't resist his Dana Cutler stories. This book starts out with what seems to be two separate, unrelated stories. Although both were interesting, I kept wondering how they would come together. One story involves Dana Cutler who is a private investigator hired to track down a stolen relic that turns out to be a dead end. When she returns to Virginia, Dana becomes involved in the second story that involves the murder of Carrie Blair just days before she would receive millions of dollars from her husband Horace according to the terms of their prenuptual agreement and he is charged with her murder. In a series of twists and turns, Margolin pulls the two seemingly unrelated incidences together. The two cases turn out to have a common thread and Dana is pitted against Horace Blair's defense attorney Charles Benedict. Dana uncovers evidence that Horace may have been framed for the murder, but Benedict blocks her attempts to get the answers she needs. This story gets really suspenseful when Dana becomes a target of the real killer as she hunts for the truth. Margolin creates a masterful book and captivates readers from the first page. This novel was very well written and the storyline was intricately put together. The character development in this story is quite in depth. You get to know a great deal about the main characters, their lives, and their histories. Sleight of Hand is very well set up. It progresses in a logical fashion from beginning to end while keeping the reader completely captivated with the many different twists found in the story. As usual, I was impressed with Margolin's ability to take multiple storylines and complex characters and bring them together in a way that held my attention.
Couldn't put it down.Rate five stars
I'd never read this author before Sleight of Hand. I loved this book and read it all in one day. The story thread just pulled me right through with interesting characters and twists and turns and a great ending. Charles is the perfect criminal. He is a genius, a magician, a killer and a lawyer. What a combination!!! I'll get more books by this author.
I thought "Phillip Margolin IS BACK"!! I loved it!!
Right up there wirh Margolin's top ones. If your a fan you won't be disappointed.
Most of Margolin's books are the best! Always keeps me on my toes and guessing, the suspense drives me crazy. This particular one was not one of my favorites, sorry to say, but still an exciting and good read.
Another Dana Cutler novel. I love this author's works. The book is worth your time.
Suspenful and complicated plot fun read
A little all over the place in the beginning, but keeps your attention and great ending
You already who killed Carrie and the best part was how the policeand the detective came togather. Loved it. The bad part was it was only 252 pages.
Fun read
For me its a little slow to start.
Not an especially great read. I certainly have read many more exciting crime stories but this definitely was not one of them.
I quit reading this when the multi millionaire (or billionaire) called an attorney who had admitted to having an affair with his wife. Said attorney is a professional hit man who panics and shoots said wife, then proceeds to blame millionaire husband. I would think the husband, being a millionaire, would have an army of legal counsel on retainer. But to choose the man who admits to cheating with your wife? I'm sure I would have many more complaints, but that was as far as I got in the book. I won't even mention when Benedict switched evidence in the courtroom. Please! people are paying good money to read a good book. This book is totally unbelievable. If you must read it, get it from the library.


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Child support, spousal support (also called alimony or palimony) and child custody orders can be very difficult to enforce and modify. Each modification or enforcement has to meet certain legal standards.
Existing terms no longer work for you
Visitation schedule needs altering
One parent becomes more or less fit to provide care for a child.
It becomes clear there is something missing from the original agreement
Frequently Asked Questions
How do I modify a child custody order?
For instance, to modify a custody order concerning children who are still under the age of 18, the party moving to modify has to show a "change of circumstances." The change of circumstances is not easy to establish in some cases. There are cases that say the mere passage of time does not, by itself, justify a custody modification. Parenting time can be modified based on other factors, and even a consultation with a lawyer can help you decide whether your case is ready to proceed.
How do I modify child support orders?
Because a change of child custody can also mean a change of child support, the modification of child custody can put particular pressure when you file court papers. Court paperwork has to show why your parenting time should be changed in the actual filing. Child support increases are usually based on changes in parenting time or changes in income. Enforcing child support requires a specific set of court papers. First, a hearing will have to be held, usually about 30-60 days after filing the court paperwork for child support, to determine whether the paying parent has met their legal obligation. Only then can the court assign a deadline to pay the full outstanding amount.
We have a non-modifiable spousal maintenance order and payments cannot be made. What happens next?
What if you have a court order for spousal maintenance? If you are supposed to pay or receive spousal support, this is another technical area. Your divorce decree should say whether your spousal maintenance obligation is non-modifiable. If it is non-modifiable, but the paying party is not paying in full or on time, or you believe it should be reduced, then the only way to change court orders is to have a judge make new orders or to agree out of court.
I want to take a different job. How will that affect my spousal maintenance payment?
If you are the paying party and you have taken a job that will earn you less income than you are capable of earning, then you could be stuck paying a higher amount of support than you can afford. But, there is case law that says that you are allowed to work at a second job and not be penalized. For instance, if you a teacher, but you work nights and weekends at Home Depot (which means you are also probably a superhero) you are only going to be "charged" for the teacher salary for purposes of computing child support or spousal maintenance.
We have a short term marriage and we each earn the same amount. Can one of us expect to receive spousal maintenance?
The spousal maintenance factors are set up to allow for spousal maintenance payments in only specific circumstances. If, for instance, you have a young or ill child, or a long-term marriage, or your age or employability make you unable to be self-sufficient, Arizona law may entitle you to a support order. But there are other factors for the court to consider in awarding support, and an attorney should talk to you about Arizona courts and spousal maintenance.
Ready for a Peaceful Transition?
Stay in Control
Preserve Your Important Relationships
We know family law issues can vary widely from one to another. We'll listen to your case needs and provide advice, resources, and any other information to ensure a peaceful transition into the next chapters of your life.
We offer a free consultation with one of our experienced, dedicated lawyers. Contact us to get started.
Read more
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Peaceful Family Law was founded in 2009 with a desire to ensure the institution of family was preserved in times of conflict. Jennifer Moshier was one of the first trained collaborative law attorneys in Arizona.
We want our clients to stay in control of their family court outcomes and be involved in making a peaceful transition in to the next chapters of their lives.


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As an entrepreneur, I have a certain affection for people and organizations that support small businesses. Today I had the privilege of being a guest on Atlanta Business RadioX and meeting two influential small business advocates, Lee Kantor and Ryan McPherson. During......Read More
Elements of a Well-Rounded Inbound Marketing Strategy
All business owners know that marketing plays an essential role in the overall growth and sustainability of their companies. Marketing works to attract new customers and also to retain existing ones. For marketing professionals, there are two different fundamental practices for marketing to consumers – Outbound marketing and inbound marketing. Outbound marketing strategies include many old-hat channels of reaching consumers by pushing out direct mail pieces, radio,......Read More
In SEO, Top Rankings Aren't Everything
The job of a search engine optimization consultant has evolved greatly over the last few years. No more is SEO just a narrowly scoped task to get clients ranked in the top 3 for their targeted keyword terms and then calling it a day. Top rankings are nice, but that's not the ultimate goal of the client who is paying the SEO consultant to his/her services.......Read More
Getting Back In Touch with Project Management Basics
Today I came across a great article titled "14 Ways to be the Worst Web Project Manager" ... It's a great article that really emphasizes the fundamentals of project management basics and the career path that I've chosen. I've been managing web redesign and SEO projects for the past 3.5 years. Every project is different with its own set of technical specs and stakeholder personalities,......Read More


