text:
Give us a call to get our secret deals 1-800-468-3578
Property Location
When you stay at LakeSide House in Keswick, you'll be in a national park and minutes from Hope Park and Keswick Mining Museum. This 4-star bed & breakfast is within close proximity of Keswick Museum and Art Gallery and Cumberland Pencil Museum.
Stay in one of 14 guestrooms featuring flat-screen televisions. Complimentary wireless Internet access keeps you connected, and digital programming is available for your entertainment. Private bathrooms with showers feature complimentary toiletries and hair dryers. Conveniences include coffee/tea makers and complimentary bottled water.
Take advantage of recreation opportunities such as a fitness center, or other amenities including complimentary wireless Internet access and tour/ticket assistance.
A complimentary full breakfast is included.
Business, Other Amenities
The front desk is staffed during limited hours.


text:
Napkins Maria Salmon Red - set of 4
$ 39.00
Maria Salmon Red
Napkins in happy colors and exotic prints are an easy way to update your table. Each napkin is hand block-printed and sewn in Jaipur, India on 100% cotton. Machine wash in warm water, tumble dry on low heat. They really do wash up beautifully 20"x20".
Set of 4 $39.00

subset: commoncrawl


text:
Do any of you think this girl is attractjve? Yes or no and on a 1 to 10 what would you rate her?? Don't worry, I don't know her personally so if you vote her ugly you won't be hurting my feelings nor hers.. I'm just having a debate with some friends and I need some other opinions 😉
- She's aightVote A
- Nah, not my type, I could do betterVote B
- DAMN I WISH SHE WAS MY GIRLFRIEND!Vote C
- Sigh bro your eyes must be broken, she ugly lolVote D


text:
Adrian Christy
Last quote by Adrian Christy
I've made 33 redundancies in total, we've gone from 24 athletes to 12, five coaches to three and no sports science - we're buying that in piecemeal.feedback
Jun 29 2017
The latest quote from Adrian Christy is: "Our development pathway is eight to 12 years, so if we lose the Tokyo cycle, we've lost 2024, too, and will have to rebuild.". It comes from the Call for UK sport to avoid two-tier funding approach article. You'll find on this page 2 articles with Adrian Christy quoted on topics such as incomprehensible and decision. Adrian Christy has been quoted 3 times in 2 articles.
Take our quote verification challenge and find out !
Quotes by Adrian Christy
Jun 29 2017
Our development pathway is eight to 12 years, so if we lose the Tokyo cycle, we've lost 2024, too, and will have to rebuild.feedback
Dec 09 2016
We are staggered by this decision, it is incomprehensible.feedback
Previous : Adrian Charles Terry
Next : Adrian Clark>
Quote Verifier
Check if the quote you read on social networks is authentic
This webpage has been created by a robot: errors and absent quotes cannot be totally avoided


text:
Top Flight offers baseball specific speed, explosion, and agility training for middle and high school athletes.
Our baseball sports performance enhancement camps develop players' throwing velocity, hitting power and exit velocity, speed (30 m fly--home to first distance), as well as upper and lower body explosiveness. We use Zepp sensors to measure hitting metrics and Freelap timing sensors for speed metrics.
0.2 s in the 60 yard dash is worth $10,000 or more in scholarship money. College programs are looking for five tool players; how fast is your fast?
Basketball Fundamentals Camps
Grades 3 - 5 and 6 - 8
Session I: 5/3, 10, 17, and 24
Session II: 5/31, 6/7, 14, and 21
Improve your game at the basketball fundamentals camp. Participants will work on their dribbling, passing, and shooting skills. Basketball specific speed, explosion, and agility training will also be a part of the camp.
Cost is $60/session if paid prior to the camp date.


text:
Don't miss this Fun and Charming Cozy Mystery by USA Today Bestselling Author Lois Winston
Kirkus Reviews dubbed her critically acclaimed Anastasia Pollack Crafting Mystery series, "North Jersey's more mature answer to Stephanie Plum." In addition, Lois is an award-winning craft and needlework designer who often draws much of her source material for both her characters and plots from her experiences in the crafts industry.
Learn more about USA Today bestselling author Lois Winston and her bestselling books on her Amazon Author Page.
For Great Book Deals sent straight to your in-box, click HERE!
Don't forget to register to WIN our GIVEAWAY!
This month we're giving away $50 to spend on Amazon.com!
a Rafflecopter giveaway Disclosure of Material Connection: This site is managed by Carmen DeSousa. Some of the links in the post above are "affiliate links." This means if you click on the link and purchase the item, I will receive an affiliate commission. Regardless, I only recommend products or services I believe will add value to my readers. I am disclosing this in accordance with the Federal Trade Commission's 16 CFR, Part 255: "Guides Concerning the Use of Endorsements and Testimonials in Advertising."
Leave a Reply.
More Free and Bargain Bestsellers!
About Us!
Dear reader, we scour the Internet every day to bring you books that are less than $5 that also have a high number of reviews with an average of four stars or higher.
August 2017


text:
By Sade Knox, Intern, Woodruff Family Law Group
King v. Giannini-King, 784 S.E.2d 237 (N.C. Ct. App. 2016).
Facts: In October 2001, Plaintiff (father) and Defendant (mother) were married and then separated, about seven years later, in early June 2008. Two minor children were born to the parties' marriage. After the separation, Defendant relocated with the minor children. Subsequently, to Defendant's relocation, Plaintiff brought an action for divorce from bed and board, child custody, and equitable distribution. Defendant filed a motion to dismiss, a motion to strike, in addition to a counterclaim for custody, child support, alimony, etc.
The trial court initially granted both parties temporary joint legal and physical custody of the minor children. The trial court, nearly a year later, entered a custody order, granting Defendant permanent legal and physical custody of the minor children, and permitted Defendant's relocation of herself and the minor children to Wilmington, N.C.
Plaintiff was granted visitation rights and relocated to Southport, N.C., where the parties began to modify their custody schedule. Plaintiff later remarried and returned to Person County. Soon after, Plaintiff filed motions alleging a substantial change in circumstance, which resulted in the trial court transferring primary physical custody of the minor children to Plaintiff, temporarily, and allowed Plaintiff to relocate the minor children to Person County.
Defendant filed a motion asking for a new trial, or set aside the temporary order, shortly thereafter, which the trial court denied. Finally, in mid-December 2014, the court entered a permanent order that granted both parties joint legal and physical custody of the minor children. Defendant appealed arguing the trial court had erred in their custody orders.
Issue: Whether Plaintiff's argument concerning the temporary custody order was appropriate for appellate review. Was there enough evidence to support a substantial change in circumstance?
Answer to Issue: No. Yes. Continue reading →


text:
This is a shot of a Courtyard in Oxford University. Our tour guide was telling us a neat story about the clock in Oxford and how it was either behind or ahead of London's time for the trains if I recall correctly. It was pretty neat.
Tag: University
Oxford University
This is a shot of Oxford University in England. I had taken a 10 day trip there in April 2016 and I loved it. The city of London is such a nice place, with countless types of architecture. It is an amazing place. Also, many of the scenes found in Harry Potter are either based off of Oxford University or filmed in it. I stood in the courtyard with the giant willow tree. I can't remember what movie it was from.


text:
Roost, a real estate search engine, on Wednesday announced the launch of its service in Southeastern Michigan, extending its coverage to the Detroit metro area (Oakland, Macomb and Wayne counties), as well as Ann Arbor and rural areas spanning from Washtenaw County to the Thumb. Roost's search engine covers single-family, new construction and for-sale-by-owner properties. Roost also offers property listings information for homes in Atlanta, Austin, Baltimore, Boise, Boston, Chicago, Dallas, Denver, Fort Worth, Houston, Las Vegas, Miami, Minneapolis, Nashville, Orange County (Calif.), Orlando, Philadelphia, Phoenix, Portland, Sacramento, San Diego, St. Paul, Tampa, California's Silicon Valley, St. Louis and Washington, D.C., among other market areas. *** What's your opinion? Leave your comments below or send a letter to the editor. ...
Most Read
by Bernice Ross | Aug 14
by Teke Wiggin | Aug 22
by Amber Taufen | Aug 18
Show Comments Hide Comments


text:
--Seth Godin, author, Linchpin
"Entrepreneurial professionals must develop a competitive advantage by building valuable skills. This book offers advice based on research and reality--not meaningless platitudes-- on how to invest in yourself in order to stand out from the crowd. An important guide to starting up a remarkable career."
--Reid Hoffman, co-founder & chairman of LinkedIn and co-author of the bestselling The Start-Up of You: Adapt to the Future, Invest in Yourself, and Transform Your Career
"Do what you love and the money will follow' sounds like great advice -- until it's time to get a job and disillusionment quickly sets in. Cal Newport ably demonstrates how the quest for 'passion' can corrode job satisfaction. If all he accomplished with this book was to turn conventional wisdom on its head, that would be interesting enough. But he goes further -- offering advice and examples that will help you bypass the disillusionment and get right to work building skills that matter."
--Daniel H. Pink, bestselling author of Drive and A Whole New Mind
"This book changed my mind. It has moved me from 'find your passion, so that you can be useful' to 'be useful so that you can find your passion.' That is a big flip, but it's more honest, and that is why I am giving each of my three young adult children a copy of this unorthodox guide."
--Kevin Kelly, Senior Maverick, WIRED magazine
"First book in years I read twice, to make sure I got it. Brilliant counter-intuitive career insights. Powerful new ideas that have already changed the way I think of my own career, and the advice I give others."
--Derek Sivers, founder, CD Baby
"Written in an optimistic and accessible tone, with clear logic and no-nonsense advice, this work is useful reading for anyone new to the job market and striving to find a path or for those who have been struggling to find meaning in their current careers."
--Publishers Weekly
About the Author
Cal Newport, PhD, is a writer and an assistant professor of computer science at Georgetown University. He also runs the popular website Study Hacks: Decoding Patterns of Success.
Dave Mallow has worked extensively in animation, video games, and audiobook narration. Among his animation credits are the voice of Baboo in Mighty Morphin Power Rangers; Angemon, Gekkomon, and Uppamon in Digimon: Digital Monsters; and Akuma in Street Fighter.


