text:
When I think about romance, I think about Italian food. Perhaps it's the fact that Garlic is an aphrodisiac. Or, maybe it's that famous pasta kissing scene from Disney's Lady and the Tramp. Either way, just in time for Valentine's Day, I'm serving you Pizzeria Mozza from Newport Beach.
Brought to you by three masterminds – chef Nancy Silverton of La Brea Bakery and the former Campanile, chef & restaurateur Mario Batali, and restaurateur & TV host Joe Bastianich – Mozza set the pace for a slew of upscale chains to hit the OC, like Fig & Olive and Red O.
Perched on PCH across from the Balboa Bay Club, Pizzeria Mozza stands tall, bright orange, and loud. The inside matches with a bustling crowd from the packed bar to the dining room.
It serves as a great buffer for a nervous first date, or a reason to request a large table in the intimate dining room to share plates with 8 of your friends, just like the hand model & I did.
- excellent array of Leeks, spicy 'n caramelized flavor with spongey yet crispy crust, Coach Farm Goat Cheese, Leek, Scallion, Garlic & Bacon Pizza $18
- bursting with juiciness, sweet 'n tart, Bacon, Salame, Fennel Sausage, Guanciale, Tomato & Mozzarella Pizza $19
- bitter, salty 'n spicy, could use more creaminess to cool it down, Rapini, Cherry Tomato, Anchovy, Olive & Chile Pizza $16
- like heaven for a vegetarian, hearty, salty 'n tangy in all the right ways, Crispy Goat Cheese with Umbrian Lentils $12
- melt in your mouth, pesto that's heavy-in-a-good-way, Mozza Caprese with sweet Roasted Tomatoes $13
- citrus burst counteracts its natural bitterness, Bruschette with Chicken Livers, Capers, Parsley & Guanciale $9
- dripping with oil and hard to wrap your mouth around, Bread (complimentary)
- like Italian Cracker Jacks on crack, fluffy 'n salty caramel dream, Caramel Copetta with Marshmallow Sauce & Salted Spanish Peanuts $10
- a menage-a-trois in your mouth, Bittersweet Chocolate Tartufo with slippery Olive Oil Gelato and sexy Sea Salt $12
Mozza's menu offers a vast variety of dishes, that even our table of 8 couldn't conquer. From fresh homemade pastas & pizzas to plates of the day like Crispy Duck Legs, there's something for everyone to love.

domain: food


text:
By John Celock
Kansas lawmakers have advanced the governor’s push to merge the state’s securities and insurance regulators but have added a new ban on securities regulators from working outside state government.
The state House of Representatives advanced legislation Wednesday that would move the state securities commissioner from under the governor’s oversight and merge it into the insurance commissioner’s office. Under an amendment the House adopted, the securities commissioner and attorneys in the securities office would be barred from holding outside employment, which is common currently.
“At present the Kansas securities commissioner makes $110,407 and he also according to the county clerk in Jefferson County serves as their county attorney where he makes $67,000 and he’s the Jefferson County counselor where he makes $36,000,” Rep. John Carmichael (D-Wichita) said in sponsoring the amendment. “Our securities commissioner is making in excess of $200,000.”
Carmichael said that he had issues with Securities Commissioner Joshua Ney or other attorneys in his office holding state posts and other jobs outside of state government. He noted that the state pays $600,000 a year for legal staff in the securities commissioner’s office.
The merger of the two offices was first proposed by Brownback in his State of the State address in January, as part of an efficiency move by Brownback. The move would place the securities commissioner’s office under elected Insurance Commissioner Ken Selzer’s (R) office. Under the legislation, the insurance commissioner would appoint the securities commissioner with the consent of the state Senate, which is currently done for gubernatorial appointees to the post. The prosecutorial role of the securities commissioner would shift to the state attorney general’s office.
The move would be similar to what several states have done in merging insurance and securities regulation into a joint agency, including New York which made the move in 2011 when lawmakers merged the Department of Banks and the Department of Insurance into the Department of Financial Services. Brownback did not propose moving the state banking commissioner or the state credit unions director from the governor’s oversight to the insurance commissioner’s.
Rep. Vic Miller (D-Topeka) proposed an unsuccessful amendment to move the securities commissioner to the attorney general’s office. Miller and Democrats said that the move would allow for the securities regulation to be separate from insurance and would work well with the attorney general’s role in consumer protection. Carmichael said that it would keep the elected insurance commissioner from being able to raise money from two industries regulated by the office.
Republicans objected to the measure, noting that it is unknown if Attorney General Derek Schmidt (R) wants to handle securities regulation and if it would work. In addition, concerns were raised about the attorney general’s office not being able to perform certain licensing functions that the insurance commissioner’s office already handles.
“We have not had any study of consolidation under the attorney general’s office,” House Judiciary Committee Chairman Blaine Finch (R-Ottawa) said. “We don’t know what cost savings it would have. We don’t know if the attorney general wants it.”

domain: news


text:
With stunning and endless views of rolling waves, the Praia D’El Rey Marriott Golf & Beach Resort is truly heaven sent, conveniently located only 45 minutes’ drive north of the country’s capital Lisbon.
From marble bathrooms to the finest linens, no detail has been spared to ensure your comfort and pleasure.
The hotel boasts 177 deluxe rooms, including 9 suites, all with private terraces or balconies offering unique views of either the Atlantic Ocean or the beautiful 18 holes championship golf course and gardens.
All guest rooms have been designed to the highest specification and are elegantly furnished and spacious. Impeccable 24-hour service is provided by the hotel’s friendly and helpful staff.
Our restaurants offer the best of Portuguese cuisine with delicious menus prepared by our talented chef Carlos Gonçalves.
This content is hosted by Google, and you did not accepted our functional cookies, you can change your cookie settings and allow functional cookies in order to see this content.
|Genre de Chambre
|Chambre lits jumeaux

domain: travel


text:
A Reminder: Don’t Be a Slave to your Dreams
Sometimes you are obsessed with what you think your life would be like if you had all the things you wished you had. Instead of enjoying your life as it is, even if it is not comfortable. But surely things will change because the situation is not permanent. NB: Even though you do not know… Continue reading
Clarity Makes Your Dreams Become Your Realities
When you are absolutely clear about what you want, you can then think about your goals most of the time. And the more you think about them, the faster they will materialize in your life. The process of asking yourself questions about your goals in each part of your life begins to clarify your thinking… Continue reading
Get Fired Up In Achieving Your Dreams
What drives you to get up in the morning and chase your dreams? Why are you working until 10PM on a weekend? Why are you at home working when you could be out partying or hanging out with friends? It’s easy to get lost in your own head and allow procrastination to put your dreams… Continue reading
Dreams Do Come True! French Star “Mbappe” Dream That Became a Reality
As we all know the world cup is over. A lot of fans were disappointed with there team, while some were surprised at the performance of young players in the tournament. No doubt France deserves the cup. From the beginning of the tournament, they put up a good fight with other teams and conquered. But… Continue reading
14 Things People Don’t Realize You’re Doing Because You’re Quietly Chasing Your Dreams #BriannaWeist #inspiration
1. Keeping to yourself. Maybe you sometimes spend Friday nights working on projects. Maybe you put traveling over home ownership; maybe you put your 10,000 hours over swiping on another dating site. Maybe you weren’t able to keep up with your high school friends in some ways, but whether or not the outside world can… Continue reading
My goal is to help you live a life of Gratitude, Inspire you to never give up, and motivate you to keep going till you succeed.
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domain: psychology


text:
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domain: library


The Freedom City shopping mall accident plunged into mourning the country which hosted its first New Year’s celebrations in three years.
“In the outskirts of Kampala, in a shopping mall there was a concert that was organised and we received information towards midnight, around midnight, that there was a stampede,” Patrick Onyango, Kampala Metropolitan Police spokesperson recounted.
“Three children died on (the) spot and six others were pronounced dead on arrival in a hospital.” He added that the youngest victim was a girl “aged 10 years and the oldest among the dead was a 29-year-old woman.”
It is alleged that the incident occurred at when the event’s MC encouraged attendees to go outside and watch the fireworks display. Once the display ended, a stampede ensued. Emergency responders arrived on the scene and transported the injured.
“The injured are recovering. There were also about 10, but they are recovering. They are not so badly off. But mostly these were children.”
The shopping mall is a popular venue for music concerts.
According to the Ugadan police force spokesperson, apart from Freedom City’s tragic incident, the End of Year and New Year celebrations which included 1618 fireworks displays, were generally successful.
On Sunday, no arrests had been made yet.Police detectives are still trying to establish the number of people who were inside the mall.