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Job Summary
- Provides multifaceted marketing support. Plans, directs, or coordinates marketing policies and programs. Uses social media platforms to promote the company. Will also work with inside sales in customer service roles as needed.
General Accountabilities
- Identifies, develops, or evaluates marketing strategy.
- Formulates, directs, or coordinates marketing activities or policies to promote products or services.
- Compiles lists describing product or service offerings.
- Coordinates or participates in promotional activities or trade shows.
- Initiates market research studies or analyzes their findings.
- Launches and grows presence on social media platforms, such as Facebook, Twitter, etc.
- Works cross functionally to ensure social media goals align with company goals.
- Writes and curates content designed to engage followers and promote the brand in a positive way.
- Creates and executes social media campaigns to promote the brand.
- Ensures customer satisfaction by responding to any complaints, questions and comments left on the company's social media pages in a timely and appropriate manner.
- Continuously monitors site analytics to see what types of content followers respond to the most and adjusts the strategy accordingly.
- Prepares reports to update internal staff on usage statistics.
- *The company reserves the right to add or change duties at any time.
Job Qualifications
- Education: Associate's degree in marketing, communications, or related field preferred
- Experience: 2-3 years of related experience
- Excellent verbal and written communication
- Coordination
- Active listening
- Social perceptiveness
- Judgment and decision making
- Persuasion
- Critical thinking
Technological Skills
- Microsoft Office
- Adobe Creative Cloud: Photoshop CC, Lightroom, Illustrator CC, InDesign CC
- Video editing experience a plus but not required
Thank you for your interest in this employment opportunity. Should you wish to pursue this, we will need to have you complete our Orientation, which will acclimate you to our One-Stop Center and services, and will get you registered with us – your first step to success. Orientations can be completed online at http://opp-inc.org/for-career-seekers/oppinc-workforce-center-services/oppinc-orientation/ as well as in our Resource Room located in the Opportunity Inc. One-Stop Workforce Center, 861 Glenrock Road, Suite 100 in Norfolk, just behind Military Circle Mall.
For your convenience, you can also complete the orientation at our Suffolk office on Mondays at 1:30PM (157 N. Main Street in Suffolk 757-514-7737) or our Franklin office (located at the Paul D Camp Community College Regional Workforce Development Center 100 N. College Drive).
Once your Orientation and Survey are completed, please come in to complete your intake process – intakes are held Mondays, Wednesdays, and Fridays from 8:30 to 12:30 or Tuesdays and Thursdays from 12:00 to 4:00.
If you are already enrolled, please contact your Career Developer, reference the Job Order number (Job Code Number), and ask that they refer you for the position. In either case, please have a resume available to target the position in which you are interested.
Thanks again, and we look forward to seeing you at Orientation!
You must sign in to apply for this position.


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(of a man)
Carry a lover to several locations inside a house (to demonstrate physical strength) before having sex.
Takuu – 1,800 speakers – Taku Atoll near Bougainville, Papua New Guinea
Piers Kelly came across this word while editing the Takuu dictionary. Richard Moyle explains that its cognates in several parts of Polynesia refer to the kingfisher bird. The kingfisher is not known on Taku Atoll, although it may have been present in the past. Even that meaning has a hidden story because the word is a compound noun: tiko 'shit', tara 'curved end of a house'.
Moyle writes: "How the term became transferred to a form of athletic foreplay remains unclear, and my dictionary collaborators were less than forthcoming."
Source: Richard M. Moyle (2011). Takuu Grammar and Dictionary. Canberra: Pacific Linguistics
Credit: Piers Kelly, Richard M. Moyle
Comments are closed.


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Auctions - next auction: Autumn 2017 2nd of September
SAHS arranges gun and militaria auctions twice a year. Auctions ares open to public. Items for sale can be studied before the auction.
Photos of most of the items are available on our webpages. You can leave your items for sale, too. Buyer pays final price added with 10% for SAHS. Cash and debit cards accepted at the auction site.
Prebidding is possible via e-mail. Last minute to leave offer is friday night before the auction. No later bids will be noticed. Please notice that you must receive verification of your offer, unverified offers have not been received in time.
Autumn auction will be held as a part of Ikaalinen Gun Show. For dates, please refer our calendar and pages below. Here you can also find auction list couple weeks before the date. Old auctionlists with prices can be found in "Previous auctions" -page.
After last bid SAHS is not responsible of items sold. Guns and ammo are sold only for collection purposes, not for use. For paperwork, you have three months to collect your licenced gun. Buyer is responsible of all expences after buying the items.
For more information, write to huutokaupat (ät) sahs.fi or call +358 (0)45 665 2330 Antti Alonen.


text: Mad is an American animated sketch comedy produced by Warner Bros. Animation. It was inspired by its unrelated predecessor, Mad TV, where each episode is a collection of short animated parodies of television shows, movies, games, celebrities, and other media using various types of animation instead of the usual animation style that Warner Bros. Animation is known for. The series premiered on the evening of September 6, 2010 on Cartoon Network. The series mostl...... more on Wikipedia


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I don't think cooking more in itself will simplify my life. Buying prepared food is easy and fast, but it's expensive (and spending more money than I have complicates my life). It would be even more expensive to have a personal chef to prepare all of my meals for me, so that's not happening any time soon. Therefore, knowing how to cook is a very useful skill.
Cooking simplifies food in the way that we know everything that went into our meals. Our diet is simplified. Cooking from scratch reduces the ingredients so we're eating basic elements of life grown from the earth, not "edible food-like substances" concocted in labs or extra ingredients to lengthen shelf time. (Michael Pollan's books and documentaries are great resources for getting back to the roots of our food and how humans eat.)
I did not learn how to cook growing up. I could prepare myself some food — like boil pasta, make myself an English muffin pizza, pour cereal, chop up a salad, etc. I could survive, but my repertoire was not very balanced or interesting. I didn't know what a roux was, how to reduce a sauce, how to make pancakes from scratch, what causes food to rise or be sticky or caramelize, or what happens when you whip in some air. I've learned a lot and I continue to learn new things all the time. (I still don't really know the difference between boiling and braising.)
I'm not taking a kitchen 101 class or a cooking course or anything. I'm just going in the kitchen and trying my hand at cooking different recipes. Sometimes they work — most of the time they work — but sometimes they flop. I learn from both outcomes. And I'm having a lot of fun doing it.
I put "cook new recipe" on my weekly to-do list for a month before I finally got around to doing it, but now I'm on a roll. I don't even have to put it on my list anymore. Every Saturday morning before work, I drink my coffee and flip through a cookbook (usually The French Women Don't Get Fat Cookbook) and pick out 2 or 3 recipes to try to that week. Then I go to the grocery store after my shift and get everything I need to cook those recipes. This has been the best form of meal planning for me so far.
Cooking is more than a hobby for me. I wouldn't really call it a passion either. It's a way of life, I guess? It's a way to life? We cannot survive without eating and being able to provide for ourselves will make ourselves better — more independent and more sustainable. And, above all, I enjoy cooking. I think it's fun and interesting and sexy and delicious.
Cooking, I believe, is love — that we give to ourselves and share with others.


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Monthly Archives: May 2011
Mobile Media Mindblast – be there!
Mobile Media Mindblast is happening on Friday 3rd June – a collaborative event that includes the big players – Vodacom, Google, MXit plus Joseph Jaffe as keynote speaker. Are you wondering how you should be using the explosive growth of … Continue reading
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Best reading material on the loo – twitter or fave magazine?
This morning I had a few precious minutes on the loo and found myself in a quandary – do I read my favourite mag, or do I scan twitter for the latest? I chose twitter … it's been a week … Continue reading
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Twitterati hungry for election news in SA
8.30pm on election night, an hour and a half after voting stations have closed … what's highlighted in twitter trends in South Africa? Our hunger for election knowledge, again … SAtwitterpolitician ballot @helenzille LGE2011 queue IEC voters thumb council district … Continue reading