text:
Freehold is an interesting read … BUT … you have to wade through the boring parts in the beginning quarter to half of the book before it really starts to grip you and the last quarter – well that part won't let you go!
Freehold centers around a woman named Kendra. At the beginning of the story, she's a supply sergeant in the UN military (the year is roughly 25XX. We're never given the specific date). When a friend warns her she's about to be arrested for selling military equipment out the back of the supply shed during her last mission (a crime she documented, not committed), she makes a run for the Freehold of Grainne, the one place in the known galaxy where the local government won't turn her over to the UN. A couple of times early on, I got lost in technobabble, unable to envision exactly what was going on. Occasionally I thought some of the activities could use a bit more explanation, especially when dealing with esoteric technology. The sex was a little more explicit that I would have liked, but thankfully not overly so.
Once she arrives on Grainne the story almost completely switches pace and in some ways travels back in time as it's a complete throwback to Heinlein and his libertarian viewpoints (take a read of books like "Moon is a Harsh Mistress" and "Alongside Night" in its detailed rendering of the economic realities of life to see what I mean). On Grainne, the idea of Welfare and government charity runs contrary to the very nature of the society. Grainne also has the highest standard of living of any human planet. In short, the UN welfare state doesn't dare allow the Grainne to go on existing because it throws a bright light on all that's wrong with the UN's socialist nanny state. I think to some extent this comparison by itself struck a real chord considering what's happening in some of the countries around the world and how they are being led – however, this is not a political blog, so I'll stop the preaching right here!
When the UN-led Earth decide that they can no longer let Grainne survive and exist, however – well that's where things get really interesting. The book takes a decidedly more militaristic bent after Kendra is indoctrinated into the Grainne armed forces and the eventual battles with the Earth forces remind me of nothing more than Tom Kratman and his Carrera Series. Another extremely violent and sexually charged series, however one in a very similar vein in my opinion.
Of course, the idea of the failing socialist state attacking the smaller but far more successful capitalist state has been done before in science fiction. It's one of the central themes of David Weber's wonderful Honor Harrington series. What makes Freehold different is how the story focuses on Kendra. Weber's Harrington novels, which have a similar political theme are high space opera. This is not. This is the story of one woman, torn from her home, exiled in a foreign land, forced to make a life there and eventually to defend her new home against her old.
Latest posts by Admin (see all)
- Mark Watches 'Voyager': S01E16 – Learning Curve - July 28, 2017
- Mark Watches 'Voyager': S01E15 – Jetrel - July 27, 2017
- Elon Musk wants to colonize Mars, no terraforming required - July 27, 2017
- How Android Wear 2.0 puts the Huawei Watch 2 ahead of the competition - July 18, 2017


text:
Just to confirm that this review is for the Schiphol airport branch of the national museum - as many reviews seem to be reviewing the national museum instead. Anyhow, the room - for that is what it is - is a very pleasant attraction with varying exhibitions - and is a good place to spend 15-20 minutes.
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.


text:
Aroma Treasures Lavender Pure Essential Oil
Rs. 147Rs. 14010ml+Braun Female Groomer FG1100
Rs. 1,895Rs. 1,514
Rs. 2,042 Rs. 1,654 19% off Slashed discount Rs. 388
1*Aroma Treasures Lavender Pure Essential Oil + 1*Braun Female Groomer FG1100
Buy these together for Rs. 1,654
Total Price: Rs. 2,04219% off
Product Discount: Rs. 388
Final Price : Rs. 1,654
Aroma Treasures Lavender Pure Essential Oil
Rs. 147Rs. 14010ml+
Rs. 2,842 Rs. 2,835
1*Aroma Treasures Lavender Pure Essential Oil + 1*Philips HP8647 Hair Straightener, Hair Dryer, Curler
Buy these together for Rs. 2,835
Total Price: Rs. 2,842
Product Discount: Rs. 7
Final Price : Rs. 2,835
- Description
Quick Overview
Physical Wellbeing : Lavender essential oil has a calming scent, which makes it an excellent tonic for the nerves and anxiety issues. Therefore, it can also be helpful in treating migraines, headaches, depression, nervous tension and emotional stress.
Lavender essential oil is also good for urinary disorders because of its stimulating effect on urine production. A regular massage with lavender oil can also provide relief from pain in the joints, backache and lumbago. Lavender oil is widely used for various respiratory problems including throat infections, flu, cough, cold, asthma, sinus congestion, bronchitis, whooping cough, laryngitis, and tonsillitis .
Beauty Applications : According to dermatologists and aromatherapists, lavender essential oil is one of the most beneficial oils in the treatment of acne, a condition that primarily affects young people as they move through puberty. Lavender essential oil is also useful for hair care and has proven very effective on lice, lice eggs, and nits .
Emotional Wellbeing : Lavender essential oil induces sleep, which has made it a common anti-depressant .
Key Features :
Essential oils are naturally extracted from plants to induce relaxation, increase energy and reduce stress. It seeks overall balance of mind, body and soul. Our essential oils are multipurpose in nature due to their antiseptic, antiviral and anti inflammatory qualities. Its healing properties assist in curing various disorders. Apart from its medicinal properties, it is also a great ingredient for emotional wellbeing, a definite anti depressant. Universally, these oils are also used for beautification .
How to use product
2-4 drops into 5 ml vegetable oil for massage. 6 - drops in bath tub/bucket. 6 - 8 drops on vapouriser with water. For external use only .
Additional Information: Aroma Treasures has a wide range of Aromatherapy products in the categories of essential oils, vegetable oils, and ready to use 100% natural oil based products, natural shampoos, skin care, spa range, vaporizers, and room freshening blends. They firmly believe that to sustain a consistent growth rate, it is imperative to improve and innovate products regularly.
Explore a wide range of Aromatherapy Oils here.
Expiry Date : Click Here August 2019
All our products are far from expiry, and procured directly from the brand/authorized resellers of the brand.
Reviews (0)
- Most Recent
- Like
- Rating
Filter By:
Sort By:
By Neha Gupta
I use it as an additional ingredient in my diy acv toner
Posted on 8/15/2017
By Shafeena
I liked the fragrance of Aroma Treasures lavender essential oil. The smell does stay for quite sometime. I mixed it with other oils I have and I got a good blend, the smell of lavender is at the top note.It does have a soothing effect slowly and gently.
Posted on 8/14/2017
By POOJA
Amazing product and amazing smell.
I am using Essential Oil for the first time, and this is amazing. I like the smell very much. It is very relaxing.
Posted on 8/13/2017
By Anagha Girme
it has very nice scent..affordable range..thank you!
Posted on 8/5/2017
By Vaishali singh
I bought it to mix a few drops of my DIY face serum. The smell is lovely. The best part of Aroma treasures is, their packaging is convenient and is of latest manufacturing date. So it can be stored for a long time. I am using this oil in my DIY body butter, my hair oil and also in my bath tub.
Posted on 8/4/2017
By Saloni Jain
The product is amazing in its work, has helped me to reduce acne problems.
Posted on 8/4/2017
By prema Latha
It has been the best thing to keep my toddler relaxed and happy and sleep well, it has helped me getting good relaxation in just few minutes after applying it, my migraine instantly got better, can feel a sense of relief in max 5-6 minutes and the aroma is enchanting and soothing.
Posted on 8/1/2017
By prema Latha
It is one of the best relaxing oil, i bought it for a my toddler for her massage-bath and to have a tantrum less sleep and it is effective, i also used to it- we can feel the difference in max 5-6mins i used it when i a terrible throbbing migraine and neck pain and i found an instant relax and also total reduction in migraine.
Posted on 8/1/2017
By priya
This is the first time m buying any essential oil..n aroma treasures lavender essential oil is just amazing..i love it's smell..its so calming...mainly bought for my insomnia but also using it for skin n hair. Awesome product..thank u nykaa!!
Posted on 7/31/2017
- Ask Nykaa (0)


text:
Listen to free music by Majority Rule
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station.
Majority Rule – A Prescription| play
Mar 23, 2011 at 9:29 pm via YouTube | AMAZON | ITUNES
rickyftw jammin' some "Majority Rule" in my headphones at work! 4 more listeners on blip and ill be at 50!!
Majority Rule – At 3 AM| play
hugel Nights like tonight remind me that I really would "turn it all in for late nights and impossible dreams."
heymeghann @a_ride Thanks!
hannagudm Þetta band er bara OF gott! Too bad að þeir eru hættir og maður geti ekki séð þá live.
DasKapital still hardcore
Artists Like Majority Rule
Blip.fm DJs who play Majority Rule
Friends: MerchantCircle | Charity Blossom | Website Load Testing


text: Videos on Red Letter Media Wiki. Media in category "Videos" This category contains only the following file. 08:10 You're Invited - The O... 18 KB Retrieved from "http://redlettermedia.wikia.com/wiki/Category:Videos?oldid=3877" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Files Add category Cancel Save


text:
Halloween and Gifted. Not a natural link? You might be surprised. Those who follow theorist Howard Gardner, a Harvard educator, would not be surprised. Gardner believes that students gravitate towards content that naturally interests them. He thinks that students have one or more dominant intelligences in the areas listed below:
• Musical Intelligence
• Bodily Kinesthetic Intelligence
• Logical Mathematical Intelligence
• Linguistic Intelligence
• Spatial Intelligence
• Interpersonal Intelligence
• Intrapersonal Intelligence
• Naturalist
• Existentialist (sometimes included)
So, it's not surprising that some of these talents would surface in the form of a Halloween costume. My eldest child's dominant intelligence is linguistic. Her Halloween costumes always had a literary connection, whether she dressed as Pippi Longstocking or donned a nun's habit and held a cow, as "Holy Cow." Now she teaches honors high school English.
It was the same with many of my gifted students. One interpersonal learner dressed up as Alexander the Great. A student who was very interested in nature wore a butterfly costume. And a child who had read many of Shakespeare's plays by third grade, dressed as William Shakespeare. A local television station got wind of his costume and featured him on a morning news program. Currently, he is in high school, but he also writes for a local newspaper.
Some schools have designed programs with Gardner's intelligences in mind. Some have identified students as gifted when they demonstrate aptitude in any of the multiple intelligences. Many teachers give students multiple intelligence inventories (questionnaires or surveys) to ascertain what content interest their students.
Recognizing that we have different talents—not just aptitudes for reading and math-- is the key point. We are diverse learners with diverse interests. Expose the interpersonal learner who studied Alexander the Great to other world leaders. Give him some leadership training of his own. Expose the linguistic learners to different genres. Have them write and develop portfolios. Expose the naturalist to different ecosystems. Give her a chance to do field research.
Don't be tricked into thinking your child has just selected an "ordinary" Halloween costume. There may be a message there. As Doug Coupland observed, "if human beings had genuine courage, they'd wear their costumes every day of the year, not just on Halloween." Be Aware.
Filed under: Uncategorized
Tags: Howard Gardner


text:
QMobile launches feature phone brand "MAXX". Affordability and simplicity is the forte of QMobile which is very much visible in Maxx feature phones. Recently, two Maxx devices have been launched titled Maxx Mobile Turbo T1 and Maxx Mobile Turbo T2 under the warranty of Digicom. QMobile launched Maxx Mobile to compete with low priced rival brands like RIVO, Voice, and GRight etc. The specifications and features of Maxx Mobile Turbo T1 and T2 is surely amazing and most significant is the price of both phones. Turbo T1 comes in 1350 PKR and Turbo T2 is for 1500 PKR only. QMobile chooses desi style marketing strategy for this low priced Maxx Mobile. Popular actress Mehwish Hayat will be seen shaking her legs on the theme song of Maxx Mobile. Maxx Mehwish Hayat is the brand ambassador and is surely making its waves across Pakistan.
Mobile Prices,
Mobile Rates Pakistan,
Mobile Phones Pakistan,
i want to buy this set at my home how is possible please reply now
Bhai is mein java setting hai
is kemat me ye zabardast mobile ha betry timing buhat achi ha
mera beta ye mobile bhut to nokia 3310 chala okay na smja
Ye mobile market ma kitne ka ha?
Whtsap hay ?
Bohat nice mobile hae rate b munasib hae aur function b achey haen,
Maxx Turbo T2 Seal Krna Ha Warnti Ha 2017 5 Monthday Ki Kise Ko Liyna Ho To Call Me 03471176318 Sargodha
Sir Jhang say kis jaga yai avlaible hai repely


text:
The drop bear is a pesky creature, represented by a "K". Clinging to the ceiling of a given level of the Caverns of Creation, they cannot be detected by the player until they attack (even if the player has Sight Beyond Sight). If they successfully attack the player, the player will often be stunned for two to six turns-- enough time, generally, for the drop bear to make its way to a wall and disappear along the ceiling once more.
The drop bear's attack is repelled with one hundred percent accuracy by the pointy hat, which is destroyed in the process. A failed attack will stun the drop bear for one to two turns, at which point it will try to escape. As they generally have low hp, they can usually be beaten by a player whose collection of pointy hats give him an edge.
Defeating the drop bear will cause him to drop an item; playing on the dual meaning of its name, the drop bear can drop literally any item in the game, including items that are one-of-a-kind. (In theory, this also includes the Sasquatch Hide Armor, but since it disappears after being dropped, it's kind of a moot point.)