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BELOIT - The League of Women Voters (LWV) of Beloit held their annual meeting on April 27.
Retiring board members recognized were Deb Fallon, Dorothy Stroud and Lynda Owens.
The Making Democracy Work award was presented to Daksha Howard from Beloit College for her work with high school and college students being involved in voting. Bette Carr received the Joyce Schoenfield Making Democracy Work award for her untiring work in all aspects of LWV.
The program presentation for the meeting was Ruth Kolpack from Justice Overcoming Boarders and Dorothy Harrell from NAACP on "Owning the Problem, the Incarceration Rates in Wisconsin."
For more information on the Beloit area League, visit my.lwv.org/wisconsin/beloit.

domain: sociology


text:
Bill Willingham has been writing, and sometimes drawing, comics for more than twenty years. During that time he's had work published by nearly every comics publisher in the business and he's created many critically acclaimed comic book series, including Elementals, Coventry, PROPOSITION PLAYER, DAY OF VENGEANCE, SHADOWPACT, JACK OF FABLES and the Eisner Award-winning Vertigo series, FABLES. Additionally, his work has been nominated for a number of other awards including the Harvey and Ignatz comic industry awards as well as the Hugo and International Horror Guild awards. He lives somewhere near a good poker room.
FLINCH BOOK ONE
The first eight issues of Vertigo’s quirky, star-studded horror anthology is collected for the first time! These stories spotlight a small-town cult, a violent gang of senior...
Enter for Your Chance to Win an Autographed Complete FABLES Collection and More!
As you may have heard, the FABLES Finale is near so we wanted to celebrate with this extremely cool collection. Take a look at these, you know you want them…...
FABLES VOL. 22
It’s the final trade paperback volume of FABLES! No, wait – it’s FABLES #150, the grand finale of the best-selling, award-winning comic book series! And it’s also an original...
FABLES DELUXE EDITION BOOK TEN
In an epic from FABLES #77-82, JACK OF FABLES #33-35, and THE LITERALS #1-3, the existence of the Fables is threatened by The Literals, who just don’t like their messy, mythical...
POLL: Who's Your Favorite Fable?
FABLES VOL. 22, the graphic novel-sized final issue of Bill Willingham and Mark Buckingham’s beloved modern...
FABLES DELUXE EDITION BOOK ELEVEN
These tales from FABLES #86-100 include the epic “Witches,” in which Bufkin, the meek and mild flying monkey, is trapped with the evil witch Baba Yaga, while Frau Totenkinder and...
FABLES VOL. 21: HAPPILY EVER AFTER
In this penultimate volume of Bill Willingham’s FABLES, the residents of Fabletown look to live “happily ever after,” but there is a steep price to pay for happiness as Rose Red...
The end of the Fables world looms as an epic battle brews between two sisters. Will Rose Red follow fairytale tradition and betray Snow once more?

domain: entertainment


Hinges are generally small movable hooks, fasteners or joints that enable a door to be fastened to a doorframe or a lid to a box. Hinges are the pivotal points on which the door or lid turn when opened or closed. There are two basic types of hinges which are concealed and exposed and these two types are further divided into several styles. Concealed hinges are also known as European hidden hinges. The most important factors for perfect hinge installation are making sure that you select the proper hinge selection and be certain to take accurate measurements.
Hinges come in reversible types and right-hand and left-hand position vertically mounted types. If you stand on the outside of the door and it opens from you to the right, you need a right-hand hinge and if it opens to the left, you need a left-hand hinge. Loose-pin hinges will let you change the handing of the hinge by taking out the pin, inverting the hinge and putting the pin back in.
Hinges are available in a wide variety of sizes and types. Many of them are made from fire, water and corrosion-resistant materials and finishes such as steel, cast iron, brass, and aluminum, etc. Exposed hinges come in 3/8" inset and variable overlay. The 3/8" inset hinges are designed to hold lip-mold doors where the door fits 3/8" into the cabinet hole. The variable overlay hinges make sure that the door covers the hole completely with some overlap. As far as concealed hinges are concerned, one must be aware of the overlay or the inset. The ½" overlay is most common type that is used.
A butt hinge is the most commonly used hinge to hang doors. The projection and parliament hinges are used when the door opens very wide (approximately 180 degrees) and clears a projection. The screw holes on these hinges are placed towards the edge of the hinge flap and they help to increase the angle of the opening. The tee hinge, band and gudgeon hinges are lightweight and weather-resistant types that are generally used on garden gates, wooden entrance gates and garage doors.
Hinges should ideally be fixed with screws that match the hinge material. For example, if the hinge is made of brass, it should be fixed with a brass screw and not an aluminum screw. Otherwise, the contact between the two dissimilar metals will cause the hinge to corrode.


text:
Prime Minister Narendra Modi, who was in Odisha to attend the BJP national executive meet, felicitated the families of freedom fighters in Bhubaneswar today.
"Today, the history was recalled with pride. It is my honour to see the descendants of martyrs. Unfortunately, the long years of freedom movement was confined in few persons and specific period. We should recall the events and contribution of everyone who participated in the freedom struggle," said the Prime Minister.
Later, the Prime Minister took to Twitter to "write about the freedom fighters" and also shared photographs of the felicitated relatives of the martyrs.
These freedom fighters participated in the struggle for Independence against the colonial British rulers. Some of them took part in the famous Paika rebellion.
WHAT WAS PAIKA REBELLION?
Paika Rebellion was an armed rebellion under Bakshi Jagabandhu against the British East India Company's rule in Odisha in 1817. The freedom fighters were known as Paiks.
The Paiks served as warriors and undertook policing during peace-time. The Paika rebellion was brutally squashed by East India Company's forces.
"From the Paika warriors to Quit India, Odisha has a rich history of freedom fighters, who fiercely resisted colonialism," the Prime Minister said.
THE FREEDOM FIGHTERS NARENDRA MODI WANTS YOU TO KNOW:
Taking to Twitter, Prime Minister Narendra Modi named more than 15 freedom fighters and paid tributes to them. While some of the freedom fighters mentioned by the Prime Minister were part of the Paika rebellion, others fought the British at different points of time. Little is known about most of them.
"India will never forget these courageous freedom fighters, who lost their lives so that fellow Indians can breathe the air of freedom," said Narendra Modi.
Here are the freedom fighters mentioned by the Prime Minister:
#Jayee Rajguru: Talking about Jayee Rajguru, Prime Minister Narendra Modi said he resisted colonialism tooth and nail. "His leadership and valour will always be remembered," he said.
History books remember him as the first person to revolt against the British Raj in the provision.
#Bakshi Jagabandhu: He integrated people from all walks of life in the struggle against colonialism, Narendra Modi said.
Bakshi Jagabandhu was the commander of the forces of the king of Khordha, and widely regarded as one of the earliest freedom fighters of India.
#Dinabandhu Samantaray Mohapatra: Narendra Modi described him as an accomplished warrior, who was responsible for protecting 12 forts. He was at the forefront in resisting the British.
#Dama Subudgi Mangaraj: He was an able organiser, said the Prime Minister. "He went village to village to mobilise people. He was punished by the British and died in jail," Narendra Modi said.
#Samant Madhab Chandra Routray: Narendra Modi described Routray as an expert at guerilla warfare. He was also known for "his care towards the poor and the needy".
#Pindiki Bahubalendra: "He was a fearless warrior, who battled the British at Cuttack and Puri" is how the Prime Minister described him. Bahubalendra was later arrested by British and martyred, said Narendra Modi.
Pindiki Bahubalendra was part of the Paika Rebellion in 1817.
#Krutibas Pattasani: "The courage of Krutibas Pattasani gave sleepless nights to the British. He even defeated the British at Banapur," the Prime Minister said.
Like Pindiki Bahubalendra, Pattasani too was part of the Paika Rebellion.
#Rindo Majhi: He created problems for the colonial forces at Kalahandi, and was eventually arrested and hanged, said Prime Minister Narendra Modi.
#Veer Surendra Sai: He fought the British from 1830s onward and his struggles continued up till 1864, when he was arrested, said the Prime Minister.
Surendra Sai eventually died in Asirgarh Jail in February 1884.
#Madho Singh: "He was determined to free India from imperialist powers. He lost his sons while battling the British," the Prime Minister said.
#Chakhi Khuntia: Describing Khuntia, Narendra Modi said, "A 'panda' at Puri, Chakhi Khuntia was drawn towards freeing India from colonialism. He worked closely with the army of Rani Laxmibai".
Khuntia was a priest at the Jagannath Temple in Puri and also a poet. He died in Puri in 1870.
#Chakara Bisoyee and Dora Bisoyee: Both fearlessly fought the British in Kandhamal, said the Prime Minister. "In that era, the prize for Chakara Bisoyee was Rs 10,000," Narendra Modi said.
#Raghunath Mohanty and Dibakar Parida: The Prime Minister described them as friends, who "fought the British in Ranpur in 1930". According to him, both were hanged in 1941.
#Laxman Nayak: "Laxman Nayak galvanised people during the Quit India movement. The British tried everything to break his morale but he remained firm," said Narendra Modi.
A civil rights activist, Nayak led a peaceful demonstration in front of a police station. He was hanged in Berhampur Jail.
#Laxmi Indira Panda: Of her, Narendra Modi said that she joined Subhash Chandrs Bose's Indian National Army and "was undeterred in the effort to ensure that India is freed from colonial rule".
Laxmi Panda died in 2008.

domain: politics


text:
I have a question about the recipe "Whole Wheat Banana & Cardamon Pancakes (American Style)" from madrascoffee.
Are you channeling your best self with this comment? (If you're not sure, check out our Code of Conduct.)
I have a question: where are the bananas? Also, if I want to use rice flour, should I follow the same process?
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The ACLU is suing Illinois for withholding changes to birth certificates for transsexuals until they've had genital surgery.
The ACLU filed the lawsuit on behalf of three transsexuals – Lauren Grey, Victor Williams and Nicholas Guarino -- who claim they were denied correct gender markers on their birth certificates because they had not undergone genital surgery.
"Illinois long ago recognized the importance for transgender people to have a birth certificate that accurately describes who they are," said John Knight, director of the lesbian, gay, bisexual & transgender project at the ACLU of Illinois. "We've been telling the department for two years that its arbitrary surgery rules clash with the medical standard of care for transgender people and make it impossible for most transgender people to correct the gender on their birth certificates."
"We took them at their word when they said they would make an appropriate change, but all we've seen is more delay," he said. "It's time that they did something to fix that."
The Illinois Public Health Department declined to comment, citing the pending litigation.
But the department's policy states that people can have their gender changed on their birth records "after undergoing surgery intended to or having the effect of reflecting, enhancing, changing, or reassigning gender."
The suit claims that before that policy was enacted in 2005, the department routinely changed the gender marker on birth certificates for people who had undergone a gender confirmation procedure that didn't include genital surgery.
The policy puts transsexuals at risk of discrimination in employment, housing, access to public services and makes them vulnerable to physical or emotional harm and embarrassment if their identities are revealed, the suit contends.
"I've struggled for too many years to be able to live as a woman only to have a birth certificate that constantly reminds me of the gender I've worked so hard to put behind me," said Grey, who has changed her gender on all of her government-issued identifications except her birth certificate. "I am who I am."
Grey said her application for Women Business Enterprise certification with the federal government was denied because her identity on her government documents did not reflect her female gender. As a result, she said she left the business and was forced to take a new position with a lower title and status.
Guarino said he has been unable to change his gender on his driver's license or other Texas identifications because of his birth certificate.
Williams said having a birth certificate that identifies him as a female thwarts his ability to seek employment as a man, gain access to other private and public services and puts him at risk of embarrassment and potential violence.