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Back again8/06/2010 03:25:00 AMBriana Latrise
I'm gon get it together. I gotta do it right this time.
I can't be livin' like I was... It was all just a lie.
I told myself that I was on, that everything would be fine.
But my plan is NOTHING, when compared to what is right.
If I would just give in... If I could just let go,
My true dreams would come to life without a reality show,
Without a slavery deal, without a "moment" to glow.
I would just live for an eternity with peace in my soul.
That's valuable!
Not the materials we've easily been sold,
Never lies that we believe because the idols say so.
Not the conditions of the system, of which some choose to tightly hold,
Or the seconds of false contentment perpetuating dopes sold...
(I need to finish but I havn't slept more than 2 consecutive hours in the last 7 days... Special Thanks to the Century Regional Detention Facility! Holla!!! Deputy Martinez... Imma pray for you... Ya filthy mongrel... Lol. And I'm out!)
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Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor-coordination, and have an attenuation of age-induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value.
G-protein coupled receptor (GPCR) signaling through Gαs/Olf and subsequent stimulation of adenylyl cyclase activity is the canonical means of cAMP accumulation in the mammalian central nervous system (Offermanns 2001). Subsequent cAMP-dependent activation of protein kinase A (PKA) and phosphorylation of proteins, such as the cAMP response-element binding protein (CREB) transcription factor, is a major regulator of synaptic plasticity associated with learning and memory (Poser & Storm 2001). The ubiquitous nature of this second messenger system, linkage to numerous neurological and psychiatric diseases, and diversity in GPCRs coupled to Gαs/Olf and adenylyl cyclases has provided fertile ground for the development of targeted therapeutics. In addition to the therapeutic potential of this signaling pathway, pharmacological and genetic gain and loss of function experiments have provided a wealth of knowledge regarding the basic biology and physiology of cAMP signaling cascades in the CNS.
Following stimulated synthesis of cAMP, temporal and spatial control of intracellular cAMP signaling is tightly regulated by cyclic nucleotide hydrolysis through phosphodiesterases (PDEs) (Conti & Beavo 2007). In mammals, there are 11 families of PDEs, and each PDE family has its own unique kinetic, regulatory partners, as well as inhibitor sensitivity (Bender & Beavo 2006). Of these PDEs, the PDE8 family is one of the more recently discovered. The PDE8 family consists of two distinct genes -Pde8a and Pde8b. PDE8A and PDE8B have a high affinity for cAMP and hydrolyze cAMP with a low Km; additionally, neither of the PDE8s can effectively hydrolyze cGMP, nor are they regulated by cGMP (Fisher et al. 1998; Soderling et al. 1998). Recent studies have indicated potential functions of PDE8B, in thyroid cells (Arnaud-Lopez et al. 2008), adrenal cells (Horvath et al. 2008; Tsai et al.2011), and pancreatic β cells (Dov et al.2008); however, little is known about the influence of PDE8s on CNS function.
Like GPCRs and adenyly cyclases, the diversity of PDE families and their distinctive distribution make them attractive therapeutic targets for numerous CNS and peripheral processes. The clinical success of PDE5 selective inhibitors has provided impetus towards the design of therapeutics targeting other PDE families (Levy et al. 2011). For example, antagonism of the PDE4 family has been tested for improving cognitive function (Barad et al.1998) and PDE10A inhibition has been targeted for the treatment of motor degenerative impairments associated with Huntington's disease (Giampa et al. 2010). Pde8b expression analysis in humans has demonstrated regionally distributed expression patterns, with the highest levels in the striatum and hippocampal formation (Lakics et al. 2010; Perez-Torres et al. 2003). Clinically, a mutation in the Pde8b gene has been implicated in autosomal-dominant striatal degeneration (ADSD) (Appenzeller et al. 2010) and higher levels of Pde8b transcript have been observed in the hippocampal dentate gyrus of Alzheimer's patients (Perez-Torres et al. 2003).
Although expression and linkage analysis suggest that PDE8B may play an important role in brain function, a thorough behavioral analysis of PDE8B function has not been demonstrated. To better understand PDE8B function in the CNS and to determine the viability of targeted therapeutics towards PDE8B for treatment of neurological disorders, we further investigated the anatomical distribution of Pde8b gene expression in the mouse brain, and through targeted gene inactivation investigated the behavioral consequences of loss of PDE8B function.
All animal usage and procedures were approved by the Institutional Animal Care and Use Committee of the University of Washington (Seattle, WA) in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Mice were housed in a 12 hour light-dark cycle and behaviors were performed during the light phase. PDE8B KO were generated as described (Tsai et al. 2011). Briefly, gene targeting was performed in ES cells (Cell Line E14; 129P2/OlaHsd; Deltagen, San Mateo, CA) derived from the 129/OlaHsd substrain and subsequently backcrossed to the C57BL/6 strain greater than fifteen generations. All mice for experimentation were obtained through a double Pde8b+/− cross. Male PDE8B KO mice between the ages of 4–12 months (unless otherwise noted) were used in all experiments, and an approximate equal number of age-matched, male wild type (WT) littermates were used as controls. All animals were handled before behavioral testing as follows: Initially, mice were handled in groups along with their cage mates for 10 min per day for 2 days. Subsequently, each mouse was handled individually for 5 min per day for 2 days. Finally, mice were housed individually, and handled twice a day, 5 min per session for 3 additional days. During the handling sessions, petting and rubbing, as well as "catch-and-release" were performed. For experiments in which mice were used in multiple assays, only one stressful paradigm was performed and was always proceeded by a non stressful procedure. All instances were mice were used in more than one assay are described within the specific methods section. All behavioral experiments were performed with the researchers blind to the genotypes.
Anesthetized animals were perfused with 0.2% heparin-PBS, followed by 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. After perfusion, brains were removed from the skull, and postfixed with the same 4% paraformaldehyde solution on ice (6 hr) then rinsed with 10% sucrose-PBS buffer, soaked in 20% (8 hr) and 30% sucrose (overnight) to remove paraformaldehyde and to cryoprotect the tissue. The tissues were then embedded in Tissue-Tek® OCT™ (Sakura Finetek U.S.A, Inc., Torrance, California, USA) then frozen and stored at −80 °C until cryostat sectioning at −20 °C. The 20 μm sagittal sections were mounted onto slides and placed in an X-gal staining mixture (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 5 mM EGTA, 20 mM K3Fe(CN)6, 20 mM K4Fe(CN)6•H2O, and 1 mg/mL X-gal in PBS, pH 7.4) at 37 °C overnight. After X-gal staining, slides were washed three times with PBS, pH 7.4, once with dH2O, then counterstained with eosin and mounted with Permount® (Thermo Fisher Scientific, Waltham, MA, USA).
The water maze was a circular feeding trough (79 cm in diameter) filled with 25°C water rendered opaque by the addition of nontoxic white paint. A rectangular escape platform (5 cm × 7.5 cm) was placed 1 cm beneath the water surface at a fixed location throughout the training period. During each training trial, mice were individually released into the maze at a random location near the edge of the pool, and allowed to swim freely until they found and climbed onto the escape platform and remained there for 30 sec. For each case in which an animal was unsuccessful in finding the platform after 90 sec, the animal was placed onto the escape platform and not retrieved until they remained there for 30 sec. The mice received 3 training trials per day with a minimal 30 min break between each trial. After all mice had sufficiently learned the task, determined by their ability to find the platform within 10 sec, a probe test was administered 24 hours after the last conditioning trial. For these experiments a total of n = 13 WT and n = 12 PDE8B KO mice were used. A portion of these mice (n = 7, WT and n = 6, KO) were utilized in a novel object recognition task one week prior to water maze conditioning. During the probe test, the submerged platform was removed and the movement of each individual animal was recorded by a video camera fixed directly above the pool. Video footage was analyzed using Ethovision software (Noldus Information Technology, Leesburg, VA, USA) to determine the number of platform crossings, time spent and frequency of entries into each quadrant. For the remote memory test, mice were trained as above, except that all mice received 4 days of conditioning. A total of 22 mice were used for this experiment (n = 10, WT and n = 12 KO). One PDE8B KO mice failed to escape onto the hidden platform within the allotted time on 10 of 12 trials and was removed from the experiment.
Contextual fear conditioning was conducted as previously described (Athos et al. 2002). First, the animals were transferred to behavioral testing room at least one week prior to the pre-handling sessions allowing the mice to acclimate. Contextual fear conditioning was performed in a plexiglass chamber with a shock grid floor and a 16″ × 16″ photobeam sensor grid (Truscan Mouse Chamber manufactured by Coulbourn Instruments, Allentown, PA, USA). During day one of the experiment, each mouse was placed in the conditioning chamber individually for 2 min to habituate, then a 0.7 mA foot shock (2 sec) has administrated through the grid floor. After conditioning, mice remained in the cage for an additional minute to facilitate the association of the shock with the conditioning chamber and were then returned to their home cage. 24 hours later, mice were returned to the same conditioning chamber to determine and the amount of freezing time during a 2 minute re-exposure. Freezing time was scored by hand (sampled every 5 sec) and by computer software based on infrared beam breaks; no difference was detected between the two methods so only computer generated scores are presented. For context-independent freezing following conditioning, mice were treated as above, except that foot shock was delivered in Med Associates ENV-300 chambers (Med Associates, St. Albans, VT, USA), a context distinct from the testing apparatus. For these experiments a total of n = 11 WT and n = 12 PDE8B KO mice were used for contextual conditioning and n = 10 WT and n = 12 PDE8B KO mice were used for context-independent testing. Mice used for context-independent conditioning were previously assayed in the Morris water maze remote memory paradigm one week prior.
Similar to contextual fear conditioning, delayed cued fear conditioning used an adverse stimulus (foot shock) paired with a 90–100dB audio tone. The tone was played during the last 30 sec of the 2 min habituation period followed by delivery of a 2 sec (0.7 mA) foot shock. As in the contextual fear conditioning, the mice remained in the cage for an additional minute to facilitate the pairing between the shock and the tone (as well as the context of the conditioning chamber). 24 hours post-training, freezing behavior was manually scored for 2 min in a novel context (a hexagonal plexiglass cage). For these experiments a total of n = 11 WT and n = 11 PDE8B KO mice were used. A portion of these mice, n = 6 WT and n = 6 PDE8B KO mice had been previously assayed in the Morris water maze, three weeks prior to fear conditioning.
In this test, naïve animals were placed one at a time into the same plexiglass conditioning chamber as used in contextual fear conditioning. A series of electrical shocks with increasing intensity (from 0.05 mA to 0.7 mA increased in 0.05 mA increments) were delivered to the mice and their reactions were observed. Mice were allowed to freely explore the chamber for 2 min, after which the first electrical shock was delivered through the floor grid for 2 sec. After a 2 min recovery period from the previous shock, a second higher intensity shock was delivered. We classified the general reactions of the mice into 4 categories - flinching, flinching and vocalization, jumping, and jump and vocalization. We evaluated the sensitivity of individual animals to a given current by their reactions. For these experiments a total of n = 3 WT and n = 5 PDE8B KO mice were used.
The elevated plus maze was conducted as described previously (Bruchas et al. 2009, Lister 1987). The apparatus was comprised of two open arms (25 × 8 × 12 cm2) and two closed arms (25 × 8 × 12 cm3) that extended from a common central area 50 cm above the ground (Med Associates, St. Albans, VT, USA). Mice naive to experimental testing were transported to a designated room (low light condition, ≈10–20 lux) one hour prior each experiment to acclimate. During each trial, a mouse was placed in the center of the maze, and allowed to freely explore for 6 min and movement was monitored by video camera. Time spent in the closed arms versus the open arms were scored using Ethovision software (Noldus Information Technology, Leesburg, VA, USA). Lighting was adjusted such that control animals spend approximately equal time in both the open and closed arms. For these experiments a total of n = 5 WT and n = 11 PDE8B KO mice were used.
The open field test was performed in a circular arena (49 cm in diameter), evenly illuminated by white overhead fluorescent lighting. The central area (≈17 cm in diameter) was designated as the center zone. Each mouse was placed in this area and left to freely explore for 10 min. We recorded the movement of each mouse using a video camera that was mounted directly above the circular arena. The video footage was analyzed using Ethovision software (Noldus Information Technology, Leesburg, VA, USA). For these experiments a total of n = 6 WT and n = 5 PDE8B KO experimentally naive mice were used.
Mice were placed on food restriction for 10–14 days until 85% of the original body weight reached and held steady for at least 3 days. All training and testing were conducted in operant conditioning chambers (ENV-307W, Med Associates, St. Albans, VT, USA). Prior to the start of conditioning, mice were given a 15 min magazine period in which one food pellet was given freely every minute or if either lever was pressed. Mice then underwent a 60 min instrumental conditioning session daily for 7 consecutive days. Two levers were presented during these conditioning session, and each lever press was reinforced with a 20 mg pellet (Bio-Serv, Frenchtown, NJ, USA) and followed by a 6 sec inter-trial interval (ITI) during which the room light turned off and both levers became inactive. The conditioning session ended either after 50 trials were completed or 60 min had elapsed, whichever came first. When animals were able to complete all 50 trials within the 60 min session, the magazine period was reduced to 5 min and eliminated on the next day. A total of n = 13 WT and n = 13 PDE8B KO mice were used. One cohort of mice (n = 7, WT and n = 6, KO) were previously utilized in a novel object recognition task and water maze conditioning task, the second cohort was used previously in the acoustic startle response paradigm described below.
Sound-attenuated startle chambers (SR-Lab, San Diego Instruments, San Diego, CA, USA) were used to measure startle responses and prepulse inhibition (PPI) as previously described (Zweifel et al. 2011). A 65 dB background white noise was maintained throughout the experiment. Following a 5 min habituation period, a series of escalating 40 ms acoustic pulses (null, 80, 90, 100, 105, 100, and 120 dB) were presented to the mice with an ITI of 30 sec. This series was presented ten times for a total of 70 trials. For these experiments a total of n = 6 WT and n = 6 PDE8B KO mice were used. For PPI, mice were given a 10 min habituation period after which they were presented with five 40 ms, 120 dB, pulse-alone trials. Mice were then presented with 50 trials of either a startle pulse-alone, prepulse presentation at either 5, 10 and 15 dB above background (70, 75, 80 dB, repectively), or a null trial in which there was no acoustic stimulus with an average ITI of 15 sec (range, 5–25 sec). A startle trial consisted of a 40 ms, 120 dB pulse of white noise. Prepulse trials consisted of a 20 ms duration prepulse of 70, 75, or 80 dB intensity, which preceded the 40 ms, 120-dB pulse by 100 ms. PPI was calculated for each prepulse level using the following formula: percentage of inhibition = [(average startle response on prepulse trial/average startle response on pulse–alone trial) × 100]. For these experiments a total of n = 8 WT and n = 6 PDE8B KO mice were used.
A rotating rod (4 cm in diameter) incrementally accelerated from 4–40 rpm over 2 min (Rotor-rod, San Diego Instruments, San Diego, CA, USA) was used to determine motor coordination. Mice were trained 3 sessions per day with ≈10 min between sessions. Rotarod performance was measured by latency to fall from or to clasp the rotating rod. For these experiments a total of n = 7 young adult (< 6 months in age) WT, n = 7 young adult, age-matched PDE8B KO mice, and n = 4 aged adult (>2 years in age) WT and n = 7 age-matched PDE8B KO mice were used.
Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). Repeated measures analysis of variance (ANOVA) was used for multivariate analysis. Student's t-tests were performed using two-tailed distribution.
To investigate the distribution of either Pde8a or Pde8b gene expression in the mouse brain, we utilized mouse lines in which Pde8a or Pde8b are inactivated by replacement of either exon 17 (Pde8a) or exons 14 and 15 (Pde8b) with a lacZ cDNA (Figure 1a and b) (Tsai et al. 2011; Vasta et al. 2006). Expression of the exogenous nls-lacZ allows visualization of β-galactosidase (β-gal) activity under the control of the endogenous Pde8a or Pde8b promoter (Tsai et al. 2011; Vasta et al. 2006). Analysis of β-gal activity on sagittal brain sections from PDE8A KO mice revealed that β-gal expression is largely undetectable in the brains of these mice (Figure 1c). In contrast to PDE8A KO mice, we observed β-gal activity in PDE8B KO mice, with the highest levels of activity in the dentate gyrus and CA1 regions of the hippocampus, the ventral striatum, and the Purkinje cell layer of the cerebellum (Figure 1d). These results consistent with observed in situ hybridization analysis reported by the Allen Institute for Brain Science (http://mouse.brain-map.org).
We have previously demonstrated that replacement of exons 14 and 15 of Pde8b, which encodes the catalytic domain of PDE8B, with a cDNA encoding lacz is sufficient to disrupt functional PDE8B activity (Tsai et al. 2011). To determine the behavioral significance of Pde8b expression in the brain, we analyzed PDE8B KO mice and WT littermates in a suite of behavioral tasks. We observed that numerous basic behaviors are unaltered by Pde8b inactivation (Table 1). However, we did observe selective enhancement of context-dependent fear, spatial memory, performance in an instrumental conditioning task, and motor coordination (summarized below) indicating that some hippocampal-, striatal-, and cerebellar-dependent behaviors are enhanced following Pde8b inactivation. These findings are consistent with several of the well-known functions of cAMP signaling in these brain regions.
Pde8b appears most highly expressed in the dentate granule cell layer and CA1 pyramidal cell layers of the hippocampus (figure 1b). These hippocampal subdivisions have been demonstrated to be critical in contextual memory recall and spatial pattern separation (Bourtchuladze et al. 1994; Rolls & Kesner 2006). Therefore, we assessed whether Pde8B inactivation would alter contextual fear or spatial memory using either contextual fear conditioning paradigm or Morris water maze.