text:
|Home | About | Journals | Submit | Contact Us | Français
Cortico–basal ganglia (BG) circuits are thought to promote the acquisition of motor skills through reinforcement learning. In songbirds, a specialized portion of the BG is responsible for song learning and plasticity. This circuit generates song variability that underlies vocal experimentation in young birds and modulates song variability depending on the social context in adult birds. When male birds sing in the presence of a female, a social context associated with decreased BG-induced song variability, the extracellular dopamine (DA) level is increased in the avian BG nucleus Area X. These results suggest that DA could trigger song variability changes through its action in Area X. Consistent with this hypothesis, we report that DA delivered to Area X weakens the output signal of the avian cortico-BG circuit. Acting through D1 receptors, DA reduced responses in Area X to song playback and to electrical stimulation of its afferent cortical nucleus HVC. Specifically, DA reduced the response to direct excitatory input and decreased firing variability in Area X pallidal neurons, which provide the output to the thalamus. As a consequence, DA delivery in Area X also decreased responses to song playback in the cortical output nucleus of the BG loop, the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Further, interfering with D1 receptor transmission in Area X abolished social context-related changes in song variability. In conclusion, we propose that DA acts on D1 receptors in Area X to modulate the BG output signal and trigger changes in song variability.
Cortico–basal ganglia (BG) circuits are thought to promote motor skill acquisition through reinforcement learning (Hikosaka et al., 2002; Graybiel et al., 2005). During reinforcement learning, individuals first explore their environment. Reinforcers shape this variable behavior until it converges on an optimum for a particular context. Thereby, repetition of a successful behavior (exploitation) replaces exploration (Sutton and Barto, 1990; Ishii Yoshida and Yoshimoto, 2002). It remains unclear which neural circuits allow the individual to switch from variable behavior underlying exploration to highly stereotyped behavior during exploitation, and how those circuits mediate the switch. However, changes in neural activity in the BG have been interpreted as representing the neural analog of explore-exploit behavior (Barnes et al., 2005).
Songbirds have a specialized portion of their BG, the anterior forebrain pathway (AFP, Fig 1C), which is needed for song learning and plasticity (Nordeen and Nordeen, 1997; Brainard and Doupe, 2002), but not for adult song production (Bottjer et al, 1984; Scharff and Nottebohm, 1991). This circuit generates song variability that underlies vocal experimentation in young birds (Ölveczky et al, 2005). In adult birds, the same circuit can modulate song variability depending on the social context (Kao et al, 2005; Kao and Brainard, 2006), or provide an instructive signal to guide adaptive changes in vocal output (Kao et al, 2008; Andalman and Fee, 2009). The neural input triggering changes in BG-driven song variability remains unknown.
In the BG, the neuromodulator dopamine (DA) plays a key role in reinforcement learning (Montague et al., 1996). Because DA delivery in the striatum signals reward prediction errors, it may provide a reinforcement signal to the BG (Schultz et al., 1993; Phillips et al., 2003; Wise, 2004). However, recent studies suggest that DA function in motor learning may go beyond such a reinforcement signal (Salamone et al., 2005; Costa, 2007). In particular, the DA system is involved in the regulation of inter-individual differences in exploration and exploitation behaviors (Frank et al., 2009). Moreover, DA is also involved beyond motor learning in the representation of social cues in mammals (Wang et al, 1999; Anstrom et al., 2009; Aragona and Wang, 2009).
In songbirds, the song-related BG nucleus Area X receives dense dopaminergic innervation from the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) (Lewis et al., 1981; Bottjer, 1993; Gale et al., 2008). Immunocytochemical data suggests that DA plays a social context-dependent role in the regulation of vocal communication (Heimovics and Riters, 2008). In particular, DA level is increased in Area X of male birds when they sing to females (Sasaki et al, 2006), a social context associated with decreased BG-induced song variability (Kao et al., 2005). Moreover, VTA neurons, possibly including dopaminergic neurons, show singing-related activity that is modulated by social context (Yanagihara and Hessler, 2006) and DA neurons express Fos after social stimuli (Bharati and Goodson, 2006). These results suggest that DA delivery in Area X could trigger changes in BG-driven song variability. More generally, such a mechanism would allow DA to regulate the balance between exploration and exploitation during and after the acquisition of motor skills.
Adult male zebra finches (Taeniopygia guttata) were obtained from a commercial supplier and used in accordance with an animal protocol approved by the University of Washington Institutional Animal Care and Use Committee. Animals were housed under a 14/10h light/darkcycle with food and water available ad libitum.
Each animal was first food deprived for 30 min and then given three intramuscular injections totaling 5–6.5 ml/kg of 20% urethane over 1h. Local anesthetic (1% lidocaine) was injected under the scalp before the animal was placed in a stereotaxic apparatus with the beak at an angle of 64° downward from the horizontal. Small craniotomies were made above the midline reference point, the bifurcation of the midsagittal sinus, and above HVC (used as proper name) and the lateral magnocellular nucleus of the anterior nidopallium (LMAN) or Area X unilaterally. Lidocaine gel was then applied to the incision at 3h intervals.
We infused drugs in the CNS using a combination of osmotic minipumps and cannulas, as in Meitzen et al. (2007). Animals were anaesthetized with 2% isoflorane and placed in a stereotaxic apparatus with a head angle of 64°. Anesthesia was maintained with 1% isoflorane for the duration of the surgery. Local anesthetic (1% lidocaine) was injected under the scalp, and small craniotomies were made above the midline reference point, the bifurcation of themidsagittal sinus, and at 6.5–7 mm anterior, 1.8 mm lateral from the reference point. The head angle was then changed to 0°. Two small bent cannulae (Alzet) were lowered to the surface of Area X (2.5–3 mm deep) and attached to the skull with dental cement. An osmotic minipump (Alzet, length, 17 mm; diameter, 6 mm; filled weight, 0.5 g; model 1002, 14 d delivery) filled with 100 μl of drug solution was then connected to the two cannulae with polyvinylchloride tubing and a Y distributor. The pumps were placed in a custom built backpack strapped to the bird's back using a harness made from surgical dressing. To mount the osmotic pump in the backpack, we used a 0.65 ml plastic microcentrifuge tube (ISC BioExpress) filled with 250 μl of sterile saline. We threaded the output tube of the pump through a hole in the cap and inserted the pump snugly into the microcentrifuge tube until the lid snapped into place. We sealed the lid and tube extrusion hole using cyanoacrylate adhesive and Parafilm(Fisher Scientific).
Glass pipettes (TW100F-3, World Precision Instruments) were pulled using a micropipette puller (Model P97, Sutter Instrument Co.), and the tips were blunted to impedances of 5 to 25MΩ. A ground electrode was placed in the cerebellum posterior to the midline reference point. A concentric stimulation electrode (FHC) was placed in HVC (0 mm rostral, 2.4 mm lateral to the midline reference point, 0.5 mm deep). The recording electrode signal was amplified 10x and low-pass filtered at 3kHz (Axoclamp2B amplifier, Molecular Devices), amplified further 100x and high-pass filtered at 300 Hz (model 440, Brownlee Precision). Recordings were monitored using an oscilloscope and an audio monitor. We searched for single-unit neuronal activity in Area X or LMAN using HVC stimulation as a search stimulus. Once a neuron was isolated, the electrophysiological signal was sampled at 20 kHz and spike times and raw traces were stored for further analysis (Spike2, Cambridge Electronic Design). Principal components analysis of the spike shapes allowed clear separation from noise and all extracted units obeyed a refractory period of 1 ms. In recordings in Area X, neurons displaying spontaneous firing above 25 spikes/s (sp/s) are referred to as pallidal neurons given their similarity with identified Area X pallidal neurons recorded at their terminals in the medial portion of the dorsolateral nucleus of the thalamus (DLM) (Person and Perkel, 2007). Moreover, we have shown in a previous study (Leblois et al., 2009) that over 66% of these neurons are antidromically activated from DLM. In contrast, Area X pallidal neurons recorded in DLM never display firing rates lower than 25 sp/s. Hence, Area X neurons displaying a firing rate lower than 25 sp/s are hereafter called putative interneurons.
Recordings were performed during HVC microstimulation (monophasic 0.2 ms single pulses), with various stimulation intensities (10 to 2000 μA). Each pulse of HVC microstimulation saturated the amplifier and occluded spiking activity for 1–2 ms in the recordings, due to the "overshoot" following saturation. Because the duration of this stimulation artifact was much shorter than the fastest responses recorded, it occluded only spontaneous activity and thus did not alter our analysis of evoked firing. Previous studies have shown that stimulation with a monopolar microelectrode at 200 uA activates about 50% of the neurons located in a shell of 0.2 mm outside radius (Ranck, 1975; Tehovnik et al, 2006). These values should be considered with caution because the current intensity necessary to activate a soma or axon at a given distance depends on a number of other variables such as the size of the cell or its biophysical properties (e.g. cellular excitability or axon myelination). In addition, concentric bipolar electrodes greatly reduce current spread, especially for higher stimulation intensities (Bagshaw and Evans, 1976; Follett and Mann, 1986), and estimates from monopolar electrodes thus provide an imprecise upper bound of the activated volume. The volume of the "activated shell" for 200 μA (inside and outside radius: 0.1 and 0.2 mm), is 0.029 mm3, and represents about 10% or less of HVC volume (0.2–0.5 mm3, MacDougall-Shackleton et al, 1998). Therefore, 200 μA pulses applied near the center of HVC through concentric bipolar electrodes are expected to activate fewer than 5% of HVC neurons.
We cannot exclude the possibility that some high amplitude stimulation led to current spread to neighboring structures. However, because of the segregation of the anterior forebrain pathway (AFP) circuit, we believe that occasional activation of neighboring structures does not modify the interpretation of the present data.
All drugs were diluted in a 0.9% saline solution with 0.5% dextran-conjugated Alexa-fluor 488 (3000 molecular weight; Invitrogen). The role of DA in Area X was examined using micro-injection of DA or related compunds (0.5–2 mM, Tocris). Drugs were pressure ejected from glass pipettes (10–20 μmtip diameter) using a Pressure System IIe (Toohey Co.; 50-ms pulses at 10–16 psi). Injected volumes were 20–100 nl. When the recording and drug injections were made in the same structure, we aimed to place the tip of the injection pipette 200–300 μm from the tip of the recording pipette. We recorded 21 neurons in Area X before, during and after DA injections in the same nucleus in 10 birds (1–4 neurons per bird). We recorded 17 neurons in LMAN before, during and after DA injections in Area X in 13 birds (1–3 neurons per bird). When multiple neurons were recorded from the same animal, we waited at least 2h between two consecutive DA injections to make sure that DA was washed out.
Slicing procedures were as described by Farries and Perkel (2000). Briefly, each animal was anesthetized with isoflurane and euthanized by decapitation. The brain was dissected rapidly into ice-cold, oxygenated artificial CSF (ACSF) containing the following (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1 NaH2PO4, 16.2 NaHCO3, 11 D-glucose, and 10 HEPES, osmolarity adjusted to 300 mOsm with sucrose. Coronal brain slices (300–450 μm thick) were prepared using a vibrating microtome (Vibratome), and slices were stored at room temperature submerged in bubbled ACSF in which HEPES was replaced with equiosmolar NaHCO3. All chemicals were obtained from Sigma-Aldrich. All solutions were bubbled with a 95% O2 and 5% CO2 mixture.
After resting for at least 1 h after sectioning, slices were placed in a recording chamber and superfused with ACSF heated to 28–30°C. Glass pipettes were pulled to a tip of < 2 μm in diameter (Micropipette Puller P-97, Sutter Instrument Co.), filled with 0.9% saline, and had a resistance of 4–8 MΩ. Signals were amplified with either an Axoclamp 2B (Molecular Devices) followed by a Brownlee Precision DC amplifier, or with a MultiClamp 700B amplifier (Molecular devices). Signals were low-pass filtered at 3 kHz, high-pass filtered at 300 Hz, and digitized at 20 kHz with a Digidata 1322A (Molecular Devices) and stored on a personal computer using pClamp 9 (Molecular Devices). A tungsten bipolar stimulating electrode was placed near the boundary of Area X in a location that distinguishes inputs from HVC versus the lateral magnocellular nucleus of the anterior neostriatum (LMAN), on the basis of a previous description of innervation patterns (Ding and Perkel, 2003). Recording pipettes were placed near and ventral to the stimulating electrode in most cases. The spatial relationship between the stimulating and recording electrodes in the other axes varied. Once a spontaneously active neuron was located, the recording electrode was approached as close as possible to obtain a signal-to-noise ratio > 3. Then, short latency spiking was evoked by electrical stimulation at 0.1 – 0.2 Hz.
At the end of each acute recording experiment, recording sites were labeled by iontophoretic injections of fluorescent dye (5% Alexa-488- or -568-conjugated 10kDa dextranamine in 0.01M phosphate-buffer PB, pH 7.4, ejected by 5 μA DC pulses of 7 s duration, 50% duty cycle for 5 min.). Animals were euthanized by intramuscular injection of sodium pentobarbital (Nembutal) and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde as fixative. The brain was then removed, post-fixed in 4% paraformaldehyde for 24h, and cryoprotected in 30% sucrose. 40μm-thick sections were then cut in the parasagittal plane on a freezing microtome and processed for histological examination to verify the location of stimulating and recording electrodes, and drug-injection sites. In addition to gross observation of electrode tracts, the brain slices were visualized using a fluorescence microscope to allow better determination of recording location. A summary of recording locations reconstructed from stereotaxic coordinates and post-hoc histological analysis is presented on Fig. 2.
At the end of cannulation experiments, animals were euthanized by overdose of pentobarbital and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brain was then removed, post-fixed in 4% paraformaldehyde for 24h, and cryoprotected in 30% sucrose. 50μm-thick sections were then cut in the parasagittal plane on a freezing microtome, mounted on slides and stained with cresyl violet. Histological examination showed that the cannula tip was in Area X or less than 500 μm away from the edge of Area X in all four birds, and that any lesion due to cannula placement and drug flow was restricted to a region not exceeding 10% of the total volume of Area X. Fig. 10B displays a 50 μm parasagittal brain section showing the track of the cannula used to infuse drugs in the behaving bird.
Birds were individually housed in sound isolation chambers (Acoustic Systems, Austin, TX) 7 days before and 20–40 days following the cannula implantation surgery. We continually recorded spontaneous vocalizations using Syrinx software (John Burt, www.syrinxpc.com). In addition, we recorded vocalizations evoked by the presentation of a female by placing a female in the cage for 3–4 min, at intervals > 20 min. Such presentation was performed less than 6 times a day and for at least three days in each condition.
Spike times were analyzed using Matlab software (version 220.127.116.112, R2007b, MathWorks, Natick, MA). For each cell, we calculated spontaneous firing rate, interspike interval (ISI) distribution, and peri-stimulus time histogram (PSTH) of the response to HVC stimulation and/or to song playback. The average firing rate during song playback was calculated. Spontaneous firing rate was calculated during a window of same duration as the song playback preceding the playback. The difference between the firing rate during and before a given song playback was calculated for each trial and averaged across trials to give a mean song response strength (Solis and Doupe, 1997). Song response strength measurements were used to calculate the discriminability statistic d′, which is used to quantify the selectivity of a neuron for a given stimulus over another (Solis and Doupe, 1997), where the differencebetween the average song response strength (RS) to two songs was normalized by the square root of the average of the variances of the song response strength (σ) measurementsfor the two songs.
Analyses of responses to HVC stimulation were performed on PSTHs smoothed as follows. For each trial, the firing-rate time course was determined with 1 ms resolution by convolving the spike train with a Gaussian kernel of width 1 ms (Baker and Gerstein, 2001). The mean and SD of the spontaneous rate were determined over the 100 ms preceding stimulation. A neuron was considered to display a significant response if at least two consecutive bins of the PSTH were beyond limits defined by the spontaneous mean ± 2.5 SD. Responses were often made up of several components (especially in Area X pallidal cells), some inhibitory and some excitatory. We defined the beginning of the response component as the time of the first of two consecutive bins of the PSTH in which the firing rate fell outside significance limits; similarly, the end occurred when two consecutive bins fell back within significance limits. Stimulation response strength, similar to the song response strength but calculated over a short time window (30 ms in Area X, 60 ms in LMAN), was defined as the difference between the average firing rate over the response window following HVC stimulation, and the average spontaneous firing rate over a window of the same duration preceding HVC stimulation. We defined excitation (or inhibition) relative area as the percentage increase or decrease in number of spikes relative to the average number of spikes, across the population of cells, expected in a response window of spontaneous firing. To this end, the area of the PSTH significantly above (or below) spontaneous firing was divided by the population average spontaneous firing rate. The result was expressed as a percentage of the population average spontaneous firing. For LMAN neurons, Area X putative interneurons and pallidal neurons displaying only excitatory responses, the lowest stimulation current intensity evoking a reliable response (at least one additional spike in each trial) was selected for further analysis. For pallidal neurons displaying some inhibition in response to stimulation, the lowest current intensity evoking an inhibitory component in their response was selected for further analysis.