domain: law


text:
A guy shared a story of how he had wanted to date a girl that he always see around in the gym and proceeded to ask another guy (A) who was always hanging out with her if she was single.
(A) told him that they were only friends and he would help him ask the girl but while he was asking halfway, the girl mistook it as he was asking her out instead and told him that she has been waiting for him to ask her out for the longest time.
As a result, the guy accidentally became (A)’s wingman instead.
Here is the story
“At my gym over the past couple of months I’ve noticed a cute girl who works out around the same time I do and I was interested in asking her out, but I’m kind of awkward and I don’t approach girls in the gym.
A few times I’ve seen her talk to a guy (A) who she seems to be friends with, I haven’t seen anything romantic or flirtatious between them and I was pretty confident they weren’t together. So my awkward self decided to talk to (A) and ask him if she was single.
He said he had no idea, that they know each other because they used to work together, and have been friends ever since. As he was talking she came walking over to us, and before I could stop him he turned to ask her:
(Paraphrasing what I remember)
(A): hey are you single? Becau-
Her: God it’s about time! I have been waiting forever for you to ask me out.
Her: …is that not what you were asking?
(A): yeah it is! Are you free Saturday?
Her: absolutely! Text me, I have to get going soon.
Throughout this I was just standing there like a third wheel watching like an idiot. After she left (A) turned to me with an apologetic look on his face and said he’d always had a crush on her but never acted because he didn’t want to hurt their friendship if she didn’t feel the same way.
I told him I completely understood and was happy for both of them.
All in all, -1 for me and +2 for them = net gain for love.”
Image source: Unsplash.com


text:
Garmin MapmyIndia v7.1
[Only registered and activated users can see links. ]
MapmyIndia version 7.1 maps cover:
•7.1 million places
•1.9 million road kilometers
•4,751 cities at street-level
•39 cities at house-level
•36 cities in full 3D
•Maps in 4 local languages
•City guides and rich points of interest
•Photographs of places to visit, stay, eat and shop in popular tourist destinations and cities
•Voice guidance in 9 regional languages
Thelocal languages will include Hindi, Punjabi Tamil and Malayalam.
MapmyIndia’s version 7.1 maps – India’s most comprehensive, exhaustive, detailed and updated map coverage – provide first- and best-in-class advanced navigation and driver assistance features with visual cues, such as: •Extended lane guidance (telling the user which lane to get into prior to a turn or exit)
•Junction views (how the upcoming intersection looks in real-life)
•Sign-posts (which major landmarks, localities and cities lie in the direction of upcoming turns)
•Day-time based turn restrictions (based on time and day, whether the particular road is 2-way or one-way in a particular directions)
•Live traffic (what is the live congestion on roads in the city)
MapmyIndia continues to offer the most localized navigation experience, with city guides and rich points of interest (which provide detailed descriptions and photographs of places to visit, stay, eat and shop in popular tourist destinations and cities) voice guidance in 9 regional languages and is also introducing regional maps in 4 local languages (which displays the map in the regional language), to aid users who prefer to communicate and interact with the navigation systems in local Indian languages and not just English.
MapmyIndia’s GPS navigation system enables accurate turn-by-turn voice-guided navigation to the building doorstep, not just a nearby landmark or the general locality. The version 7.1 MapmyIndia maps continue to provide best-in-class full 3D coverage of India, with digital elevation models and terrain across India, thousands of full-scale 3D landmarks across India, which offer life-like 360-degree views of popular tourist and commercial landmarks, and full 3D city models containing 3D buildings in residential and commercial areas for 36 major cities across India, including all the major metropolitan areas.
↓↓↓ Don't post thanking posts. Use the "Like this post" link below instead.
And if you download a torrent I have posted, please keep seeding, as I´m only seeding a few days until it´s well seeded
Hello everyone, I'm new here!
The map and Garmin India 2013.30 which is more detailed?
Navigate the streets with confidence. This product provides detailed road maps and points of interest for your device, so you can navigate with exact, turn-by-turn directions to any address or intersection.PHP Code:Please Login or Register to see the links
- Includes more than 1.38 million km (860,000 mi) of roads throughout India, including NH-8 from Delhi to Mumbai, and NH-7 from Varanasi to Hyderabad.
- Displays more than 7.1 million points of interest throughout the country, including restaurants, lodging, banks, ATMs and shopping.
- Gives turn-by-turn directions on compatible devices.
- Speaks street names (example: "Turn right on Main Street").
- Includes navigational features, such as turn restrictions, roundabout guidance and speed categories.
- Contains traffic data for compatible devices that use traffic receivers.
And the MapMyIndia:
With MapmyIndia Navigation map data productPHP Code:Please Login or Register to see the links
- Get the most reliable and accurate directions to over 10 million destinations of India
- Richest understanding of local addressing and naming nomenclature
- Accurate routing with help of over 100 navigation attributes within the road database like alternate names, turn restrictions, one way streets, physical barriers etc.
- Extensive coverage of points of interest making it easy to find and reach your favourite restaurants, closest ATMs, fun-filled tourist destinations, emergency services, convenience stores and more locations spread out over multiple categories
- Enriched with advanced features make it a world-class navigation mapping product
- Door-to-door navigation with house number data
- 3-D city models and 3-D landmarks
- Regional content with map of 9 regional languages
- Phonetic database to enable Text to speech and Speech recognition functionalities
- City guide and guided tours
- Junction views and signposts
- ADAS (Advanced-driver assistance support) features
- Field proven maps where MapmyIndia maps are used by millions of people who help keep our data updated and continuously improve the datasets
MapmyIndia has a strong track record of releasing regular updates and maintaining *version controls. The latest navigation map version released is version 8.0.
Coverage version 8.0
- Street level navigation
- 1.90 Mn kms of attribute-rich road network coverage connecting all towns and villages across the country
- All 4,787 cities of the country at street level covering major/minor streets and destinations within these cities
- 5,79,000 villages with complete road connectivity between them
- 10.33 million points of interest spread across 64 categories, customised especially for navigation in India
- Door-to-door navigation
- 46 cities with house number level data covering over 3.39 millions addresses
- Premium navigation feature coverage
- 3D features
- All India Digital elevation model
- 3D city models for 46 cities
- 1250 textured 3D landmarks
- Maps in 9 regional languages
- Phonetic dictionary enabling Text-to-speech and Speech recognition
- City guide and Guided tours
- Junction views and Signposts
This numbers seems the answer is the MapMyIndia is better in coverage… But I never been on India; so, one with experience must answer this question!
Last edited by Pungente; 16th October 2013 at 17:59.
What maps is best for Goa?
Какая из двух будет лучше для Goa?
Hi Buddy, File does not exits on above post:
Anyone can help to get that files for my device ...?
use other links in thread.
Other link does not contains good map ... I downloaded and tried


text:
What’s the difference between a clinical psychologist and clinical neuropsychologist?
People often ask me this question after i tell them i am a neuropsychologist, “So, what’s the difference between a psychologist and your neuro-thing?”
good question! Firstly, there are many types of psychologist, and the most common type of psychologist that people think of is what we call a “Clinical Psychologist“.
A Clinical psychologist is someone who can assess, diagnose and treat psychological and mental health problems. Some common mental health problems include anxiety and depression.
A Neuropsychologist, on the other hand, is someone who can assess, diagnose and treat of psychological disorders associated with conditions affecting the brain. For example, we can look at thinking and behaviour changes after stroke or head injury, assess someone who might have dementia, and assess for learning difficulties.
The simplest (but not necessarily the most professional) way to describe it, is that clinical psychologist deals with the ‘mind’ (or heart), and a neuropsychologist deals with the ‘brain’.
Of course, clinical psychologists and neuropsychologists often work closely together.
Ok, so are your assessments different?
A Neuropsychological assessment is quite different to a clinical psychology assessment. A neuropsychologist uses a series of tests to examine thinking areas such as memory, attention, learning, processing speed and abstract reasoning. We link this information to the person’s brain changes, and provide information about the impact of their thinking changes on the person’s day-to-day living.
…can a clinical psychologist also do this assessment?
That’s similar to asking, “My mum can drive, could she race in F1 competition?”
Kind of yes….mostly, no.
Neuropsychologists are specifically trained to make these interpretations and reports. Neuropsychologists have a deeper understanding of brain and thinking processes that clinical psychologists do not. Conversely, Clinical psychologists have a deeper understanding of mental health issues, and how to treat such conditions.
We are even quite different in the ‘repetoire’ of tests that we use! Neuropsychologists have tests that look at thinking changes associated with damage to certain areas of the brain, we have different types of memory tests, different types of attention test etc.. Clinical psychologists have assessments that look at severity of depression, personality tests etc..
So, the ‘brain’ eh?
That’s right. So whenever you think, see or hear about someone who has thinking changes from some sort of possible brain injury…that person is likely to see a neuropsychologist. Many things can affect the brain (for more information see here), so you may hear about us more often!