To determine if genetic inactivation of Pde8b alters contextual fear conditioning, we assayed freezing behavior in WT control and PDE8B KO mice, as described (Athos et al. 2002). Briefly, mice were conditioned with a single foot shock (0.7 mA, 2 sec) in a novel context and assayed for context-induced freezing 24 hours later in the same context. PDE8B KO mice exhibited an increase in freezing time compared to WT littermate controls (Figure 2A; t-test, t21=2.24, p<0.05). Increased freezing behavior was not due to altered sensitivity to foot shock (Figure 2b), nor to mobility (data not shown). To determine whether enhanced freezing in PDE8B KO mice is context specific, we monitored freezing behavior in separate cohorts of control and PDE8B KO mice that were conditioned in a context distinct from the testing apparatus. There was no difference between the two groups (WT, 17.58 ± 1.68 vs. KO, 21.61 ± 3.59; t-test, t20=0.93, p=0.18).
To address the specificity of the enhancement in contextual fear conditioning in PDE8B KO mice, we utilized a delayed cue fear-conditioning task which is less dependent on the hippocampus and more dependent upon the amygdala (Phillips & LeDoux 1992). Mice were conditioned with a single pairing of a 30 second auditory cue (90–100 dB) that co-terminated with foot shock (0.7 mA, 2 sec). Cue-evoked freezing was assessed in a context independent of the conditioning chamber 24 hours later. Learning in this task was indistinguishable between PDE8B KO and WT control mice (Figure 2c; t-test, t20=0.37, p=0.36).
Next, we investigated whether functional PDE8B activity might be important in spatial memory using a Morris water maze. Mice were given three training trials per day for 2–3 days to learn the location of a hidden platform. Mice were tested 24 hours following the last conditioning day with the hidden platform removed to assess memory. During training there was no difference between PDE8B KO and control mice in escape latencies, or swim speed (Figure 2d and data not shown; acquisition, two-way repeated measures ANOVA: genotype effect, F1,23=1.27, p=0.27; effect of trial, F5,115=18.31, p<0.001).
During the probe test, PDE8B KO mice made significantly more entries into the target area (NE quadrant) than controls (Figure 2e; two-way repeated measures ANOVA, genotype X quadrant interaction, F3,69=3.28, p<0.05). Increased frequency of entry into the target area was associated with a trend towards increased time spent in the target quadrant; though this measure did not reach statistical significance (Figure 2f; two-way repeated measures ANOVA: genotype X quadrant interaction, F3,69=1.52, p=0.22). No differences in the latency to first cross (WT, 22.93 ± 6.59 vs. KO, 19.44 ± 4.42, t-test: t23=0.44, p=0.67) or frequency of crossings (WT, 3.92 ± 0.81 vs. KO, 5.08 ± 0.43, t-test: t23=1.29, p=0.10) of previous platform location were observed.
We next performed a remote memory test on cohorts of mice distinct from those described above. Mice were trained to find the hidden platform as above, except all mice received 4 days of conditioning. Analysis of acquisition revealed a significant effect of trial (Figure 2g; two-way repeated measures ANOVA: trial, F11,198=22.01, p<0.001), no genotype effect was detected (two-way repeated measures ANOVA: trial, F1,18=2.29, p=0.14). Similar to the probe test performed 1 day following conditioning described above, a probe test 10 days following conditioning revealed a significant genotype x quadrant interaction on the frequency of quadrant entries (Figure 2h, two-way repeated measures ANOVA: genotype x quadrant, F3,57=4.22, p<0.01). Again, this increased frequency was associated with a trend towards increased time spent in the target quadrant by PDE8B KO mice (Figure 2i; two-way repeated measures ANOVA: genotype X quadrant interaction, F3,57=2.5, p<0.07). No differences in the latency to first cross (WT, 68.20 ± 17.45 vs. KO, 42.39 ± 11.07, t-test: t19=1.27, p=0.10) or frequency of crossings (WT, 4.30 ± 1.42 vs. KO, 7.27 ± 1.76, t-test: t19=1.30, p=0.10) of the previous platform location were observed.
Both PDE8B KO and control mice appeared to take longer to first cross the previous platform location in the remote memory test compared to the 1 day probe test (WT 1 day, 22.93 ± 6.59 vs. WT remote, 68.20 ± 17.45; KO 1 day, 19.44 ± 4.42 vs. KO remote, 42.39 ± 11.07). Statistical analysis across all groups revealed a significant difference in control mice during the remote memory test compared to the 1 day test, this was not significant in PDE8B KO mice (one-way ANOVA: F3 =4.62, p<0.01; Bonferroni's Multiple Comparisons test, WT remote vs. WT 1 day, p<0.05 and WT remote vs. KO 1 day, p<0.01).
Analysis of β-gal activity in PDE8B KO mice demonstrated a high level of enrichment of Pde8b expression in the ventral striatum. Because PDEs are important for hydrolysis of cAMP generated in response to dopamine D1 receptors in the striatum and this signaling is important for reward learning and motivated behavior, we hypothesized that appetitive instrumental learning, motivation, or both might be altered in PDE8B KO mice. First, we tested PDE8B KO and WT control mice in a fixed ratio (FR) schedule in which a single lever press resulted in delivery of a reward pellet (FR1). Two levers were activated at the start of each trial signaled by the illumination of the chamber house light. Following a successful trial the house light was turned off, the pellet was dispensed, and the levers where inactivated; six seconds later the house lights were re-illuminated and the levers were re-activated. Both active and inactive lever presses where recorded over 50 trials or for 1 hour, whichever came first. Total lever presses per day did not differ significantly between groups (Figure 3a; two-way repeated measures ANOVA: genotype X day interaction, F6,144=0.74, p=0.62); however, there was a trend towards an overall effect of genotype (two-way repeated measures ANOVA: genotype F1,167=3.62, p=0.059).
To determine the precision of lever pressing behavior we scored non-rewarded lever presses across days. Analysis of non-rewarded lever presses revealed an overall genotype effect (Figure 3b; two-way repeated measures ANOVA: genotype, F1,167=5.55, p=0.02), with PDE8B KO mice making more non-rewarded presses, particularly during the first days of training; however, no significant genotype X day interaction was detected (two-way repeated measures ANOVA: genotype X day interaction, F6,144=2.01, p=0.06).
Following the fourth day of training all mice were completing 50 lever presses within the 1 hour time window; moreover, total lever presses and total non-rewarded lever presses were equivalent indicating both groups were equally precise in their performance (Figure 3a and B, days 5–7). Intriguingly, PDE8B KO were faster at completing the task at this later stage compared to control mice (Figure 3c, days 5–7). To elucidate further the extent to which PDE8B KO mice are performing better than control mice we analyzed cumulative reward acquisition on days 5–7. PDE8B KO mice performed significantly better than controls on all three days (Figure 3d-f; two-way repeated measures ANOVA: day 5, genotype X time, F29,725=3.47, p<0.0001; day 6, genotype X time, F29,725=8.86, p<0.0001; day 7, genotype X time, F29,725=5.22, p<0.0001).
To assess whether increased performance in instrumental conditioning by the PDE8B KO mice reflects an enhancement of motivated behavior, we tested PDE8B KO and control mice in a progressive ratio task. Break point analysis, as assessed by the total lever processes in the last trial completed, did not differ between PDE8B KO and control mice (Figure 3g). Finally, to determine whether differences in performance between PDE8B KO and WT mice are due to differences in instrumental learning, we performed a two-lever discrimination task. Mice were given block-training sessions for 6 days to associate a cue light above one of two levers with the active lever. Block trials of 10 light-lever pairings were given on each lever. On day seven mice were assayed for two-lever discrimination based on their ability to discriminate which of the two levers were active; active levers were assigned pseudo randomly. No difference was observed between control and PDE8B KO mice, with both groups performing at or above 80% accuracy (Figure 3h).
Expression of Pde8b in the cerebellum, together with previous observations linking Pde8b to ADSD, a progressive motor degenerative disorder, suggests that Pde8b inactivation may be associated with altered motor coordination (Appenzeller et al. 2010; Perez-Torres et al. 2003). To investigate this further, we monitored latency to fall from a rotating rod using an escalating rotarod task in both young (Figure 4a) and aged (Figure 4b) adult mice. Analysis of young adult mice revealed a significant effect of training (two-way repeated measures ANOVA: trial, F8,96=8.00, p<0.001), but not genotype (two-way repeated measures ANOVA: genotype, F1,12=4.16, p=0.06). In contrast, Pde8b inactivation had a significantly increased latency to fall in aged adult mice (two-way repeated measures ANOVA: genotype, F1,8=17.91, p=0.003). Analysis of average latency to fall across all groups revealed a significant decrease in performance in aged WT mice compared to young adult mice that was not observed in PDE8B KO mice (Figure 4c, ANOVA, F3 = 37.42, followed by Bonferroni's Multiple Comparisons Test: WT vs WT aged p<0.01). Additionally, this analysis revealed a significant difference in young adults between WT and PDEB KO mice (Bonferroni's Multiple Comparisons Test: WT vs PDE8B KO p<0.05).
Improved memory in contextual fear conditioning and a spatial learning task, along with improved performance in instrumental conditioning and motor coordination suggests that suppression of PDE8B activity overall improves cognitive and motor abilities. Given that alterations in the accumulation of cAMP are linked to numerous psychiatric disorders and the desire to identify PDEs for targeted therapeutic treatment of mood disorders (Xu et al., 2011), we asked whether genetic inactivation of Pde8b is associated with changes in affective state. To address this we monitored anxiety-like behavior using two standard laboratory methods, open field and elevated plus maze. Due to the difficulty in detecting increased anxiety levels in these tests under normal lighting conditions, we utilized low-light testing conditions which result in significantly higher percentages of time spent in the center of the open field arena and the open arms of the elevated plus maze (Bruchas et al. 2009). PDE8B KO mice spent significantly less time in the center of the arena than WT controls (Figure 5a; t-test, t9=2.41, p<0.05). Similarly, PDE8B KO mice spent significantly more time in the closed arms of elevated plus maze than the open arms (Figure 5b), contrary to what was observed in WT control mice (PDE8B KO; closed vs. open arms, t-test, t8=4.24, p<0.01)).
Alterations in cAMP levels are associated with perturbations of sensory motor gating, such as those measured by prepulse inhibition (PPI) of the acoustic startle reflex, which is a frequent endophenotype of psychiatric disorders such as schizophrenia (Clapcote et al. 2007). To determine whether Pde8b inactivation alters PPI, we first monitored acoustic startle responses (ASR) across multiple startle pulse intensities to determine whether baseline differences in ASR exist between PDE8B KO and WT control mice. ASR curves between the two groups were indistinguishable (Figure 5c, two-way repeated measures ANOVA: genotype effect, F1,10=0.03, p=0.86). To monitor PPI, we measured acoustic startle at 120 dB in the presence or absence of 70, 75, and 80 dB prepulses. No significant differences in PPI between PDE8B KO and WT control mice were detected (Figure 5d, two-way repeated measures ANOVA: genotype effect, F1,12=0.02, p=0.89).
The present study confirms Pde8b expression in the brain and illustrates a regionally restricted enrichment. Consistent with the expression pattern of Pde8b, behavioral analysis of mice with genetic inactivation of both Pde8b alleles demonstrates: 1) enhanced contextual fear conditioning; 2) improved spatial memory; 3) increased performance during instrumental appetitive conditioning; 4) attenuation of age-induced motor coordination decline; and 5) increased basal anxiety levels.
Previous analysis of Pde8b in humans has demonstrated regionally restricted patterns of expression, with the highest levels in the striatum, hippocampal CA1 and dentate gyrus, cortex, and cerebellum (Lakics et al. 2010; Perez-Torres et al. 2003). Our findings utilizing a lacZ reporter assay from brain sections of PDE8B KO confirm a similar pattern of expression in mice and are consistent with in situ analysis of Pde8b expression performed by the Allen Brain Institute for Brain Science (http://mouse.brain-map.org).
Hippocampal-dependent learning and memory, such as contextual fear and spatial pattern recognition, are known to be regulated by cAMP-dependent processes (Poser & Storm 2001; Sweatt 2001). We observed high levels of expression of the Pde8b within two major subdivisions of the hippocampus, CA1 and dentate gyrus that are regions critical for memory recall and spatial pattern recognition (Rolls & Kesner 2006). Consistent with the importance of cAMP in hippocampal function and the high level of Pde8b expression in these brain regions, we observed that the PDE8B KO mice had significantly enhanced contextual fear and spatial memory. Though these findings are consistent with cAMP signaling in the hippocampus, similar signaling pathways in the ventral striatum are also known to play a role in spatial learning and memory (Ferretti et al. 2010); thus, the contribution of inactivation of Pde8b in the ventral striatum to enhanced spatial memory reported here cannot be excluded. Similar to the effects of Pde8b inactivation of reported here, antagonism of PDE4 and analysis of PDE4D KO mice has have also demonstrated improved performance in several learning paradigms (Li et al. 2011), as well enhance early-phase long-term potentiation (Rutten et al. 2008). We have previously demonstrated that PDE8B can act synergistically with PDE4 in other tissues (Tsai et al. 2011); thus, it will be interesting to determine if a similar synergy might exist in the hippocampus.
Similar to cAMP signaling in the hippocampus, striatal regulation of cAMP levels downstream of dopamine receptor activation is critical for numerous aspects of motor function, reward learning, and motivated behavior (Nishi et al. 2008). We find that Pde8b is expressed in the striatum as previously reported; however unlike in the human studies (Perez-Torres et al. 2003), we find that expression of Pde8b in mice is higher in the ventral striatum compared to the dorsal striatum. Increased performance by PDE8B KO mice in a simple instrumental conditioning task are opposite those reported for PDE10A KO mice (Piccart et al. 2011), suggesting that PDE10A and PDE8B may be regulating distinct neuronal populations within the striatum that serve opposing roles, such as the direct and indirect pathway. Interestingly, we find that although the overall performance of PDE8B KO mice is faster than that of WT mice in completing the simple instrumental conditioning task, these mice made more errors (non-rewarded lever presses) during the first days of training. This finding indicates, at least early on in conditioning, faster performance comes at the cost of diminished accuracy. Nonetheless, with further conditioning PDE8B KO mice made as few errors as control mice, yet still completed the task more quickly. Finally, although our data suggests that Pde8b in mice is more highly expressed in the ventral as opposed to the dorsal striatum, they do not discount the known importance of the dorsal striatum in performance and learning of simple instrumental responding (Shiflett & Balleine 2011).
A frameshift mutation in Pde8 that results in loss of PDE8B function has been genetically linked to the autosomal dominant striatal degenerative disorder, ADSD (Appenzeller et al. 2010). Although we find that Pde8b is expressed in the striatum, loss of PDE8B function in mice does not impair motor performance, even in aged animals. In fact, we find the opposite result. Inactivation of Pde8b not only improved motor performance in young adult animals, it was protective against age-induced decline in motor coordination. Consistent with our rotarod findings we observed that PDE8B KO mice were slightly or even significantly better in tasks that required motor coordination; as evidenced by reduced latencies to find the platform in the Morris water maze and reduced latency to complete the instrumental conditioning task. The reason for these apparently disparate results is not clear; however, one possibility is that we find the highest levels of Pde8 expression in the ventral striatum and cerebellum in mice, which contrasts with the expression pattern in humans where Pde8b appears highest in the dorsal striatum (Appenzeller et al. 2010; Lakics et al. 2010; Perez-Torres et al. 2003).
The potential therapeutic value of developing PDE8B elective inhibitors for treatment of hypothyroidism, Cushing's disease, testosterone deficiency, or cognitive and motor impairments is tempered somewhat by our observation that loss of PDE8B function significantly increases anxiety-like behavior. In addition to our findings, others have demonstrated that increasing cAMP levels either by introducing a constitutively active Gαs to the striatum (Favilla et al. 2008), by treating animals chronically with a PDE4 inhibitor, rolipram, (Heaslip & Evans 1995; Imaizumi et al.1994; Li et al. 2009; Silvestre et al. 1999), or by genetically inactivating PDE10A is sufficient to trigger anxiety-like behaviors. Given the prevalent nature of findings relating to the anxiogenic properties of attenuating PDE activity, the therapeutic value of PDE antagonism must be weighed against potential alterations in affective state. Due to the robust improvements observed in motor coordination and cognitive function, PDE antagonism coupled with anxiolytic therapies may be a strategy that could circumvent this potential confounding side effect.
We would like to thank members of the Zweifel, Beavo, and Storm laboratories and Dr. Ali D. Guler for helpful discussion on this manuscript. This work was supported by grant from the National Institutes of Health. P30MH089887 (LSZ, LLT, SNN, and GLJ). R01GM083926 and R01AR056221 (JAB, LLT, SNN, and MSA). RO1MH073601 (DRS and GCKC).
The authors have no conflicts of interest to report.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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HELSINKI—When William Nylander went down with a suspected concussion in the tournament opener, it was left to the rest of Team Sweden to try to replace the player many believe to be the best at the world junior hockey championship.
And who stepped up? None other than William's younger brother and Toronto condo roommate, Alex.
Alex Nylander scored the lone goal — on a nifty breakaway — in Sweden's 1-0 win over the United States (1-1) on Monday as Sweden (2-0) went atop the group playing at Helsinki Ice Hall.
"We're a really good team and we can go far in this tournament," said Alex Nylander, who plays for the Mississauga Steelheads of the OHL. "We work hard every day. We have good chemistry through the whole team."
With the goal, the younger Nylander — who is in his draft year — led Sweden in scoring with a goal and four assists for five points.
"I try not to think about that," said Nylander. "I just try to work hard every day, try to play my game. When you do that, I think the points will come."
William Nylander — the Maple Leafs top prospect — remains sidelined. He did not come to the rink to cheer on his teammates, spending another day of quiet at the hotel. The Swedes are bearing down without him.
"William is what you call a franchise player," said Sweden coach Rikard Gronborg. "He's a guy that attracts a lot attention from opponents. What happens now is other guys have to play more ice time."
Alex Nylander, playing with another Leaf prospect Dmytro Timashov and now centre, Andreas Englund, has stood out in his brother's absence.
"Of course I'm impressed," said Gronborg. "He's an excellent young player. He did what we need him to do: Score that goal.
"I'm impressed he's played as well as he has. Hats off."
As for William Nylander, the hope is he will return to the ice before the tournament is over.
"Right now I have a big red flag saying he's not playing," said Gronborg. "That's all I know. I talked to him, he looked fine, so far so good. He's feeling well and doing well. That makes me feel pretty good."
The tournament was supposed to be a chance for the Nylanders to play together. Alex hopes that still happens.
"He's feeling good, a lot better from the hit," said Alex Nylander. "I hope he can get back as soon as possible. Maybe for the (medal round) he'll be back."
Linus Soderstrom made 46 saves for the shutout as Sweden beat the U.S. at the world juniors for the first time since 1996, ending a 12-game losing streak.