Songs were sorted and analyzed using custom Matlab programs. Zebra finch songs are highly stereotyped, making them especially well suited for in-depth analysis. The acoustic structure ofsong is arranged in a hierarchy, with 5–50 ms vocal units known as syllables strung together in a stereotyped sequence called a motif. Each song consists of one or several motifs, preceded by introductory notes and separated from each other by <100 ms of silence. We designed a program to sort motifs and songs from all sound files continuously recorded using the syrinx software. Briefly, the program detected putative motifs based on peaks in the cross correlation between the recorded sound file and a clean pre-selected motif. Such putative motifs were then sorted based on their spectral similarity with the pre-selected clean motif, using thresholds set by the experimenter. For motifs for which such analysis did not allow unambiguous distinction, an additional PCA analysis on the spectrograms of putative motifs allowed us to sort motifs from other sounds. This analysis allowed us to successfully sort >90% of the motifs sung by a bird on a given day (assessed by comparing hand sorting with the automated sorting by the program).
Song analysis consisted of determining the fundamental frequency of harmonic stacks and the frequency of the lowest harmonic in all sub-syllabic elements displaying clear frequency modulation. The latter feature is strongly correlated with fundamental frequency for sub-syllabic elements displaying a harmonic stack structure (cf below), and can also be calculated for non-harmonic elements. Fundamental frequency was calculated based on peaks in the auto-correlation function, as in Kao and Brainard (2006). The frequency of the lowest harmonic was estimated as follows. For each sub-syllabic element considered, we selected a time window where the frequency of the lowest harmonic was stable. The spectrogram of the corresponding note was calculated and the frequency of its lowest harmonic was estimated within this time window. To improve the resolution of this frequency estimate, we performed a piecewise cubic spline interpolation of the mean spectrogram around its peak. We evaluated variability in the frequency of the lowest harmonic by calculating the standard deviation of its distribution over all clean renditions of the motif in each condition. The number of motif renditions considered was: 81 ± 38 at baseline, 73 ± 62 during SCH23390 infusion and 57 ± 28 during saline infusion for female evoked vocalizations (range 22–159); 3300 ± 2500 at baseline, 1800 ± 1600 during SCH infusion, and 1800 ± 1100 during saline infusion for spontaneous vocalizations (range 470–5441).
For the sub-syllabic element displayed in Fig. 10, we compared our estimate of the lowest harmonic with fundamental frequency estimates. There was a strong correlation between the results of the two methods (R2=0.98, n=1000 randomly sampled songs).
Both context and treatment might affect the location of the bird when singing and its variability, inducing fluctuations of signal amplitude. To rule out an influence of the location of the bird on song variability, we calculated the correlation between our measure of the frequency of the lowest harmonic and random fluctuations of signal amplitude. We found no correlation between the signal amplitude and the frequency of the lowest harmonic for the sub-syllabic element displayed in Fig. 10 (R2= 0.07, n=1000 randomly sampled songs).
Numerical values are given as mean ± SD, unless stated otherwise. Response latency, strength and duration before and after drug injections were compared using a paired t-test. In addition, for each cell, spontaneous activity over multiple trials was compared before and after drug injection using a paired t-test. For the behavioral experiment, CV of the frequency of the lowest harmonic of sub-syllabic elements and relative frequency variability were compared across different conditions using paired t-tests. For each paired t-test applied, we report the associated p-value (the probability of observing the given result, or one more extreme, by chance if the null hypothesis is true), the value of the test statistic (t), and the degrees of freedom of the test (df).
We first investigated the effects of DA on responses evoked by song playback in Area X pallidal neurons, most of which project to the thalamic nucleus DLM (Leblois et al., 2009). As described previously (Doupe, 1997; Solis and Doupe, 1997; Person and Perkel, 2007; Gale and Perkel, in press), pallidal neurons increased their firing rate in response to playback of BOS, with an average response strength of 8.4 ± 5.1 sp/s (n=13). Other sound stimuli (noise, conspecific song and BOS played in reverse) evoked weaker responses. We measured the selectivity of responses to BOS in pallidal neuron with the discriminability index d′, which averaged 1.6 ± 0.9 (n=10), significantly greater than 0 (p<0.001, t=5.2, df=9).
Injection of DA into Area X suppressed responses to BOS playback in Area X pallidal cells (Fig. 1A). Overall, the BOS response strength after DA injection was significantly lower than under control conditions (from 8.9 ± 5.6 to 2.2 ± 1.3 sp/s, n=7, p=0.01, t=3.5, df=6; Fig. 1D), and the selectivity of the remaining responses was significantly decreased (d′ from 1.4 ± 1 to −0.2 ± 0.4, p=0.02, t=3.2, df=6) and not different from 0 (p>0.1, t=−1.4, df=6). Similarly, injections of the D1 receptor agonist SKF38393 in Area X suppressed song responses in Area X (BOS response strength: from 10.1 ± 3.9 sp/s to 2.9 ± 1.4 sp/s, n=3, p=0.04, t=5, df=2; d′: from 2.1 ± 0.5 to 0.1 ± 1.6, p=0.1, t=3, df=2). Responses returned after washout of the drug in 6/7 neurons following DA (BOS response strength: 9.7 ± 7.6 sp/s, d′: 0.7 ± 1.5) injection and 1/3 neurons following SKF38393 injection (BOS response strength: 5.8 sp/s, d′: −1). In contrast to dopaminergic drugs, injection of saline did not modify the response of pallidal neurons to BOS playback (BOS response strength: from 5.5 ± 4.1 to 6.7 ± 3, n=3, p>0.1, t=−1.3, df=2; d′: from 2.3 ± 3 to 1.6 ± 0.9, p>0.5, t=0.5, df=2; Fig. 1E). Changes in song response under DA were often accompanied by increased spontaneous activity (Fig. 1A, see next section for extensive analyses of changes in spontaneous activity). It is possible that elevated firing rates contributed in some cases to decreased responses in pallidal neurons due to a ceiling effect. Responses to BOS playback were strongly suppressed in Area X pallidal neurons, however, even when spontaneous activity was not affected (n=3/7 under DA and 1/3 SKF; Fig. 1B). Moreover, there was a complete lack of correlation between increase in spontaneous firing rate and reduction in song response (R2=0.002).
In summary, DA injection in Area X of urethane-anaesthetized zebra finches strongly dampened responses of Area X pallidal neurons to song playback.
We further examined the effects of DA on spontaneous firing of Area X neurons using a combination of in vivo and in vitro electrophysiological recordings. In vivo, Area X pallidal neurons displayed high spontaneous activity (mean firing rate: 58 ± 15 sp/s, n=24). Spontaneous activity was significantly increased by DA injection in Area X in most pallidal neurons (10/15, Fig. 3A) and decreased in only one neuron. Overall, DA significantly increased spontaneous activity by 5 sp/s (to 63 ± 14 sp/s, n=15, p=0.001, t=−3, df=14), which recovered after washout (60 ± 13 sp/s, p=0.04, t=−2.2, df=14). Concerning putative Area X interneurons, the effect of DA on spontaneous firing rate was not consistent (Fig. 3B). Spontaneous activity was increased in two such cells, increased in one and unchanged in two. In vivo injections of the D1 receptor agonist SKF38393 appeared to have similar effects on pallidal spontaneous activity as DA, increasing spontaneous firing rate in most neurons, although the increase was significant in only 4/9 cells, and the overall change in average firing rate was not significant (Fig. 3C).
In vitro, DA increased the intrinsic firing rate of putative pallidal cells, defined as cells displaying a spontaneous firing rate above 10 sp/s. DA significantly increased the firing rate from 19 ± 8 sp/s to 22 ± 9 sp/s, (n=18, p=0.005, t=−3.2, df=17, Fig. 3D). Because these recordings were made in the presence of the ionotropic glutamate and GABAA receptor blockers kainic acid and picrotoxin, respectively, the effect of DA was likely direct on pallidal neurons rather than through indirect circuit effects. This increase in firing rate was mimicked by application of the D1 receptor agonist SKF38393 (from 19 ± 3 sp/s to 27 ± 17 sp/s, n=11, significant only when the cell with the largest increase was excluded: p=0.001, t=−5, df=8). The effect of DA was blocked by prior application of the D1 receptor antagonist SCH23390 (from 19 ± 8 sp/s to 20 ± 10 sp/s, n=7, p=0.2, t=0.8, df=6). The D2 receptor agonist quinpirole had no effect (from 17 ± 6 sp/s to 17 ± 6 sp/s, n=5, p=0.9, t=−0.1, df=4).
These results indicate that DA, acting via D1 receptors, increases the intrinsic spontaneous activity of Area X pallidal neurons.
Area X receives auditory input from nucleus HVC. Area X pallidal neurons receive direct excitatory input from HVC as well as feed-forward inhibition via Area X interneurons (Farries et al., 2005). The reduction of song-evoked responses in Area X pallidal neurons might rely on change in the responsiveness of Area X neurons to their direct input from HVC. To test whether DA modulates the responsiveness of Area X neurons to their input from HVC, we recorded the response of Area X neurons to HVC electrical stimulation (see Leblois et al. 2009) before and after DA injection in Area X.
DA suppressed the rapid excitation evoked by HVC stimulation in Area X pallidal neurons (Fig. 4A and B) sometimes revealing or increasing an inhibitory component of the response (Fig. 4A). DA injection suppressed the peak of the population average of responses to HVC stimulation (Fig. 4C). Over all neurons, the average peak response dropped from 363 ± 133 sp/s to 190 ± 103 sp/s after DA injection (n=16, p<10−4, t=6, df=15) and recovered to 357 ± 143 after washout. Moreover, the stimulation response strength over a 30 ms window following HVC stimulation dropped from 49 ± 24 sp/s to 3 ± 23 sp/s (p<10−6, t=10.8, df=15), and recovered after washout to 47 ± 27 sp/s (Fig. 4D). To provide a fuller description of the data, we also examined the relative area (see methods) and duration of the excitatory and inhibitory components of the responses. For each response feature, a t-test revealed a significant change between the baseline and DA conditions. A False Discovery Rate analysis indicates that some of the tests are at the threshold for statistical significance (threshold p value of 0.04). DA decreased both the relative area and the duration of the excitatory components in the response of pallidal neurons to HVC stimulation (excitation relative area: from 112 ± 61 % to 32 ± 27 %, p=10−5, t=6.6, df=15, Fig. 4E; excitation duration: from 18 ± 11 ms to 9 ± 5 ms, p=0.004, t=3.4, df=15, Fig. 4F). These values recovered after washout (excitation relative area: 90 ± 54 %; excitation duration: 16 ± 9 ms). The inhibitory components in response to HVC stimulation were larger and longer in duration after DA injection (inhibition relative area: from 3 ± 5 % to 11 ± 11 %, p=0.03, t=2.4, df=15, Fig. 4G; inhibition duration from 2 ± 3 ms to 5 ± 4 ms, p=0.04, t=−2.1, df=15, Fig. 4H), and recovered after washout (inhibition relative area: 5 ± 7 %; inhibition duration: 3 ± 4 ms). Overall, DA decreased the response of pallidal neurons to HVC stimulation, reducing the excitatory component of the responses and emphasizing their inhibitory component.
In putative interneurons, DA decreased the rapid evoked excitation to a lesser extent (Fig. 4I). In these neurons, the stimulation response strength decreased only slightly on average, from 44 ± 44 sp/s at baseline to 23 ± 53 sp/s (n=5, p=0.03, t=4.1, df=3, Fig. 4J), and recovered after washout to 40 ± 45 sp/s. DA did not cause a significant change in the response peak in interneurons (from 373 ± 183 sp/s to 268 ± 256 sp/s).
DA thus reduced the responses of all Area X neurons to HVC electrical stimulation, and the response reduction was more pronounced in pallidal neurons.
To dissect the mechanisms by which DA reduces the response of pallidal neurons to song and to HVC electrical stimulation, we studied the effects of DA in an in vitro preparation. Stimulation of HVC fibers innervating Area X led to a rapid and strong increase in firing in Area X putative pallidal neurons (see Materials and Methods). These responses were suppressed by application of the glutamate AMPA receptor antagonist CNQX (Fig. 5A). CNQX strongly decreased the stimulation response strength from 49 ± 13 sp/s to 9 ± 14 sp/s (n=4, p=0.03, t=5.4, df=3, Fig 5B). Stimulation response strength recovered after washout (27 ± 13 sp/s). The response peak also decreased in the presence of CNQX, from 140 ± 40 sp/s to 33 ± 25 sp/s (p=0.02, t=4.7, df=3), and recovered after washout to 85 ± 34 sp/s. Conversely, responses were unaffected by the cholinergic receptor antagonists mecamylamine and atropine (stimulation response strength from 32 ± 19 sp/s to 27 ± 14 sp/s, n=4, p>0.5, t=0.9, df=2; response peak from 92 ± 53 sp/s to 75 ± 51 sp/s, p>0.05, t=1.2, df=2; Fig. 5C). Therefore, responses of pallidal neurons to HVC fiber stimulation in vitro were mediated by glutamate, similar to responses to HVC stimulation in vivo (Leblois et al., 2009).
As observed in vivo, DA application also diminished responses of pallidal neurons to stimulation of HVC fibers in vitro (Fig. 5D). Stimulation response strength was decreased from 20 ± 18 sp/s at baseline to 9 ± 8 sp/s under DA (n=8, p=0.01, t=3.5, df=7, Fig. 5E), and recovered after washout (17 ± 15 sp/s). Response peaks also strongly decreased, from 102 ± 40 sp/s at baseline to 54 ±31 sp/s under DA (p=0.002, t=4.8, df=7), and recovered to 84 ± 44 sp/s after washout. In the presence of the GABAA blocker picrotoxin, DA still diminished pallidal responses to HVC fiber stimulation (Fig. 5F). Stimulation response strength decreased from 30 ± 35 sp/s at baseline to 13 ± 19 sp/s under DA (n=13, p=0.01, t=2.3, df=12, Fig. 5G), and recovered to 36 ± 49 sp/s after washout. Similarly, peak response decreased from 94 ± 60 sp/s at baseline to 59 ± 44 sp/s under DA (p=0.02, t=2.6, df=12) and recovered after washout (94 ± 60 sp/s). Therefore, the DA induced decrease in the response of pallidal neurons to HVC stimulation was not solely due to a change in the feedforward inhibition received by pallidal neurons. Instead, DA decreased the effect of direct excitatory input from HVC on the firing of pallidal neurons.
We then sought to determine the DA receptor type underlying the effect of DA on the response of pallidal neurons to HVC inputs. Prior application of the D1 receptor antagonist SCH23390 blocked the effect of DA on the HVC electrical stimulation excitation of Area X pallidal neurons (Fig. 6A). Over all, the stimulation response strength was unchanged by DA in the presence of SCH23390 (from 29 ± 20 sp/s to 26 ± 26 sp/s, n=9, p=0.9, t=0.06, df=8, Fig. 6C). The response peak was also unchanged (from 134 ± 47 sp/s to 129 ± 52 sp/s, p=0.6, t=0.8, df=8). Moreover, the DA D1 receptor agonist SKF38393 mimicked DA and reduced the response to HVC fibers stimulation in pallidal neurons (Fig. 6B). SKF38393 strongly decreased the stimulation response strength (from 10 ± 10 sp/s to 5 ± 8 sp/s, n=10, p=0.01, t=2.3, df=9, Fig. 6D) as well as the response peak (from 77 ± 37 sp/s to 53 ± 35 sp/s, p=0.03, t=2.7, df=8). Both values recovered after washout (stimulation response strength: 7 ± 12 sp/s; response peak: 59 ± 40 sp/s). On the contrary, the D2 receptor agonist quinpirole did not change either the stimulation response strength (from 21 ± 21 sp/s to 17 ± 17 sp/s, n=5, p=0.3, t=1.1, df=4, Fig. 6E) or peak (from 120 ± 17 sp/s to 117 ± 10 sp/s, p=0.8, t=0.2, df=4) of pallidal responses to HVC fiber stimulation.
Therefore, the DA induced decrease in HVC-driven responses of Area X pallidal neurons is mediated by D1 receptors.
Because irregular firing in Area X pallidal neurons can drive firing in downstream thalamic neurons in nucleus DLM, we then investigated how DA affected the firing variability of Area X output pallidal neurons in vivo. We compared the variability in ISI duration in three different conditions: in spontaneous firing, in the response to song playback, and in the response to HVC electrical stimulation. In all cases, DA led to an increase in firing regularity as measured by a narrowing of the ISI distribution. Neurons whose firing rate increased after DA treatment displayed a shift in the peak of their ISI distribution (Fig. 7A). More importantly, the ISI distribution was much narrower after DA injection than it was at baseline or after washout, and the shortest and longest ISIs did not occur when DA was applied. Similarly, neurons that did not undergo a change in mean firing rate nevertheless displayed a narrowing of the ISI distribution after DA injection, with fewer short and long ISIs (Fig. 7B). Thus, over the entire population of neurons tested, DA application caused a narrowing of the mean ISI distribution over all Area X pallidal neurons examined (Fig. 7C), and the number of shorter and longer ISI was significantly decreased (ratio of ISIs < 8ms: from 6 ± 4 % to 3 ± 4 %, p=0.005, t=4.8, df=14; ratio of ISIs > 25 ms: from 14 ± 18 % to 6 ± 10 %, p=0.01, t=2.1, df=14). DA application decreased the coefficient of variation (CV) of the ISI distribution in spontaneous activity in all Area X pallidal neurons (from 0.35 ± 0.06 to 0.24 ± 0.06, n=15, p<10−5, t=9.6, df=14, Fig. 7D). The CV recovered after washout (to 0.3 ± 0.05).