domain: medical


The Federal Reserve raises interest rates while claiming officials have no idea what caused the collapse of Silicon Valley Bank! The White House refuses to answer questions about payments from communist China to members of President Biden’s family. HHS Secretary Xavier Becerra cannot seem to answer any question about anything while in front of Congress. Piers Morgan sits down with Ron DeSantis and discusses Donald Trump and Vladimir Putin. A self-proclaimed time traveler predicts that aliens will invade the Earth sometime later today. Joe Biden has weakened America’s alliances, not strengthened them.
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Calcium Acetate Anhydrous is a form of Calcium Acetate. Calcium acetate is a chemical compound which is a calcium salt of acetic acid. It has the formula Ca(C2H3O2)2. Its standard name is calcium acetate, while calcium ethanoate is the systematic name. An older name is acetate of lime. The anhydrous form is very hygroscopic; therefore the monohydrate (Ca(CH3COO)2•H2O) is the common form.
We are one of the leading manufacturers, supplier, exporter and stockist of Calcium Acetate across India.
Applications of Calcium Acetate Anhydrous
Calcium Acetate Monohydrate E263 is used in candy and confections, cake batters and mixtures, pie fillings, puddings, certain cheeses, gelatine, snack foods, sweet sauces, bread and baked foods, pet products.
Functions of calcium acetate in food as follows:
- growth inhibitor for certain bacteria
- texturizer, thickening and firming agent
- buffer in controlling the pH of food during processing (acidity regulator)
- stabilizer when salts
In Beverage: Calcium Acetate can be used in beverage.
In Pharmaceutical:Calcium acetate E263 binds phosphate to lower blood phosphate levels in kidney disease.
In Health and Personal care:It is also used as Fragrance Ingredient; Masking; Viscosity Controlling in cosmetic.
In Agriculture/Animal Feed/Poultry:Calcium Acetate Monohydrate E263 can be used as calcium supplement in pet foods.
Specification of Calcium Acetate Anhydrous
|Appearance||White Fine Powder|
|Assay||99 % Min|
|Ph of 5%||6.5 – 9|
|Arsenic||2 PPM MAX|
|Heavy Metal||5 PPM max|


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Book the Erie Canal Bike Path/vacation Home - Situated in the historical can be seen at Canajoharie Creek Falls and Glimmerglass State Park. Map There is a deck out back for relaxing a quiet side yard should you choose to sit outside.
A fun easy ride the whole family can do. Perfect length at about 24 miles round trip give or take a couple miles.
Might be a little boring for mountain bikers looking for some up and downs or single track but overall it's got what you need to puppet bike out on your bike to enjoy a full day. Zoar, Ohio. Show More Show Less. Point to Point.
Reviews Photos Recordings Sort By. Terry Jomes. Rodrigo Montes Roberto. Excellent ride.
Adam Sykut. Mark Munoz. Ajla Karalic. Janet Fledderjohann.
Wednesday, July 26, Savannah Taylor. Nice hike.
Bryan Stallard. Elena Tate. Alan Bourne. Scott Densmore.
Brittany D. Mairah Maavich.
Thursday, July 28, Michele Moscorelli. Denny Williams.
Monday, February 29, Kevin F. Gene Eminhizer. Julie H. Tuesday, July 01, Grace Troyer.
Thursday, April 17, Cart 0. All of your saved places can be found here in My Trips.
Log in to get rveiews updates and message other travelers. Profile Join. Travel feed: Log in Join. Erie Canalway Trail, New York: Kanedas bike, Erie Canalway Trail Reviews: Erie Canalway Trail.
Review Highlights. Reviewed August 8, Reviewed July 28, All photos Full view.
Traveler Overview. Journey along the scenic and historic Erie Canal between Buffalo and Albany, a National Heritage Corridor, and enjoy great scenery, interesting history, and unparalleled cycling. Suggested duration: More than 3 hours.
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Is there a recommended dress code for this place or activity? Can this place or activity comfortably accomodate people using a wheelchair? Boundary Marker — historic structures — are grouped together south of the Crystal Springs Park Trailhead.
erie canal bike trail reviews Be careful finding the big rock along Erie Avenue that once marked the western border of our country! You can then stop at the Lake Avenue Trailhead srie rent bicycles or canoes, have a bike repaired, get ice cream or a bite to eat, or pick up a new pair of running or hiking shoes.
It should be noted that there are no trailside services between Navarre and Bolivar and that this stretch of the Towpath Trail also is open to equestrians. Home Explore The Towpath Trail.
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domain: geography


Greystone Development and Barry Rice architects designed this lovely condominium on the Upper West Side in 2109. There are 7 floors and 14 units. There is a doorman, outdoor terrace and lounge, bike and building storage and a gym. The building was from another time but has been thoughtfully remodeled to fit the modern lifestyles of today's New Yorker.
There are white gourmet kitchens and grey marble baths. Wood floors and large floor to ceiling picture windows. The Upper West side is home to many fine restaurants, entertainment and shopping.164 West 74th Street is an address located on the Upper West Side of Manhattan, New York City. Here's some key information about 164 West 74th Street:
1. Location: Situated on West 74th Street between Columbus Avenue and Amsterdam Avenue, 164 West 74th Street is located in a desirable residential neighborhood on the Upper West Side. The Upper West Side is known for its charming tree-lined streets, historic brownstones, proximity to Central Park, and cultural institutions like Lincoln Center.
2. Architecture and History: The architectural style of 164 West 74th Street may vary depending on the specific building or structure at that address. The Upper West Side is characterized by a mix of architectural styles, including pre-war brownstones, townhouses, and apartments, showcasing the neighborhood's historical and architectural charm.
3. Residential Units: 164 West 74th Street likely houses residential units, which can include apartments, condos, townhouses, or possibly a combination of these housing types. The specific layout, size, and features of the residential units would depend on the building's design and any subsequent renovations or modifications.
4. Amenities and Services: The amenities and services available at 164 West 74th Street can differ based on the building or housing complex. Common amenities in the Upper West Side may include features like a doorman, elevator, laundry facilities, storage, and possibly shared outdoor spaces or common areas.
5. Neighborhood and Accessibility: The Upper West Side is a vibrant and walkable neighborhood, offering a variety of restaurants, cafes, shops, and cultural attractions. It is also home to Central Park, providing recreational opportunities and beautiful green spaces. The Upper West Side benefits from convenient access to public transportation, such as subway lines and bus routes, facilitating commuting to other parts of the city.
For detailed information about available units, pricing, and specific requirements for purchasing or renting a unit at 164 West 74th Street, it is recommended to consult a reputable real estate agent who specializes in the Upper West Side or contact the building's management directly. They will have the most accurate and up-to-date information tailored to your needs and preferences.


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2011 133 am Relate
2011 1:33 am Related News After missing two deadlines,s claims, : Employees found to have sublet their official quarters for monthly rent of Rs 3, 2011 3:54 am Related News ?Written by AmritaJain | Pune | Published: December 31 ?
The police have reportedly asked them to evacuate their houses in two days? the Air Force official said.But at the same time we do not want the lives of the boys to be under any kind of danger.Written by Amrita Chaudhry | Ludhiana | Published: January 1 two current national security officials rejected the White House explanation, Bannon’s seat on the NSC’s “principals’ committee, Those attacks killed 130 and 17 more died in two January attacks. For all the latest World News, 2011 3:21 am Related News An Iranian model recounts her journey from Tehran to the Delhi runway Manizeh Karimi is Iran?Written by Alia Allana | Published: April 5
DGP Paramjeet Singh Gill said: ?Written by Amrita Chaudhry | Ludhiana | Published: April 28 This grand tradition must be preserved and following,which carries names of brave soldiers, An official said the universities had been asked to review the process on a quarterly basis and send reports to the government. The government has asked educational institutions that were expected to get the NAAC accreditation by March 2007 to send in their proposals and complete the process by November 30, Minimum hourly wages as decided by the board,Thatte said, while in 2017 it will be 2.he is turned down on the grounds that since the scheme to provide financial assistance was a centrally funded scheme.
Thus when a chronically ill patient is guided to get financial assistance from Punjab? leaving another 130 injured. killing hundreds of militants in aerial strikes and a major ground offensive.the Haryana government had expressed its reluctance stating that the Haryana Police already has such a wing. 2012 3:02 am Related News The Punjab and Haryana High Court,2 respectively.2 degrees, who spoke on condition of anonymity. “The nation of Qatar unfortunately has historically been a funder of terrorism at a very high level.


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David Ochanda, Ericsson Kenya's business development director speaks at the LTE Media workshop
An open access model is the best for the deployment of a 4G or LTE network in Kenya as it will ensure all parts of the country are covered, thereby enhancing the country’s overall broadband penetration.
David Ochanda, Ericsson Kenya’s business development director, told attendees at an Ericsson workshop on LTE yesterday (Wednesday) this model will make it possible for LTE to provide services to clients other than the owners for free.
“The open access model – if adopted by the Kenya government and by extension the IT industry – will see the network being deployed countrywide,” said Ochanda.
“When left to deploy the LTE network as proposed in the consortium model, the MNOs (mobile network operators) will only focus on high potential areas where they can get maximum revenues, thereby further minimising access to broadband services by underserved regions.”
The director added that Kenya was meant to be the pioneer in open access, with other countries in the region meant to learn from it.
“However, this has not been realised, with Rwanda now expected to lead the region by deploying its LTE via the open access model,” he said.
The government through industry regulator the Communications Commission of Kenya (CCK) has formed a nine-member consortium to lead the deployment of an LTE network.
Kenya’s proposed consortium includes the government (through the Treasury), as well as Safaricom, Telkom Kenya-Orange, Airtel, Essar Telecom’s YU Kenya, KDN, MTN, Alcatel-Lucent and Epesi Communications. It was set up to implement the initial phase of the country’s LTE project at the cost of KSh8.4 billion (US$96 million).
Industry statistics indicate that by 2020 the ICT industry will have moved to 5G technology, with much of the data traffic expected to come from smart devices rather than phones and tablets.
Already, Ericsson has deployed LTE/4G networks in several African countries – among these being Nigeria, South Africa, Tanzania, Angola and Botswana.