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Muted Colors
Muted colors are those which lack vibrancy or saturation. They tend to lean toward the more subdued end of the spectrum, while still creating a chic look with their subtlety. It seems that the muted colors of 2017 are mellow, but with a little more pigment than the pastel-looking hues that were in last year. In fact, the colors are perhaps more dusky in nature which emits a rich and warm feel to a space. Let's take a look at how some of these serene and soothing colors can still manage to warm up a room.
Warm neutrals:
People have a tendency of finding new ways to bring elements from the outside into their homes with a sense of style. There is something about combining earthy colors into a space that is not only reminiscent of nature, but that creates a calm and collected atmosphere. A perfect example of these colors is taupe and a muted earthy green. Especially taupe or similar hues would be a great choice for upholstery on beds, sofas, chairs and even rugs! It's a color that will remain timeless, and therefore would be a great investment when it comes to furniture.
Dusky blues:
Among the other popular muted colors this year is dusky blue and in a variety of hues. It is slightly muted and quite sophisticated and reminiscent of the beach, but without falling into a kitschy category. Although this shade of blue is far from being bold, it would still be wise to use to accessorize your home. Opt for bedding,decorative pillows, throws, ottomans, etc. These pieces entail a much smaller investment.
Not just on the runway, but in home décor, shades of gray are the getting the attention they deserve. Grays in particular create a refined look in the home. Despite being a neutral tone, it is quite rich. Just like earthy tones, gray is equally a good choice for furniture pieces that you intend to keep for years to come. However, it has more of a modern feel to it.
What do you think about muted colors in your home? Is it something you would consider?
Content provided by: @tailbase
Green, Green & More Green
Green, Green & More Green!
From the runway straight to your home, green is making an entrance into 2017 and becoming one of the most prevalent colors yet. With endless tones and styles to choose from, green can easily be paired with any style preference. Unlike fashion, the world of home décor may hold onto a trend for quite some time, so no need to worry about green being pulled out from the home anytime soon. When green is incorporated into your furniture, it adds a touch of sophistication to your space, as it seems to restore new energy. Let's take a look at how Pantone's color of the year has made its way into your home.
Green seating:
The urban design of Ashley's "Gavelston" dining chair features beautiful textured green upholstery that will surely make a statement in your dining room! Along with the espresso wood finish, this dining chair embodies the beauty found in natural aesthetics. Without having to look after plants, bring the same effect of freshness and rejuvenation into your space.
Green Rugs:
Sink your feet into a gorgeous "Caci" green shag pile rug that will have you feeling comfortable and delighted. The choice of color, along with the style of the rug is enough to make quite the statement! If you're really feeling wild, then you may want to sport the crisp lime and cream "Kerry" area rugin a beautiful Moroccan trellis design. Without a shadow of a doubt, this rug rolls out refreshment and hits bullseye when it comes to being on trend.
Green Table Lamps:
Brighten up your room with the organic-inspired style of the "Sheaon" table lamp. The understated chic ceramic lamp features a rich green and a subtle-shaped base, topped with a textural linen-colored shade to capture the essence of elegance. There's no denying that it's timeless!
Including green into your home is definitely a wise choice! What are your thoughts on this color trend?
Content provided by: @tailbase
Jewel Tones
Jewel tones
This year has said its goodbyes to earthy beiges and welcomes pure drama with open arms. Beautiful deep jewel tones such as emerald green, sapphire blue, amethyst and some pastel colors have emerged to bring life and richness in your home. By layering the colors and textures, a once cold space will definitely transform into a warm one, with just a few design elements! Your jewel tones do not necessarily have to match. So long as they are of a similar visual weight, then they will be visually harmonious. Let's take a look at how we can incorporate jewel tones into your home, besides on your walls or window coverings.
Accent chair:
The "Clarinda" accent chair is the perfect blend between a classic slipper chair and a traditional wingback chair. This beautiful modern design features tapered legs and clean lines, along with a subtle tufting for added flair. While the deep seat and winged back offer comfort, this accent chair's gorgeous sapphire blue color will surely make a statement in your room.
Are you looking for a piece that can add a lot of drama in your space? Then why not consider a jewel-toned sofa? The rich vibrant color of the "Sagen-Teal" sofa will awaken a certain regal vibe in your living room. You will feel like royalty when you have a seat on the plush cushions and bold shape of this contemporary design.
Turn up the warmth in your space with a beautiful burgundy "Ashton" throw. The woven herringbone pattern, along with the thick fringe will add visual texture to a bed, sofa or chair! In fact, since the throw has so much personality, it would be best to pair it with shades of gray as a backdrop.
As you can see, vibrant colors are here to stay. Jewels tones are not only meant to be worn! Dress your home in the colors of royalty, so that you feel a certain distinction right within your own space.
Content provided by: @tailbase
Faux-Fur On Your Furniture
Faux-fur on your furniture
The winter is a season that brings out our primal instincts and leads us to preserve ourselves from the wretched bitter cold. So how exactly do we manage to do so in harsh weather conditions? We keep warm of course! One way of putting this into practice is by layering. In fact, the same concept is true for the home. You may find yourself pulling out extra rugs for layering, or your winter throw to keep you and your loved ones warm, while you spend time together in the family room. In 2017, these elements have made a comeback in the form of trendy ultra-soft and cozy furniture. Furry furniture is making its appearance either way, so why not embrace its fuzzy return?
The faux-fur throw:
One way that faux-fur has made its comeback is with the throw. The trend does not just imply neatly folding a fuzzy throw and placing it in a corner. Better yet, it serves as a tool to embellish your furniture! The beautiful texture will create a pop of aesthetic to your living room's sofa or chair that's reminiscent of nature's beauty. Cover up your seating with the throw and you'll have a new look in no time!
The faux-fur pouf:
Another way to give into your animal instinct is to add a luxurious faux-fur pouf to your home. Whether it is in your living room, bedroom or entrance, this piece will surely bring warmth to your space. Just see for yourself. Lay your feet on this fuzzy pouf and feel the incredibly soft and plush texture beneath your skin. This piece is truly a dream!
Nothing says warm like draping your home with cozy faux-fur. This can be done with faux-fur throws and poufs, which come in a variety of colors and textures. Not only does it capture wild beauty, but a jaw dropping feeling of luxury.
Content provided by: @tailbase