DA also decreased firing variability in response to song playback or in response to HVC stimulation. In response to playback of the BOS, the average ISI distribution narrowed (Fig. 7E), shorter ISIs became less frequent (ratio of ISIs < 8ms: from 38 ± 8 % to 10 ± 11 %, p=10−7, t=21.2, df=6), and the ISI CV was decreased (from 0.36 ± 0.07 to 0.2 ± 0.06, n=7, p=0.008, t=3.8, df=6, Fig. 7F) after DA application. Similarly, DA application caused shorter ISIs in response to HVC stimulation to become less frequent (ratio of ISIs < 8ms: from 29 ± 12 % to 9 ± 11 %, p=10−6, t=18.4, df=14, Fig. 7G), and the ISI CV increased over all Area X pallidal neurons (from 0.59 ± 0.1 to 0.42 ± 0.12, n=15, p=0.002, t=4.2, df=14, Fig. 7H).
Overall, DA decreased the spontaneous and evoked firing variability in Area X pallidal neurons in vivo.
Each thalamic neuron in nucleus DLM receives a tonic inhibitory input from a single Area X pallidal neuron. Its post-inhibitory rebound properties makes it likely to fire when a series of short ISIs in its pallidal input is followed by a longer ISI (Person and Perkel, 2005, 2007; Kojima and Doupe, 2009; Leblois et al., 2009). Therefore, thalamic firing, and, more generally, information transmission from HVC to LMAN, relies on a high variability in ISI duration of Area X pallidal neurons. Because DA decreases ISI length variability in Area X pallidal neurons, it is expected to impede information transmission along the AFP.
To test this hypothesis, we recorded evoked activity in nucleus LMAN before and after injecting DA in Area X. Consistent with our prediction, we found that DA reduced the response to song playback in LMAN neurons (Fig. 8A). Responses to playback of BOS were suppressed in all LMAN neurons recorded after DA injection in Area X (BOS response strength: 2.6 ± 1.8 sp/s to 0.4 ± 1.1 sp/s, n=10, p=0.0004, t=5.4, df=9, Fig. 8B), and recovered after washout (BOS response strength: 2.2 ± 2.1 sp/s, n=7). Not surprisingly, the selectivity of any remaining responses was also reduced (d′: 1.4 ± 1.0 to −0.3 ± 0.8, p=0.0006, t=5.1, df=9, Fig 8C), and recovered after washout (d′: 1 ± 0.9). In contrast, saline injections in Area X did not modify the strength or selectivity of responses to BOS playback in LMAN neurons (BOS response strength: 2.1 ± 1.6 sp/s to 2.3 ± 1.5 sp/s, n=3, p=0.7, t=−0.6, df=2; d′: 1 ± 1.2 to 1.3 ± 1, p=0.4, t=−0.2, df=2; Fig 8D, E).
Responses of LMAN neurons to HVC electrical stimulation were also decreased after DA injections in Area X (n=6/7; Fig. 9A). Overall, the stimulation response strength was 8 ± 9 sp/s at baseline and 3 ± 5 sp/s following DA injection in Area X, and recovered to 8 ± 7 sp/s after washout (Fig. 9B). Moreover, the response peak was 48 ± 44 sp/s at baseline and 23 ± 20 sp/s under DA, and recovered to 48 ± 34 sp/s after washout (Fig. 9C). These decreases in stimulation response strength and response peak were not significant due to one neuron that showed an increased response after DA was injected in Area X. Interestingly, DA injections in Area X significantly decreased the duration of the response to HVC electrical stimulation in LMAN neurons (from 28 ± 13 ms to 14 ± 10 ms, n=7, p=0.02, t=3.4, df=6, Fig. 9D).
Spontaneous activity in DLM and in LMAN is expected to decrease following DA injection due to the shorter and less variable length of ISIs in DLM inhibitory input from Area X. Surprisingly, spontaneous activity in LMAN was not affected by DA injection in Area X (from 2.3 ± 1.4 sp/s to 2.3 ± 1.6 sp/s, n=17, p=0.9, t=0.7, df=6), suggesting that LMAN spontaneous activity may not rely on its input from DLM. Alternatively, the relation between spontaneous activity in DLM and in Area X output neurons may be more complex than expected.
Levels of extracellular DA measured in Area X differ in different social contexts (Sasaki et al., 2006). Because DA can modulate the amplitude of the output of the AFP through its effects in Area X, mostly through D1 receptors, it is a good candidate to trigger variability changes depending on the social context. To test this hypothesis, we blocked D1 receptors in Area X in behaving birds by slowly infusing the D1 receptor antagonist SCH23390 into Area X. To measure differences in variability associated with different social contexts, we measured the frequency of harmonic components of syllables that displayed a clear harmonic structure (Fig. 10A). Consistent with previous reports (Kao et al., 2005; Kao and Brainard, 2006; Sakata et al., 2008), we found that the frequency of the lowest harmonic was more variable when the male sang alone in the cage than when a female was present in the cage. The presence of a female decreased the CV of the frequency of the lowest harmonic (CVFLH) in 12 out of 13 sub-syllabic elements from 4 birds. Overall, the CVFLH was 0.019 ± 0.01 when a male sang alone, and 0.012 ± 0.01 in the presence of a female (p=0.001, t=4.4, df=12; Fig. 10D).
In line with our hypothesis, we found that infusion of the D1 receptor antagonist SCH23390 abolished changes in variability related to social context (Fig. 10C). Indeed, the CVFLH of all sub-syllabic elements considered did not display any change in variability with social context when SCH23390 was infused in Area X (0.020 ± 0.011 when singing alone versus 0.021 ± 0.01 in the presence of a female, p=0.5, t=0.7, df=12; Fig. 10D). Even when considering only sub-syllabic elements showing context-dependent variability before surgery (12 out of 13), no difference was found between the two social contexts (CVFLH of 0.19 ± 0.007 alone versus 0.18 ± 0.008 with a female, p=0.4, t=0.8, df=11). The CVFLH during both social contexts after D1 receptor blockade was not significantly different from the CVFLH in the absence of a female before drug infusion (in presence of a female: p=0.9, t=0.2, df=12; in the absence of a female: p=0.4, t=1, df=12).
As a control, we infused saline in Area X before (1 bird) or after (3 bird) SCH23390 infusion. During saline infusion, the context-dependent song variability was restored, and the CVFLH was significantly lower in the presence of a female (0.014 ± 0.01 versus 0.017 ± 0.01 alone, p=0.02, t=3.9, df=12).
For each sub-syllabic element displaying context-dependent variability in frequency, we compared the SD of the lowest harmonic frequency when the bird was singing to a female relative to the SD of the lowest harmonic frequency when singing alone before surgery. This relative song variability measure was strongly increased under SCH23390 infusion, from 57 ± 16 % to 96 ± 29 % (p=0.0004, t=5, df=11), and partially recovered under saline infusion to 77 ± 23 % (Fig. 10E).
We also calculated the mean CVFLH over all sub-syllabic elements displaying context-dependent variability in frequency in each individual bird. Before surgery, the average per bird CVFLH (n=4) was significantly different in the absence or presence of a female (0.017 ± 0.004 versus 0.010 ± 0.002, p=0.04, t=3.5, df=3). During SCH23390 infusion, no significant difference was found between the two social contexts (average CVFLH of 0.018 ± 0.004 versus 0.016 ± 0.005, p=0.7, t=0.6, df=3). The variability was significantly lower in the presence of a female during saline infusion (average CVFLH of 0.015 ± 0.002 alone versus 0.012 ± 0.002 with a female, p=0.02, t=5, df=3).
In addition, we were able to extract the fundamental frequency from a subset of sub-syllabic elements that exhibited harmonic stack structures. Under control conditions, the fundamental frequency of those 8 elements, from 3 birds, was significantly lower in the presence of a female (0.01 ± 0.003) than when the bird was alone (0.018 ± 0.004; p=0.0006, t=5.8, df=7). When D1 receptors were blocked by infusion of SCH23390 in Area X, there was no longer any difference between the two social contexts (0.02 ± 0.01 versus 0.021 ± 0.006; p=0.6, t=0.5, df=7). The difference in variability was retained after saline infusion in Area X, with a CV of the fundamental frequency of 0.012 ± 0.003 in the presence of a female, and 0.015 ± 0.004 when the bird was alone (p=0.03, t=2.8, df=7).
Other than the changes in song variability, there was an overall trend for mean frequency of the lowest harmonic to increase when a female was present in the cage (9/13 sub-syllabic elements showed an increase, 4/13 a decrease). The frequency was therefore 17 ± 27 Hz higher (+ 0.8 ± 1%, p=0.04, t=2.4, df=12) when a female was present in the cage than when the male sang alone. Although previous studies in zebra finches did not report any change in average song features (Kao et al., 2005; Kao and Brainard, 2006), this increase in frequency is consistent with an increase in fundamental frequency reported in Bengalese finch singing to females (Sakata et al., 2008).
In contrast to the variability measures, changes in the mean frequency of the lowest harmonic induced by SCH23390 infusion in Area X were not consistent among the 13 sub-syllabic elements considered. During SCH23390 infusion, 9/13 sub-syllabic elements sung in the absence of a female displayed an increase in their mean frequency of the lowest harmonic (8/13 in the absence of a female), while 4/13 displayed a decrease in mean frequency (5/13 in the absence of a female). Changes were not significant in either social context (p>0.5, t=−0.3, df=12).
These results indicate that the D1 receptor antagonist SCH23390 abolished social context-related song variability when infused in Area X without altering average song features.
We report here that delivering DA in the BG reduces the output of the AFP, a BG-thalamo-cortical circuit known to regulate song variability. DA acts through D1 receptors to reduce the response to HVC excitatory inputs in pallidal neurons, which provide BG output to the thalamus. Interfering with D1 receptor transmission abolishes social context-related changes in song variability. Our data indicate that DA triggers variability changes in song by modulating the amplitude of the AFP output signal through its action on D1 receptors in Area X.
DA modifies neuronal activity and synaptic transmission in the BG in mammals (Calabresi et al., 2000) and in birds (Ding and Perkel, 2002; Ding et al., 2003). Because the striatum receives the largest DA input in the BG in mammals (Smith and Villalba, 2008), previous studies have concentrated on DA effects on striatal neurons. Endogenous DA reduces responses to glutamate (Kiyatkin and Rebec, 1996, 1999) or to behaviorally relevant stimuli (Rolls et al., 1984; Nicola et al., 2000) mainly by acting on D1 receptors in mammalian striatal neurons. Similarly, in songbirds, D1 receptor activation depresses glutamatergic synaptic current in Area X spiny neurons (Ding and Perkel, 2002). Consistent with this effect, we found that DA decreases the response to HVC glutamatergic input in Area X putative interneurons, possibly including but not restricted to spiny neurons (which are thought not to project out of the nucleus). Area X inhibitory interneurons induce feedforward inhibition on pallidal neurons, shifting single spikes (Leblois et al., 2009). Therefore, the decreased responsiveness of Area X inhibitory interneurons under DA may partially underlie the increased regularity of evoked pallidal firing reported here.
Although DA innervation is much weaker in BG output nuclei than in the striatum in mammals, DA can directly affect the activity of BG output neurons (Kliem et al., 2007; Zhou et al., 2009). In vitro, DA increases spontaneous activity and firing regularity in nigral neurons through D1 receptors (Zhou et al., 2009), an effect similar to the effects reported here on avian pallidal neurons. In contrast, Kliem et al. (2007) reported an increase in GABA levels and a decrease in spontaneous activity mediated by D1 receptors in pallidal and nigral neurons in vivo.
All previous studies focused on localized effects of DA in each BG structure, and it is difficult to predict how simultaneous DA release in several BG nuclei would globally modulate information transmission through the circuit. Because Area X includes both striatal neurons and pallidal-like output neurons, our study provides new insight on the integrated action of DA on the BG network as a whole.
DA plays an important role in the modulation of social behavior, including display of social status (Miczek and Gold, 1983), defensive/submissive behavior (Puglisi-Allegra and Cabib, 1997), and mating behavior (Young and Wang, 2004). In particular, DA release in the BG is crucial for pair-bond formation during mating (Wang et al., 1999; Aragona et al., 2003). In songbirds, DA affects the motivation to sing (Schroeder and Riters, 2006), and its role in song regulation is likely dependent on social context (Heimovics and Riters, 2008). Moreover, Sasaki et al. (2006) have shown that DA level in Area X is increased only in male birds singing toward a female, a social context associated with stereotyped song production. Our results suggest that higher DA levels in Area X in the presence of a female may be responsible for the decrease in AFP-driven song variability in this social context.
BG circuits in general and the AFP in particular are proposed to introduce variability necessary for exploration during motor learning (Graybiel, 2005; Ölveczky et al., 2005). In adult birds, song variability is modulated with social context, and song is less variable when directed to a female than when the same song is sung in isolation (Sossinka and Böhner, 1980). A neural correlate of this behavioral modulation is found in the AFP; firing is reduced and less variable during directed singing in Area X and LMAN (Hessler and Doupe, 1999; Kao et al., 2008). Immediate early gene expression in Area X is also differentially modulated (Jarvis et al., 1998). Lesion or inactivation of the AFP output nucleus LMAN substantially reduces song variability, suggesting that the AFP regulates both developmental and contextual modulation of song (Kao et al., 2005; Ölveczky et al., 2005).
Recent anatomical and physiological data shed light on a loop circuit linking Area X to DA neurons in the SNc and VTA (Gale et al., 2008; Gale and Perkel, in press). Through this circuit, song playback activates dopaminergic neurons, which in turn trigger strong DA release in Area X. Like neurons in the AFP, VTA neurons in adult zebra finches show singing-related activity that is modulated by social context (Yanagihara and Hessler, 2006), though it is unknown whether the neurons recorded were dopaminergic. Extracellular DA level in Area X is higher when adult zebra finches sing to a female, but not when they do not sing or sing alone (Sasaki et al., 2006). Together, these results suggest that the dopaminergic input to Area X could trigger changes in song variability depending on social context. Consistent with these ideas, we have shown that DA decreases the output signal of the AFP through its action on D1 receptors in Area X and that interfering with D1 receptor transmission in Area X abolishes social context-dependent changes in song variability. Moreover, we found that LMAN firing evoked by HVC electrical stimulation was shorter in duration when we applied DA in Area X. Such reduced and more precise AFP activity when DA was delivered to Area X resembles AFP song-related activity during directed singing (Hessler and Doupe, 1999; Kao et al., 2008).
In addition to introducing variability, the AFP may provide patterned signals to guide changes in motor output (Kao et al., 2008; Andalman and Fee, 2009). Such signals would most likely come from HVC and be transformed in the AFP, potentially through DA-dependent mechanisms. Indeed, DA modulates the intrinsic excitability of Area X spiny neurons and the strength of their glutamatergic inputs and is necessary for long-term plasticity of these synapses (Ding and Perkel, 2002, 2004; Ding et al. 2003). Here we show that DA also modulates direct excitatory input from HVC to Area X pallidal neurons. Moreover, we show that DA increases spontaneous activity in pallidal neurons and shortens their ISIs. This ISI shortening is likely to modify information transfer through the AFP (Person and Perkel, 2005; Leblois et al., 2009). In addition to the increased spontaneous activity, the reduction in firing variability under DA in Area X pallidal neurons is likely to reduce evoked and/or spontaneous firing in DLM (Leblois et al., 2009), and is most likely responsible for the decrease in song response in LMAN.
Interestingly, reward processing and social interaction processes share common neural substrates (Caldu and Dreher, 2007). In particular, DA release in the striatum signals reward prediction error, and may provide a reinforcement signal to the BG (Schultz et al., 1993; Phillips et al., 2003). As a reinforcer, phasic DA delivery could gradually shape motor behavior, thereby inducing a slow shift from motor exploration to the repetition of a successful behavior (exploitation, Sutton and Barto, 1990; Ishii Yoshida and Yoshimoto, 2002). Here, we highlight another possible role for the DA system in motor learning. According to our results, a change in DA level in the BG might rapidly trigger a switch between exploration and exploitation in learned behaviors. The proposed role of DA in balancing exploration and exploitation by reducing BG output signal is in line with several previous findings. First, DA-regulating genes are responsible for inter-individual differences in exploration and exploitation behaviors (Frank et al., 2009). Secondly, animals treated with drugs enhancing the DA signal such as cocaine or amphetamines induce behavioral stereotypy (Canales and Graybiel, 2000), and a recent study found that cocaine injections leading to stereotyped behavior in rats reduced evoked responses in BG output neurons (Aliane et al., 2009). Finally, differences in movement latency associated with different reward values, possibly reflecting a transition from exploration (when the action outcome is uncertain) to exploitation (when the action outcome is desirable), rely on D1-receptor transmission in the BG in primates (Nakamura and Hikosaka, 2006).
The BG are involved in several movement disorders, including Parkinson's disease (Chesselet and Delfs, 1996). In parkinsonian patients and animal models of the disease DA depletion is associated with increased firing variability in the output neurons of the BG (Boraud et al., 2002). The mechanisms described here and that allow DA to modulate firing variability in BG output may be at least partly responsible for these pathological changes.
DA acts on D1 receptors in Area X to reduce firing variability in pallidal neurons, decreasing AFP output. In social contexts associated with increased DA release in Area X, AFP output is diminished, leading to stereotyped song production driven by the monosynaptic HVC-RA pathway. On the other hand, in social contexts associated with low DA levels in Area X, the AFP output signal is enhanced and leads to strong AFP-driven song variability. Such modulation of the balance between exploration and exploitation may be a critical role of DA in motor learning.
We are grateful to Max Sizemore, Abigail Person and Sam Gale for valuable comments on the manuscript. This work was supported by NIH grants R01MH066128, R03DC009686 and P30DC004661.
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.