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Report: Kaepernick turns down invitations to Oscars
ATLANTA (KGO) -- San Francisco 49ers quarterback Colin Kaepernick is shying away from the spotlight just weeks after his first Super Bowl appearance.
The Sacramento Bee reports Kaepernick received invitations to both the Grammys and the Oscars, but turned them down. He's also apparently been offered a book deal. Instead though, Kaepernick is training in Atlanta. His workouts usually start at 8 a.m. and last until the late afternoon. Kaepernick's father says his son is in Atlanta because he would never get anything done if he stayed closer to home.
Kaepernick is a native of Turlock.
colin kaepernick, san francisco 49ers, nfl
- Several aftershocks felt after 5.7 quake in Greenville 30 min ago
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- abcnews: New bill would allow pets on Amtrak
- roundup: Bicyclist killed in SF; Body in canal ID'd
- weather: Bay Area weather forecast for Friday


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Norrington, Sir Roger
Sir Roger Norrington, © Photo: Manfred Esser
Roger Norrington came from a musical family in Oxford, England and played the violin and sang from a young age. He studied History at Westminster School, and English Literature at Cambridge University, where he was a choral scholar. Several years’ wide experience of top class amateur music making, while working as a publisher of scholarly books, ended with a return to musical studies at the Royal College of Music in London and the start of his professional career as a singer and conductor.


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Initial Public Offering Market: Ways of Development in Ukraine
Gladchuk Kseniia Nikolaevna
Senior Teacher of the Bank Investments Department
Vadym Het’man Kyiv National Economic University
The article reveals preconditions for Ukrainian IPO market activization, as well as factors restraining its development at present. The history of Ukrainian IPOs on foreign stock exchanges was analyzed. As a result, key trends dominating the IPO market of Ukrainian companies abroad were specified. Practical guidelines of Ukrainian IPO market activization and the structural model of its functioning were suggested.
IPO, Ukrainian IPO market, securities market, stock exchange, issuer, investor, IPO market trends, structural model of IPO market functioning.
Full article text is available only in Russian.
Please select from the menu Russian language and continue reading.

domain: economics


"You know what? I was quoted saying I was a virgin, but I absolutely did not say that. That's nobody's business but my own."
Former 'Lizzie McGuire' star Hillary Duff, 21, talking to Maxim about an interview she gave in 2006. Maybe she was still Disney property two years ago and her corporate virginity pledge was still binding.