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Featured property
Featured property
Featured property
£825,000 Offers Over
5 Bedrooms Detached House for sale
West Coates, Castle Terrace, Berwick-upon-Tweed, Northumberland
Elegant victorian home on castle terrace set in 1.5 acres with swimming pool. west coates is a most impressive stone built victorian detached family home set within its own private…
£650,000 Guide Price
Seabank Development, The Crescent, Berwick-upon-Tweed, Northumberland
The seabank development site is located at the south end of main street in spittal, above the promenade. the development site extends to just under an acre (0.39 hectares) and has…
£635,000 Offers Over
4 Bedrooms Detached House for sale
High Letham Farmhouse, High Letham, Berwick-Upon-Tweed, Northumberland
An elegant georgian farmhouse with holiday cottage, paddock and fabulous sea and countryside views. (contd...)
£635,000 Guide Price
The Townhouse, Ravensdowne, Berwick-upon-Tweed, Northumberland
Coming soon march 2018. maden eco are a well-known, reputable local building company and are in the process of refurbishing a 4 bedroom georgian townhouse to exceptional modern days…
£450,000 Guide Price
4 Bedrooms Terraced House for sale
Ravensdowne, Berwick-upon-Tweed, Northumberland
This impressive 3 storey double fronted grade ii listed georgian town house with west facing garden within the heart of the most historic corner of berwick on ravensdowne, voted one…
£445,000 Offers Over
5 Bedrooms Detached House for sale
Mordington Holdings, Mordington, Scottish Borders, TD15 1XA
Fantastic family home in enviable rural location within a short drive of the a1 and berwick-upon-tweed
£425,000 Guide Price
3 Bedrooms Detached House for sale
The Coach House, Ravensdowne, Berwick-upon-Tweed, Northumberland
Coming soon march 2018. maden eco are a well-known, reputable local building company and are in the process of building a brand new 3 bedroom coach house with private parking and garden…
£395,000 Offers in Excess of
5 Bedrooms Detached House for sale
Kirkbank, Paxton, Berwickshire, Scottish Borders
Kirkbank is a spacious period detached stone built five bedroom house located in the charming village of paxton and benefits from spectacular southerly views over the tweed valley…
£375,000 Guide Price
Mirandas Guest House, Church Street, Berwick-upon-Tweed, Northumberland
Mirandas guest house is a fantastic business oppertunity in the heart of historic berwick-upon-tweed. (contd...)
3 Bedrooms Detached House for sale
Wallace Green Hall, Berwick Upon Tweed, Northumberland TD15…
A delightful 3-bedroom, 4-bathroom grand, terraced manor house situated in the charming town of berwick-upon-tweed. this spacious property is recently refurbished and is only 0.5 miles…
4 Bedrooms Detached House for sale
31 Lamberton Shiels, Berwickshire, Scottish Borders
A substantial and impressive four bedroom detached property occupying an elevated position, standing in large mature gardens and enjoying superb country and coastline views,yankari…
£325,000 Guide Price
3 Bedrooms Equestrian Facility Character Property for sale
Lamberton Holdings, Lamberton, Berwickshire
Detached country house with 5 acres, outbuildings and views.this fantastic detached country property is a spacious 3 bedroom family home which has been fully renovated and now offers…
£325,000 Guide Price
3 Bedrooms Semi Detached House for sale
Grange Loaning, Castle Terrace, Berwick Upon Tweed, Northumberland
2 grange loaning is an attractive semi-detached 3 bedroom family home in immaculate condition with off-street parking, private garden all within walking distance of berwick town centre…
£325,000 Guide Price
5 Bedrooms Detached House for sale
Cantys Brigg, Cantys Bridge, Berwick-upon-Tweed, Northumberland
New to the market.period detached home with private riverbank, fishing rights outbuilding.canty's brig is a large period detached family home positioned on the banks of the whiteadder…
4 Bedrooms Semi Detached House for sale
Bridge Street, Berwick-upon-Tweed, Northumberland, TD15 1AQ
This is a rare opportunity to buy a family home and versatile business premises on one of berwicks best-loved streets. the house offers bright and spacious accommodation with 3-4 bedrooms…
Marketed by:
McEwan Fraser Legal Solicitors & Estate Agents, Edinburgh
Claremont House
130 East Claremont Street
Tel: See phone number 0131 524 9797
Website: Go to Agent Website