text:
June 9 to 16 // Something different for music lovers
Here's your guide to over 70 festivals in Winnipeg and around Manitoba.
When it comes to summer festivals, we have an abundance of choice here in Manitoba.
This, our last regular-ish paper of the year, is somewhat unconventional.
In the modern era, kinbaku is no longer used to bind one's enemies. Rather, it has been popularized as a form of sexual bondage or BDSM (an umbrella term that refers to bondage/discipline, domination/submission, and sadism/masochism) often referred to as shibari, which means "to decoratively tie."
Many dive into the animal rescue community in Manitoba expecting it to be filled with big-hearted people, then find the humans involved are not treated well at all.
Women, queer and non-binary people can find empowerment and self-sufficiency through learning and practising bicycle mechanics.
Traditionally, we've been taught about the environment in a way that's broken down and broken apart. Educating within the confines of four brick walls separates us from the world we're trying to learn about and other humans we're learning with.
Heritage museums exist to tell a story and to preserve local history. It is the job of museum curators to make that history accessible to everyone that visits.
Homelessness and addiction, and the resources that help
There are many reasons to be anxious right now. Midterms are approaching. There is still more winter ahead. Tweets are declaring the end of the world. The news is depressing with accusations of fascism in America and comparisons between the Trump administration and Germany's Nazi regime.
How can we stave off the panic when even the media is panicking?
At what point does our situational anxiety become a medical condition?
It's not always easy being green, but several Winnipeg organizations are working to make environmental initiatives more accessible across the board.
The far-right: what it is and how to stop it
The Middle Coast // Deep Dark Cave // Adam Hanney & Co. // Fox Lake // Ashley Bieniarz
If there's a more quintessential origin story than TUSK's, it would be tough to find it.
The duo behind Rosebud took a rather casual approach to forming the two-person band, and that's how they continue to operate.
June Killing Stones is a locally grown band united in their value of community and positivity. Brothers Joshua and Jake Letkeman were working on their craft for years before putting together a group.
Awaiting the Answer has come together with the common goal of bringing a new sound to Winnipeg's metal scene.
Kakagi (pronounced ke-KA-gi) is a new band whose name is derived from a small lake in northwestern Ontario.
Choosing just five new local bands to showcase in our annual Uniter Fiver contest is a pretty tall order. This year, we started with 16 amazing submissions and put them to readers and local music lovers to choose the final five.