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2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-α (DAGLα), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGLα was predominantly expressed in Purkinje cells. DAGLα was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGLα was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGLα was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLα was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLα was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLα is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.
- diacylglycerol lipase
- Purkinje cell
- hippocampal pyramidal cell
Endogenous cannabinoids (endocannabinoids) are released from postsynaptic neurons and act retrogradely on presynaptic cannabinoid receptors, causing short- and long-term suppression of transmitter release. Endocannabinoid-mediated retrograde suppression (ERS) was found originally in the cerebellum (Kreitzer and Regehr, 2001a; Maejima et al., 2001) and hippocampus (Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001) and later in other brain regions (Alger, 2002; Wilson and Nicoll, 2002; Piomelli, 2003). ERS can be triggered by three distinct modes: membrane depolarization that elevates intracellular calcium concentration ([Ca2+]i) to a micromolar range (Brenowitz and Regehr, 2003), activation of the Gq-protein-coupled metabotropic receptor/phospholipase Cβ (PLCβ) pathway leading to the production of diacylglycerol (DAG) (Maejima et al., 2001; Kim et al., 2002; Ohno-Shosaku et al., 2003), and cooperative action of [Ca2+]i elevation and Gq-protein-coupled receptor activation (Varma et al., 2001; Ohno-Shosaku et al., 2002; Hashimotodani et al., 2005; Maejima et al., 2005). Of these, the third mode appears physiologically most relevant, because ERS is induced by weak activation of Gq-protein-coupled receptors and physiological [Ca2+]i increase to a submicromolar range, either of which is subthreshold for ERS by itself (Hashimotodani et al., 2005; Maejima et al., 2005).
Cannabinoid receptor CB1 is expressed predominantly in the nervous system, whereas CB2 is present in the immune system (Matsuda et al., 1990; Howlett et al., 2002). Immunohistochemical, biochemical, and electrophysiological studies using CB1 knock-out mice have provided evidence that CB1 is the major presynaptic cannabinoid receptor at inhibitory and excitatory synapses in various brain regions (Katona et al., 1999, 2001; Farquhar-Smith et al., 2000; Hajos et al., 2000, 2001; Wilson and Nicoll, 2001; Yoshida et al., 2002; Bodor et al., 2005; Kofalvi et al., 2005; Kawamura et al., 2006). Intriguingly, CB1 is expressed in distinct interneuron subclasses in the hippocampus and also expressed in variable amounts at inhibitory and excitatory synapses in given neural regions (Katona et al., 1999; Hajos et al., 2000; Kawamura et al., 2006), suggesting its importance in information processing and integration (Freund, 2003).
2-Arachidonoyl-glycerol (2-AG) is one of the two major endocannabinoids (Devane et al., 1992; Mechoulam et al., 1995; Sugiura et al., 1995) and is synthesized on demand from DAG by sn-1-specific diacylglycerol lipase (DAGL) (Stella et al., 1997). Experiments with 2-AG synthesis inhibitors showed that 2-AG indeed mediates short- and long-term ERS (Melis et al., 2004; Safo and Regehr, 2005). Therefore, it is of great importance to clarify the site of DAGL expression to determine the site of 2-AG synthesis. Here we demonstrate that DAGLα, one of the two DAGLs cloned to date (Bisogno et al., 2003), is essentially targeted to postsynaptic spines. DAGLα is highly enriched at the base of spine neck but excluded from the main body of spines in cerebellar Purkinje cells, whereas DAGLα is distributed at the spine head, neck, or both in hippocampal pyramidal cells. The different fine localization in given neuron types suggests that specificity and efficiency of ERS depend not only on CB1 expression levels in presynaptic elements but also on the distance between postsynaptic 2-AG production site and presynaptic CB1.
Materials and Methods
Animal and section preparation.
C57BL mice at the adult stage (2–4 months of age) and postnatal day 10 (P10) were used in the present study. Under deep pentobarbital anesthesia (100 mg/kg body weight, i.p.), brains were freshly removed from the skull and frozen in powdered dry ice for in situ hybridization. Frozen sections were prepared on a cryostat (20 μm in thickness; CM1900; Leica, Nussloch, Germany) and mounted on silane-coated glass slides (Muto-Glass, Tokyo, Japan). For immunohistochemistry, anesthetized mice were fixed transcardially with 4% paraformaldehyde in 0.1 m sodium phosphate buffer (PB), pH 7.2, for light microscopy or 4% paraformaldehyde/0.1% glutaraldehyde in PB for electron microscopy, and microslicer sections (50 μm) were prepared (VT1000S; Leica).
In situ hybridization.
Fresh frozen sections were treated at room temperature with the following incubations: fixation with 4% paraformaldehyde in PB for 10 min, 2 mg/ml glycine–PBS, pH 7.2, for 10 min, acetylation with 0.25% acetic anhydride in 0.1 m triethanolamine-HCl, pH 8.0, for 10 min, and prehybridization for 1 h in a buffer containing 50% formamide, 50 mm Tris-HCl, pH 7.5, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin (BSA), 0.6 m NaCl, 0.25% SDS, 200 μg/ml tRNA, 1 mm EDTA, and 10% dextran sulfate. Hybridization was performed at 42°C for 12 h in the prehybridization buffer supplemented with 10,000 cpm/μl [33P]dATP-labeled antisense oligonucleotide probes. Probes were synthesized against nucleotide residues 3016–3060 of the mouse DAGLα cDNA (GenBank accession number NM198114) and 1996–2040 of the mouse DAGLβ cDNA (GenBank accession number BC016105) and labeled using terminal deoxyribonucleotidyl transferase (Invitrogen, Carlsbad, CA). Slides were washed twice at 55°C for 40 min in 0.1× SSC containing 0.1% sarcosyl. Sections were exposed to BioMax film (Eastman Kodak, Rochester, NY) for 4 weeks.
We used affinity-purified primary antibodies raised against the following molecules (species immunized): mouse DAGLα (rabbit and guinea pig, see below); mouse protein kinase Cγ (PKCγ) (rabbit and guinea pig; see below); mouse calbindin [goat (Miura et al., 2006)]; mouse microtubule-associated protein-2 (MAP2) [goat (Miura et al., 2006)]; mouse parvalbumin [guinea pig (Nakamura et al., 2004)]; mouse PLCβ4 [rabbit and guinea pig (Nakamura et al., 2004)]; mouse 3-phosphoglycerate dehydrogenase (3PGDH) [guinea pig (Yamasaki et al., 2001)]; rat vesicular glutamate transporter 1 (VGluT1) and 2 (VGluT2) [guinea pig (Miyazaki et al., 2003)]; mouse vesicular γ-aminobutylic acid transporter (VGAT) [guinea pig (Miyazaki et al., 2003)]; mouse cannabinoid CB1 receptor [guinea pig (Fukudome et al., 2004)]; and mouse type 1α metabotropic glutamate receptor (mGluR1α) [rabbit (Tanaka et al., 2000)].
For production of antibodies, cDNA fragments, which are preceded with a BamHI site and encode C-terminal 42 amino acids of DAGLα (1003–1044 amino acid residues; GenBank accession number NM198114) or C-terminal 14 amino acids of PKCγ (684–697; GenBank accession number L28035), were obtained by PCR or by annealing of sense and antisense oligonucleotides, respectively. After the thymidine/adenosine (TA) cloning using a pGEM-T Easy Vector System I kit (Promega, Madison, WI), cDNA fragments was sequenced and excised by BamHI and EcoRI. These fragments were subcloned into BamHI/EcoRI site of pGEX4T-2 plasmid (Amersham Biosciences, Piscataway, NJ) for expression of glutathione S-transferase (GST) fusion proteins. GST fusion proteins were purified using glutathione-Sepharose 4B (Amersham Biosciences), according to the instructions of the manufacturer, and used for immunization to New Zealand White rabbits and Hartley guinea pigs. Antigens were emulsified with Freund’s complete or incomplete adjuvant (Difco, Detroit, MI) and injected subcutaneously at intervals of 2 weeks. After the sixth injection, Ig specific to antigens were affinity purified using GST-free peptides coupled to cyanogen bromide-activated Sepharose 4B (Amersham Biosciences). GST-free peptides were prepared by in-column thrombin digestion of GST fusion proteins bound to glutathione-Sepharose 4B media.
The hippocampus and cerebellum were homogenized using a Potter homogenizer with 15 strokes at 1000 rpm in 10 vol of ice-cold homogenizer buffer containing 0.32 m sucrose, 1 mm EDTA, 1 mm EGTA, 10 mm Tris-HCl, pH 7.2, and 0.4 mm phenylmethylsulfonyl fluoride. The protein concentration was determined by the Lowry’s method. The homogenates were denatured by 50 mm (±)-dithiothreitol at 65°C for 15 min. Proteins (15 μg/lane) were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes (BioTraceNT; Pall Gelman Laboratory, Ann Arbor, MI). After blocking with 5% skimmed milk for 30 min, membranes were incubated for 2 h with rabbit or guinea pig DAGLα antibody (1 μg/ml). Tris-buffered saline, pH 7.5, containing 0.1% Tween 20 was used for diluent and washing buffer. Immunoreaction was visualized with the ECL chemiluminescence detection system (Amersham Biosciences).
All immunohistochemical incubations were done at room temperature in a free-floating state. For immunofluorescence, cerebellar sections were incubated with 10% normal donkey serum for 30 min, a mixture of primary antibodies overnight (1 μg/ml), and a mixture of Alexa Fluor-488-, indocarbocyanine (Cy3), and indodicarbocyanine (Cy5)-labeled species-specific secondary antibodies for 2 h at a dilution of 1:200 (Invitrogen; Jackson ImmunoResearch, West Grove, PA). For hippocampal sections, immunofluorescence was performed by two-step method, as follows. First, sections were incubated with 10% normal donkey serum for 30 min, rabbit DAGLα antibody (0.5 μg/ml) overnight, biotinylated secondary antibody for 2 h, and streptavidin–peroxidase complex for 30 min using a Histofine SAB-PO(R) kit (Nichirei, Tokyo, Japan), followed by visualization using a tyramide signal amplification kit [TSA-DIRECT (Red); NEN, Boston, MA]. After blocking with 10% normal rabbit serum, conventional immunofluorescence was then used as above. PBS containing 0.1% Tween 20 was used for diluent of antibodies and washing buffer. Images were taken with a fluorescence microscope (AX-70; Olympus Optical, Tokyo, Japan) equipped with a digital camera (DP70; Olympus Optical) or with a confocal laser scanning microscope (FV1000; Olympus Optical).
For immunoperoxidase electron microscopy, microslicer sections were incubated successively with 10% normal donkey serum, rabbit DAGLα antibody (0.5–2 μg/ml), biotinylated secondary antibody, and streptavidin–peroxidase complex as above. Immunoreaction was visualized with 3,3′-diaminobenzidine. For preembedding immunogold electron microscopy, microslicer sections were dipped in 5% BSA/0.02% saponin/PBS for 30 min, and incubated overnight with rabbit DAGLα (0.5–2 μg/ml), guinea pig CB1 (1 μg/ml), or rabbit PKCγ (1 μg/ml) antibody diluted with 1% BSA/0.004% saponin/PBS and then with anti-rabbit IgG linked to 1.4 nm gold particles (Nanogold; Nanoprobes, Stony Brook, NY) for 2 h. Immunogolds were intensified with a silver enhancement kit (HQ silver; Nanoprobes). Sections labeled by immunoperoxidase and silver-enhanced immunogold were treated with 2% osmium tetroxide for 30 min, stained in block with 2% uranyl acetate for 30 min, dehydrated, and embedded in Epon 812.
For postembedding immunogold electron microscopy, microslicer sections were cryoprotected with 30% sucrose/PB and frozen rapidly with liquid propane in an EM CPC unit (Leica). Frozen sections were immersed in 0.5% uranyl acetate in methanol at −90°C in an AFS freeze-substitution unit (Leica), infiltrated at −45°C with Lowicryl HM-20 resin (Lowi, Waldkraiburg, Germany), and polymerized with UV light. After etching with saturated sodium-ethanolate solution for 3 s, ultrathin sections on nickel grids were treated successively with 2% human serum albumin (Wako, Osaka, Japan)/0.1% Tween 20 in Tris-buffered saline, pH 7.5 (HTBST) for 30 min, rabbit DAGLα antibody (15 μg/ml) in HTBST overnight, and colloidal gold (10 nm)-conjugated anti-rabbit IgG (1:100; British Bio Cell International, Cardiff, UK) in HTBST for 2 h. Finally, grids were stained with 2% uranyl acetate for 20 min and with mixed lead solution for 30 s. Photographs were taken with an H-7100 electron microscope (Hitachi, Tokyo, Japan).