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Food Safety 2014 is going to start on 06 Feb and finish on 17 Dec 2014.
Food Safety 2014 will be in Maryland, Maryland USA but the venue of the event is not determined yet.
Food Safety 2014, Food Safety Risk Analysis Program will cover aspects like Health, Education, Risk Analysis, Life and Food Risk.
Food Safety Risk Analysis Program is twice a year(6) Workshop.
Travel Guide
Other Events in Washington, D.C.
37th Annual Convention & Exhibition of the American Fire Sprinkler Association
Washington, D.C., WA, USA
30 September - 03 October 2018
American Association of Teachers of Slavic and East European Languages National Conference
Washington, D.C., WA, USA
01 - 04 February 2018
64th Annual Meeting & Exhibition of the American College of Nurse Midwives
Washington, D.C., WA, USA
17 - 21 May 2019
13th Annual Meeting of Pediatric Cardiac Intensive Care Society
Washington, D.C., WA, USA
06 - 08 December 2017
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President Trump ended the week with new threats against North Korea and now, Venezuela. Jonathan Alter, Ned Price, and Betsy Woodruff join Joy Reid to discuss.
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Donald Trump Surprises By 'Not Ruling Out' Military Option In Venezuela | The Last Word | MSNBC
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Amanda, member
"As a mother of 3, I always strive to be fit not only for myself, but for my kids. Since joining EDX, I've noticed rapid improvement in my physical fitness and also my mental well-being. I'm so thankful to be a part of such a wonderful community! The owners have made this such a great place & for that I can't say thank you enough!"
-Amanda, EDX member