text:
Friday - Jan 10, 2014
The Chief Minister of Goa has acted in a manner becoming of a dictator in much the same manner of high-handedness as allegations of his party's Prime Ministerial candidate when he decided to walk all the over the farmers of Merces in order to use their fields for Narendra Modi's 'Vijay Sankalp Rally.
Friday - Jan 10, 2014
After the Canacona tragedy struck last week, Sports Minister Ramesh Tawadkar publicly misled the people of Goa by falsely claiming that he had bought and has the sale deeds of the two flats he owns at the now controversial Ruby Residency in Canacona It has now been revealed that Ramesh Tawadkar infact
Thursday - Jan 9, 2014
Disgraced Justice A.K. Ganguly's resignation as Chairman of the West Bengal Human Rights Commission is too little too late. His sordid saga has sadly irreparably stained and blemished the entire Indian Judiciary. That such a person rose to be a Judge of the country's highest temple of Justice leaves
Wednesday - Jan 8, 2014
The January 12th Narendra Modi rally in Goa "Vijay Sankalp" is being organized by the BJP near Panaji on the agricultural fields along the Merces - Ribandar bypass. A political party mela being organized with blatant all out misuse of government machinery. Without the consent of the farmers owning those fields the BJP hounds last week high-handedly barged and trespassed
Wednesday - Jan 8, 2014
Similalar to the Commonwealth Games since 2006 there is sporting event held called The Lusophony Games which involves athletes coming from Lusophone (Portuguese-speaking) countries, namely those belonging to the CPLP (Community of Portuguese Language Countries), but also others where there are significant
Monday - Jan 6, 2014
The feast of the Epiphany also known as the Three Kings Feast is celebrated at Cansaulim with great reverence and devotion. Three young boys from the villages of Cansaulim, Arossim and Cuelim are selected to enact the Three Kings also known as the Magi or the Wise Men from the East, who followed the guiding star to worship the Child Jesus over 2000 years ago
Monday - Jan 6, 2014
The Bharatiya Janata Party (BJP) which is equally embroiled in rampant corruption of the highest order cannot dare take the high moral ground. In Goa, over the last 22 months we have all been witness to how corrupt and nepotism driven the Manohar Parrikar government has been. Despite his all out efforts
Monday - Jan 6, 2014
Honorable MLA, as a member of your constituency I would like to let you know that it is now almost 2 years since we voted for a change in Government. I had even come down just for 2 days to vote in the March 2012 assembly elections. However, today I am deeply disappointed to note that the change for the better has not happened. Benaulim/Colva is in the same state as it was 2 years back; if not worse
Monday - Jan 6, 2014
The concern regarding the credentials and genuineness of the Aam Aadmi Party (APP) is on account of it being strategically steered and geared by some Hindu extremist Rashtriya Swayamsevak Sangh (RSS) pracharaks. We are witnessing it in Goa too. Understandably perception is growing of it being the undercover incognito B team of the BJP with some naïve altar boys
Sunday - Jan 5, 2014
It is intriguing that we Goans cannot stand together with other Goans and form a strong united front to save Goa. Why should we join non Goan Parties instead of forming a good, honest strong Goan party? Why not revive the old UNITED GOANS party once formed by the Dr. Jack de Sequeira and his friends?