For quantitative analysis, silver-enhanced immunogold particles for DAGLα on somatodendritic and spine membranes or for CB1 on parallel fibers were counted on electron micrographs. Furthermore, tangential and perpendicular distributions of DAGLα in spines were examined from 57 spines of parallel fiber–Purkinje cell synapses (postembedding immunogold) and 49 spines of CA1 pyramidal cell synapses (preembedding immunogold) and analyzed using IPLab software (Nippon Roper, Tokyo, Japan).
Specificity of DAGLα antibody and immunohistochemistry
We produced polyclonal rabbit and guinea pig antibodies against C-terminal sequence of mouse DAGLα. After affinity purification, both antibodies selectively recognized protein bands at 105 kDa in the hippocampus and at 120 kDa in the cerebellum (Fig. 1A). The molecular mass of the cerebellar band was almost similar to that reported previously (Bisogno et al., 2003). By immunofluorescence with parasagittal brain sections, the highest immunoreactivity was detected in the cerebellar cortex and hippocampus (Fig. 1B). Moderate levels were detected in the cerebral cortex, olfactory bulb, caudate–putamen, and thalamus. The spatial patterns of immunostaining were essentially the same with use of rabbit and guinea pig antibodies. Preabsorption of antibodies with antigen protein abolished the immunostaining (Fig. 1B, inset).
This immunostaining was compared with the distribution of DAGLα and DAGLβ mRNAs. DAGLα mRNA was expressed at the highest level in the hippocampus and cerebellar cortex, in which autoradiographic silver grains were observed on hippocampal pyramidal cells, dentate granule cells, and cerebellar Purkinje cells but not on hippocampal nonpyramidal cells or cerebellar granule cells (Fig. 1C–E). Moderate expression of DAGLα mRNA was noted in the cerebral cortex, olfactory bulb, and thalamus. DAGLβ mRNA also showed wide expression in the brain with particular abundance in the cerebral and cerebellar cortices, olfactory bulb, and dentate gyrus (Fig. 1F). However, its distribution differed from that of DAGLα mRNA in that DAGLβ mRNA was expressed at the highest level in the cerebellar granular layer and relatively low in the hippocampal pyramidal cell layer and thalamus. These signals were abolished almost completely by addition of an excess amount of cold probes in the hybridization mixture (data not shown). Because Purkinje cells were predominantly immunolabeled in the cerebellum (see below), the pattern of immunostaining is thus similar to DAGLα mRNA expression, suggesting that antibodies produced in the present study are specific to DAGLα.
We applied these antibodies to examine cellular expression and subcellular distribution in the cerebellum (see Figs. 2⇓⇓⇓⇓–7) and hippocampus (see Figs. 8, 9). In multiple labeling, DAGLα is in red and others are in green or blue throughout the present study.
Somatodendritic surface expression of DAGLα in Purkinje cell
In the cerebellum, DAGLα staining was virtually confined to the molecular layer, in which a tubular or cylindrical pattern of immunostaining was evident (Fig. 2A1). DAGLα expression in Purkinje cells was examined by double immunofluorescence with calbindin (Fig. 2A–D). DAGLα staining was most intense and continuous on spiny branchlets (or distal dendrites), but spines emitting from spiny branchlets appeared to lack the labeling (Fig. 2A1, inset, B). The intensity became lowered and intermittent on proximal dendrites (Fig. 2A1, inset). In the soma of Purkinje cells, weak and spotty labeling was occasionally detected on the somatic surface (Fig. 2C). In contrast, DAGLα labeling was not detected in axon terminals of Purkinje cells, which form synapses on neurons in the deep cerebellar nucleus (Fig. 2D). Expression of DAGLα in molecular layer interneurons was examined by triple immunofluorescence for calbindin (blue), parvalbumin (green), and DAGLα (Fig. 2E1). It is known that Purkinje cells express both calbindin and parvalbumin (light blue), whereas molecular layer interneurons express parvalbumin only (green). By subtracting the light blue color (representing Purkinje cell dendrites) from Figure 2E1, dendrites, cell bodies, and axons of interneurons were selectively visualized as green elements, but they lacked DAGLα (Fig. 2E2). Possible expression of DAGLα in Bergmann glia was examined by double immunofluorescence for DAGLα and 3PGDH, a serine biosynthetic enzyme expressed in Bergmann glia but not in Purkinje cells or other neurons (Yamasaki et al., 2001). 3PGDH-positive Bergmann fibers were devoid of DAGLα immunofluorescence (Fig. 2F). These results demonstrate that, in the cerebellum, DAGLα is predominantly expressed in Purkinje cells and localized to somatodendritic elements, particularly at high levels in spiny branchlets.
Close apposition of DAGLα to other endocannabinoid signaling molecules
Endocannabinoids are effectively synthesized in Purkinje cells by conjunctive synaptic activation of ionotropic AMPA receptor and mGluR1 (Maejima et al., 2001, 2005; Brown et al., 2003; Brenowitz and Regehr, 2005). Synaptic distribution of DAGLα was examined by multiple immunofluorescence for presynaptic markers and postsynaptic signaling molecules (Fig. 3). DAGLα was hardly detected in VGluT2-labeled climbing fiber terminals, VGluT1-labeled parallel fiber terminals, or VGAT-labeled interneuron terminals (Fig. 3A–C). Nonoverlapping pattern with presynaptic markers was also observed by double immunofluorescence for cannabinoid receptor CB1 (Fig. 3D), which is expressed in these afferents (Kawamura et al., 2006). Such nonoverlapping pattern was also seen for mGluR1α (Fig. 3E), which is concentrated on dendritic spines, particularly at the perisynaptic annulus surrounding the edge of synaptic junction (Baude et al., 1993; Nusser et al., 1994). Consequently, when triple immunofluorescence was performed for DAGLα, mGluR1α, and CB1, the three immunofluorescent signals were distributed in a mutually exclusive manner but closely apposed to one another (Fig. 3F). PLCβ4 is a major PLCβ subtype in Purkinje cells of the rostral cerebellum (Kano et al., 1998; Watanabe et al., 1998b) and was distributed in both spines and dendritic shafts (Nakamura et al., 2004). Immunofluorescence signals for PLCβ4 overlapped with those for mGluR1α in spines and with those for DAGLα in dendritic shafts (Fig. 3G). Therefore, DAGLα is localized close to CB1-carrying presynaptic elements and mGluR1α/PLCβ4-carrying postsynaptic spines but apart from both of them.
Enriched localization of DAGLα at the base of spine neck and its adjacent somatodendritic membrane in Purkinje cell
Ultrastructural localization of DAGLα in Purkinje cells was investigated by immunoelectron microscopy (see Figs. 4, 5). By using the immunoperoxidase method, spiny branchlets were most strongly labeled (Fig. 4A), whereas proximal dendrites (Fig. 4B) and somata (Fig. 4C,D) were labeled at low to moderate levels. Even with a diffusible peroxidase substrate (i.e., diaminobenzidine), immunoreaction products were often concentrated in the cytoplasm just beneath the somatodendritic cell membrane. Strong immunoreaction was observed in the neck of spines forming asymmetrical synapses with parallel fiber terminals (Fig. 4A), which were relatively small in size and contained a small number of synaptic vesicles accumulating near the synaptic junction. No immunoreaction was seen in the spine head (Fig. 4A) or at symmetrical synapses formed onto dendritic shaft (Fig. 4B). In perikarya, immunoperoxidase labeling was considerably rare but occasionally encountered (Fig. 4C). When carefully pursuing the labeled regions on serial sections, we found spines protruding from immunolabeled somatic membrane (Fig. 4D). Interestingly, somatic spines formed asymmetrical synapses with climbing fiber terminals, which were large in size and contained a large number of synaptic vesicles and dark cytoplasm. In such somatic spines, DAGLα labeling was also restricted to the neck region and its adjacent somatic membrane (Fig. 4D).
By using the preembedding silver-enhanced immunogold method, the cytoplasmic side of somatodendritic membrane was labeled. In spiny branchlets, metal particles representing DAGLα were associated with the cell membrane at the base of spine neck, whereas other regions of spines, including the head region, were little labeled (Fig. 4E). In proximal dendrites, DAGLα was distributed along the cell membrane but was excluded from the postsynaptic site of symmetrical synapses (data not shown). Spines emanating from proximal dendrites were much lower in density than those from spiny branchlets and were identified on serial sections to form synapses with climbing fiber terminals (Fig. 4F). Also in such spines, DAGLα was detected at the base of spine neck and its adjacent dendritic membrane. Using randomly taken electron micrographs, the number of metal particles per 1 μm of the cell membrane was measured for the soma, proximal dendrite, spiny branchlet, and spine. The highest density was shown in spiny branchlets, and the density was declined sharply toward the soma (Fig. 4G). Most particles that fell on the spine membrane were attributable to labeling in the neck portion.
Postsynaptic molecules, particularly those condensed at postsynaptic membrane or postsynaptic density, are often resistant to preembedding immunohistochemical detection because of limited antibody accessibility (Fritschy et al., 1998; Watanabe et al., 1998a; Fukaya and Watanabe, 2000). This can be overcome by the postembedding immunogold method (Baude et al., 1995; Ottersen and Landsend, 1997). We confirmed the lack of DAGLα labeling at synaptic junction by the postembedding immunogold method (Fig. 5A,B). Furthermore, this method disclosed that DAGLα was highly accumulated at the base of spine neck or the dendrite–spine border. This observation was quantitatively evaluated by counting the number of gold particles as a function of distance from the dendrite–spine border (Fig. 5C). The peak distribution was found at the 0–100 nm bin in the spine side, and labeling was steeply decreased toward both the spine head and dendritic shaft (n = 57 spines). Then, we examined perpendicular distribution of DAGLα by measuring the distance from the midpoint of plasma membrane to the center of gold particles. The peak distribution was found at the 0–10 nm bin toward the intracellular side of the plasma membrane (Fig. 5D). Moreover, 82.3% of the total immunogold particles were located within 35 nm from the midpoint of the cell membrane, suggesting preferential cell membrane-associated distribution of DAGLα with the C-terminal epitope intracellularly. These results demonstrate that DAGLα is distributed on the somatodendritic cell membrane, particularly at high levels in the base of the spine neck. In contrast, DAGLα is selectively excluded from the spine head, on which excitatory afferents form asymmetrical synapses.
Because 2-AG synthesized by DAGLα presumably diffuses in the extracellular space and binds to CB1, it is important to clarify how CB1 is distributed on axons and terminals in relation to the 2-AG production site. Recently, we have shown that CB1 is highly accumulated on perisynaptic portion of parallel fibers (<500 nm from the edge of synaptic junction) (Kawamura et al., 2006). In the present study, we analyzed parallel fiber synapses sectioned longitudinally to compare the distribution of CB1 on parallel fibers between synaptic and opposite sides. We found that CB1 labeling was preferentially detected on the axolemma facing Purkinje cell spines (Fig. 6). There was almost twice the number of metal particles in the synaptic side of axolemma (69.2%) compared with the opposite side (30.8%; n = 65 total metal particles obtained from 17 parallel fiber synapses).
Targeting of DAGLα to spine and excitatory synapse in developing and adult Purkinje cells