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Our collection of yellow colored 100% wool woolens. Note our wools collection is ever changing.
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- Plan to meet internal and external customer requirements
- Ensure the delivery of quality products and services
- Monitor, adjust and review customer service
We are here for you!
The Dolph team is a proud group of like-minded individuals who care about our students' needs. We are passionate about teaching, and helping you create a better tomorrow for yourself, today.


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Fire'd Up is a casual-dining restaurant serving up creativity through its innovative menu items. It offers a unique environment and provides a range of inspired dishes, from oven-baked pizzas, to creative salads, pastas, entrees, soups and sandwiches. This website allows users to experience all that Fire'd Up Pizzaria has to offer.
I have created the website and logo design. The website includes five pages with multiple images and creative typography.The logo reflects the nature of the restaurant with vibrant warm colors. An animated logo was also created to be used in digital advertisements.
Tools: HTML / CSS
Date: August, 2013


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Usually when people around nonprofits talk about how important it is to recognize contributions of time, the Nonprofiteer smells a rat. "But I give my time!" is often the excuse made by nonprofit Board members for their failure to give or raise money, to which her response is, "You can't pay the light bill with time. You can't pay your staff with time. There are all kinds of volunteers at nonprofit agencies, but Board members are the volunteers who get to govern the group because they pay for it."
However, this piece from the indispensable Nonprofit Quarterly is actually making a different, and far more legitimate, point: that there are plenty of capable people out there who may be interested in adding value to your nonprofit with their time and their skills, and that nonprofits unprepared to make use of these people are throwing money down the drain. When the Nonprofiteer isn't being cranky about Board responsibilities, she's a great booster of high-skills volunteers--people who, without seeking a governance position, wish to help the agency redesign its Website or rewrite its fundraising appeals or rethink its fundraising events so they're fun AND newsworthy AND profitable.
If you don't have a wish list of "projects we'd get done if someone with the right skills came along," write one. That way, when someone frankly tells you she wants to give her time but not her money, you'll have something to offer other than, "Join the Board and we'll make an exception for you."
Type your email address in the box and click the "create subscription" button. My list is completely spam free, and you can opt out at any time.
Filed under: charity, Fundraising, nonprofit, nonprofit Board, not for profit, personnel, volunteer
Tags: 501c3, charity, fundraising, NFP, nonprofit, nonprofit Board, not for profit, personnel, volunteer


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2005 Chevrolet Aveo - Consumer Review
Build Quality
Reliability Value
Build Quality
Reliability Value
My Kalos!
By limee on
Vehicle2005 Chevrolet Aveo LS 4dr Hatchback (1.6L 4cyl 5M)
ReviewGot a great deal on this little car which is one I had been researching for a while now. It was the Aveo or the Scion Xa. The Aveo won out on price. Have owned a number of GMs in the past with lousy build quality and reliability. This is a Daewoo and appears to be well put together with tight seems and no sqeaks/ rattles, etc. It is a blast to drive, especially in the city where it excels. It's amazingly quiet at speed after it has been wound up, and cruises comfortably at 80 with minimal fuss. Not that impressed with the fuel economy at 27 MPG (I get that from my V6 Honda Accord and feel the push in the back of the seat!) It makes me feel like I'm living in London again. The size is ideal.
Best FeaturesIdeal city size. No problems getting a parking space. Suspension is soft and comfortable and compliant. Italian design
Worst FeaturesBetter quality sound system. Center arm rest. Cruise control, better fuel economy
Recommend (0) (0)
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text: Is it critical that I get a 1080p HDTV? I am currenty researching TV's and Ive heard that this is the wave of the future. I need a 60" hdtv for the new house which will be finished the end of July. Should I spring for the extra bucks for a sep 1080p input(ala Sony XBR) or is still ok to go with a 720? How much of a difference is there between them?Thank you for all your respones. By the way my budget is $2500--any suggestions for a 60 HDTV??