text:
A criminal appeal is a formal request to rehear a case that has already been decided -- a request that a new court reconsider the decision of the first court. When one or both sides of a case that has already been decided think there was a mistake made at trial, they can file an appeal. An appeal is entirely different than a jury trial. There is no testimony taken. The court of appeals decides the case entirely upon the written briefs filed by your attorney and the offie of the Attorney General who represents the prosecution and asks that the conviction be upheld.
Copake is a town in Columbia County, United States. New York, United States. The population was 3,278 at the 2000 census. The town derives its name from a lake, which was known to the natives as "Snake Pond. " The town borders Taconic State Park. The Town of Copake is on the eastern border of the county.
Criminal Appeals Law Lawyers In Copake New York


text:
Storm Window Coffee Tables
This was my second project, made entirely from used materials (except for a few wooden dowels to attach the legs to the windows. I used some old storm windows for the tabletop and old railing spindles for the legs, all of which I purchased at Construction Junction. I even topped it all off with some coasters made from old tiles. All in all, a beautiful project that was relatively cheap.


text:
Once there are annotations then the whole PDF (even non-annotated sections) becomes gibberish when copied (select text and cmd+c) and pasted into a text editor. Post them below or email us! Thanks mucho. Do you know how to fix this? Source
Deleted! Reply With Quote 01-08-2010,02:56 AM #2 VegasGeorge View Profile View Forum Posts Visit Homepage Member Since Sep 23, 2006 Location Las Vegas, NV USA Posts 893 Specs:iMac, and Macbook Pro Try When i paste the command to the terminal , it prompt me for a password . One by one I am disabling annoying features of Lion and this was one of the worst one.
Patrick Reply Ian Johnston says: September 15, 2011 at 1:54 am Patrick - thank you very much for your input here. DaveCowen 10:10 PM EDT, Jun. 25th, 2013 @Scott Mc Laughlin - I just tried a quick test on a pdf that I can copy/paste from. One thing though…I can't get rid of Reader as it does not render every PDF correctly, particularly complicated ones generated by Adobe programs with layers… it always shows all layers, even I've noticed that my annotations don't always show up on Windows machines that use Adobe Reader.
But there seems to be no support for it being changed, translated or saved in Preview, regardless of file type. Reply Snow Leopard: SpotLight « How to get it done says: September 19, 2009 at 6:43 am […] Reference: Spotlight won't work? […] Reply spambot says: October 21, 2009 at 3:17 It was working up until a couple of days ago, I cannot isolate what I have done differently. Tried the "sudo" command and it did it for me.
i have the same problem, some figures are not displayed, obviously they are ok in acrobat reader. Now there is a go around I have been using. aaaaaaaargh Reply spambot says: October 21, 2009 at 3:21 am … and tip 2 gives "error no index found for volume" Reply Thomas says: November 5, 2009 at 3:46 pm Wow, http://apple.stackexchange.com/questions/14855/finder-cover-flow-icon-preview-not-working However, since I've purchased my MacBook Pro I've had continuous problems with Spotlight so shy away from it.
Reply Rosco says: August 10, 2010 at 05:13 Hi Scott, I believe the problem you are experiencing is different from (but similar to) what is described in the article. Plus, when they broke (because they always do eventually), our server could provide some useful information about what was there at some previous point in time (a cached version of the Let's say you're using Snow Leopard and thus Preview 5.0 (501), and you open one of our Take Control ebooks - the 2.1 version of "Take Control of Mac OS X A bugged print screen is a great Mac feature?
Installed MainMenu, tried few commands. find this I copied the Snow Leopard version to my applications folder as "PreviewSnow", and set it as the default and then copied Preview (Lion) back to my applications in the off chance New From Clipboard Greyed Out In Preview Yep, you can see why it's pure genius. Photoshop Clipboard Not Working Your issue may have been corrected with an updated version of the software.
Patrick Reply Patrick Kinsella says: April 1, 2008 at 7:03 pm Sorry: you need to know that the visible/invisible switch works only after a relaunch of Finder (via Command-Alt-Esc). this contact form Adam Engst 2009-09-08 08:05 That's an excellent point, Matt, and something we've certainly thought about over the years. What next? If that doesn't fix it, I'd probably reboot and test.
The blue thing came up after restart… it said it was going to take 2+ hours, but then it ended in like 30 minutes. ioT Botnets – There's Only So Much Apple Can Do Ireland to Formally Appeal EU Ruling on Apple Taxes, As Expected iOS 10: Airplane Mode Doesn't Turn Off All Radios (GPS, Can you tell me how you're going about copying the album artwork to the clipboard in iTunes? have a peek here Follow us if you like.
Home Mac OS X iPhone iPad Tips & Tricks Jailbreak News iOS Spotlight won't work? That also does nothing on the HD, only on inserted USB Flash drives. Unless you decide to really start fresh and not migrate and user settings.You can create a new User go to System Preferences >> Accounts >> "+" (make it an admin acct)
Draws attention and crowds. All Rights Reserved. John Martellaro | November 11, 2016 Stick It In (or On) Your Ear Bob LeVitus | November 9, 2016 TMO Weekly Sponsor Black Friday Giveaway From MacXDVD. This is the default configuration for Preview's toolbar.
But, it would be nice to have short URLs in some situations, and particularly short URLs to which we could assign some useful alias information. I updated my MacBook Pro 15″ 2.33 ghz, everything worked great. Fix a broken Spotlight menu with these troubleshooting tips Feb 15, 2007 - 114 Comments Spotlight is probably the greatest feature to hit the Mac OS in years, once you get http://vicnot.net/not-working/leopard-spotlight-not-working.html I'm trying to even just transfer Lion's version of preview onto Mountain Lion and it's not working.
Reply Leave a Reply Click here to cancel reply. I have used all the tips above, repaired permissions but no joy. We use your email address only to send you a one-time verification message confirming that you posted this comment. If you drag the attachment to the desktop and then open the clipping using Preview it should just open.
Reply iphonermac says: February 24, 2010 at 7:14 am Never mind…It seems that trying various combinations of these methods fixed the problem. scott mc laughlin 7:33 PM EDT, Jun. 25th, 2013 One infuriating thing I'e found is that if I use annotations in Preview then I lose the ability to copy text from Would also like to use the "old" Sow Leopard Preview in Mountain Lion. Press the enter key when all the filenames and paths have been entered.
thanks for making me feel stupid :D Reply Graham says: September 5, 2012 at 7:49 am If you can't fix problem #2 for one reason or another using any of the Reply May Tha-Hla says: July 24, 2012 at 7:33 am As a complete amateur when it comes to Terminal etc., would someone kindly explain in baby steps just what to do Reply alapdia says: November 9, 2009 at 10:36 am I would like to spam you but i am not good at ait Reply TubeUil says: November 25, 2009 at 6:42 am the other solution Activity Monitor >highlight UL System Server> hit the red button … that did not work No red button !
Privacy / DMCA contact / Affiliate and FTC Disclosure Fixed | Fluid | Fluid HD Logged In! Do you want me? When I checked permissions, it indicated that Preview was owned by the system file and even when I changed the permissions, was still the default app. WHen I figure it out, I'll post it.
Reply Philinda says: December 12, 2011 at 7:36 pm Damn, I wish I could think of somethnig smart like that! This behavior may be due to the quarantine flag the system places on newly downloaded items, including pictures.