Figure 4D demonstrates highly localized distribution around spines in the soma, although such somatic spines were very rare at the adult stage. This observation strongly suggests that DAGLα expression is linked with the formation of spines or excitatory synapses. To test this possibility, we applied triple immunolabeling to the cerebellum at P10, when climbing fibers predominantly innervate somatic spines before translocation to dendrites (Altman, 1972; Chedotal and Sotelo, 1992). At P10, many spots of DAGLα labeling were found and discontinuously surrounded the somatic surface of almost all Purkinje cells (Fig. 7A,B). This spotty labeling was observed just beneath VGluT2-labeled large terminals [i.e., climbing fiber terminals (Fig. 7B1,B2, green)] and also beneath calbindin-labeled somatic spines (Fig. 7B1,B3, blue), indicating spine-associated or climbing fiber synapse-associated localization of DAGLα in the soma. Compared with the intense labeling on the soma and proximal dendrites, levels of DAGLα labeling were apparently low in distalmost dendrites (Fig. 7A), which grow actively at the border between the external granular layer and molecular layer (Yamada et al., 2000). At a higher magnification, low but spotty labeling was already found on such distalmost dendrites (Fig. 7C1,C2) but was rarely associated with VGluT2-positive small terminals (Fig. 7C3), representing immature parallel fiber terminals (Miyazaki et al., 2003). This was also true for adult Purkinje cells (at 14 weeks of age), in which DAGLα immunolabeling on the somatic surface was localized in close association with VGluT2-labeled climbing fiber terminals (green) and calbindin-labeled somatic spines (Fig. 7E, blue). Together, these results support the aforementioned notion that DAGLα is targeted to particular somatodendritic sites of Purkinje cells in which spines and excitatory synapses are formed.
Spine-selective and diffuse intraspine distribution of DAGLα in hippocampal pyramidal cell
Because ERS occurs at synapses in various brain regions (Alger, 2002; Wilson and Nicoll, 2002; Piomelli, 2003), it is important to examine whether the features of DAGLα distribution found in cerebellar Purkinje cells are also applied to neurons in other brain regions. We therefore examined DAGLα distribution in hippocampal pyramidal cells, the neuron type most intensively studied so far. We found that tiny punctate labeling was predominant in the hippocampus (Fig. 8A1), which was apparently different from preferential somatodendritic surface labeling in the cerebellum. In the stratum radiatum of the CA1, DAGLα-labeled puncta were distributed densely around MAP2-labeled thin dendrites (Fig. 8A2). In contrast, DAGLα labeling was weak in intensity and few in number in the stratum lacunosum-moleculare (Fig. 8B). Punctate labeling for DAGLα was not overlapped with, but apposed close to, both VGluT1-labeled glutamatergic terminals and VGAT-labeled inhibitory terminals (Fig. 8C). Likewise, DAGLα labeling was not overlapped but often apposed closely to CB1 labeling (Fig. 8D). These results suggest that DAGLα is localized to certain postsynaptic structures in hippocampal pyramidal cells as well, but its fine distribution appears distinct from that in cerebellar Purkinje cells.
By immunoperoxidase (Fig. 9A–C) and silver-enhanced immunogold (Fig. 9D,E) electron microscopic analyses, we revealed in the CA1 stratum radiatum that DAGLα labeling was strong in dendritic spines but low or undetectable in the somatodendritic membrane. In addition, labeling pattern was variable from spine to spine. Some spines were labeled at the neck (Fig. 9A,D) or head (Fig. 9B,E) portion, whereas others were labeled at both portions (Fig. 9C). These patterns of immunogold labeling were quantitatively assessed in CA1 pyramidal cells. Labeling density in the cell membrane of spines was apparently higher than that of somata, shaft dendrites, and thin dendrites, and the densities in the latter elements were even higher than the background level as determined from presynaptic membrane (Fig. 9F). The relative tangential distribution on spine membrane was plotted from the dendrite–spine border (0%) to the edge of the postsynaptic density (100%) (Fig. 9G). Labeling was peaked twice, one at the base of spine neck (i.e., dendrite–spine border) and the other at the spine head.
These results suggest that DAGLα distribution is concentrated in spines of hippocampal neurons, but, within a spine, it is more widely or variably distributed compared with cerebellar Purkinje cells. However, essential features that DAGLα is closely associated with postsynaptic spines and excitatory synapses are applicable to both neuron types.
In the present study, we have disclosed subcellular distribution of DAGLα in cerebellar Purkinje cells and hippocampal pyramidal cells. Characteristic distribution revealed in the two neurons will provide a molecular–anatomical basis for the scheme of activity-dependent synthesis of 2-AG in postsynaptic neurons and its retrograde action onto CB1-carrying presynaptic elements (Fig. 10).
Base of the spine neck is the major site of 2-AG synthesis in Purkinje cell
Distribution of DAGLα immunolabeling was highest in Purkinje cells, in which the following features were documented. First, DAGLα was detected with the C-terminal antibodies in the cytoplasmic side of the plasma membrane (Fig. 5D). This is consistent with the predicted molecular configuration of DAGLs that has four putative transmembrane domains with both termini inside the cell (Bisogno et al., 2003). Second, DAGLα was selective to somatodendritic elements, with the highest level in spiny branchlets and progressively decreasing toward the soma. Third, DAGLα was highly enriched at the base of spine neck but virtually excluded from the rest of the spine membrane. This particular enrichment was clearly demonstrated by the postembedding immunogold method but appreciated insufficiently by immunofluorescence and preembedding immunogold methods. The facilitated detection by the postembedding immunogold method suggests that molecular assembly at the base of the spine neck is tightly organized in Purkinje cells, probably for the shaping of dendritic spines and the anchoring of particular molecules, such as DAGLα.
In Purkinje cells, ERS is induced at both parallel fiber and climbing fiber synapses by either depolarization of Purkinje cells and the resultant elevation of [Ca2+]i increase to ∼15 μm (Kreitzer and Regehr, 2001a; Brenowitz and Regehr, 2003) or strong activation of mGluR1 (Maejima et al., 2001; Brown et al., 2003). The induction of ERS is greatly enhanced when parallel fiber stimulation is combined with climbing fiber stimulation (Brenowitz and Regehr, 2005) or when subthreshold mGluR1 activation is combined with [Ca2+]i increase to a submicromolar range (Maejima et al., 2005). Parallel fibers and climbing fibers innervate spines of spiny branchlets or proximal dendrites, respectively. AMPA receptors are densely localized at the synaptic junction on the spine head and mediate fast excitatory synaptic transmission (Nusser et al., 1994), whereas mGluR1α and its downstream signaling molecules, Gαq/11 and PLCβ4, are all concentrated at the perisynaptic region on the spine head and produce DAG (Baude et al., 1993; Tanaka et al., 2000; Nakamura et al., 2004). CB1 is highly enriched at the perisynaptic region of parallel fiber terminals (Kawamura et al., 2006). Thus, the base of the spine neck is close to both the glutamate receptor-carrying postsynaptic site and the CB1-carrying presynaptic site (Fig. 10A). In addition, DAGLα is most densely accumulated at the base of the spine neck among DAGLα-possessing somatodendritic elements. These results suggest that the base of the spine neck is the major site of 2-AG synthesis in response to excitatory synaptic activity. This characteristic distribution would contribute to the sharpening of input specificity for endocannabinoid signaling. Our present finding that CB1 is preferentially accumulated on the synaptic side of axolemma appears important. Because the 2-AG-degrading enzyme monoglyceride lipase (MGL) is selectively localized in axons and terminals (Dinh et al., 2002; Gulyas et al., 2004), this preferential distribution would enable 2-AG to activate CB1 without passing through MGL-rich axoplasm and thereby contributes to the increasing of efficiency for endocannabinoid signaling.
Then why is DAGLα excluded from the spine head? DAG also functions as an activator for PKC. Like DAGLα, PKCγ is particularly abundant in Purkinje cells and activated downstream to the mGluR1 cascade (Kose et al., 1988; Nishizuka, 1992). The major mGluR1 signaling cascade in Purkinje cells, i.e., mGluR1–Gαq/11–PLCβ4–PKCγ, is crucial for climbing fiber synapse elimination and motor coordination (Kano et al., 1995, 1997, 1998; Offermanns et al., 1997). Our immunogold electron microscopy revealed wide distribution of PKCγ in Purkinje cell spines, including the head and neck portions (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). One possibility is that DAGLα is excluded from the spine head so that DAG can efficiently activate PKCγ within spines, before its catalysis into 2-AG. When DAG production is increased enough to spread over to the base of the spine neck, 2-AG synthesis would be initiated and induce ERS. If DAGLα were distributed on the spine head as well, ERS would occur more preferentially and potently at the nearby excitatory synapse than at remote inhibitory synapses. Thus, the restricted localization is also likely to coordinate induction threshold and magnitude of ERS at excitatory and inhibitory synapses.
Somatodendritic surface of Purkinje cells is an additional site for 2-AG synthesis
The surface of dendritic shafts and soma is mainly innervated by inhibitory interneurons, i.e., stellate cells and basket cells. CB1 is expressed in these interneurons (Mailleux and Vanderhaeghen, 1992) and localized abundantly in their axons and terminals (Tsou et al., 1998; Kawamura et al., 2006). ERS can be induced at these inhibitory synapses after strong depolarization of Purkinje cells (Kreitzer and Regehr, 2001b; Diana et al., 2002; Yoshida et al., 2002; Brenowitz and Regehr, 2003) and by activation of group I mGluR (Galante and Diana, 2004). We show that the cell membrane of dendritic shafts and soma possess DAGLα, although at a much lower level than the base of the spine neck. The wealth of CB1 on inhibitory axons and terminals and the distribution of DAGLα on nearby somatodendritic membrane suggest that ERS is induced at inhibitory synapses by 2-AG, which is synthesized at local somatodendritic surface as well as at the base of the spine neck (Fig. 10A).
In adult Purkinje cells, the density of spines is much higher at spiny branchlets than at proximal dendrites, which reflects innervation territories of parallel fibers and climbing fibers (Ichikawa et al., 2002). In contrast, spines are extremely rare in the soma, because this domain is predominantly innervated by basket cell axons in adults. By pursuing occasional spotty labeling on somatic surface, we could find somatic spines and selective DAGLα labeling around the spines (Figs. 4C,D, 7D,E). At P10, when somatic spines are numerous, a number of DAGLα labeling was observed on the somatic surface just beneath climbing fiber–somatic spine synapses. Conversely, distalmost dendrites of Purkinje cells at P10 are poorly developed in terms of spinogenesis and synaptogenesis and extend filopodium-like processes instead of mature spines (Yamada et al., 2000). DAGLα was already expressed as spotty labeling on such immature dendrites (Fig. 7C). Therefore, DAGLα expression appears to be linked closely with the formation of spines and/or excitatory synapses, i.e., DAGLα is targeted to around spines or synapses when they are formed and matured but disappears when they are lost. This targeting mechanism will explain the distal-to-proximal gradient of DAGLα expression along the somatodendritic surface of adult Purkinje cells.
Spine is the major site of 2-AG synthesis in hippocampal pyramidal cell
CB1 and 2-AG mediates ERS in the hippocampus as well (Hajos et al., 2000; Varma et al., 2001; Wilson and Nicoll, 2001; Hashimotodani et al., 2005). We found postsynaptic expression of DAGLα in hippocampal pyramidal cells, but its subcellular distribution is different from that of Purkinje cells. DAGLα labeling was mainly present in dendritic spines. Within spines, DAGLα was detected in either the spine head or neck in some spines, whereas both portions were labeled in others. Furthermore, DAGLα labeling was dense in the stratum radiatum, whereas the labeling was considerably low in the stratum lacunosum-moleculare. This result suggests that trafficking of DAGLα is regulated in an input-selective manner in given CA1 pyramidal cells. The input-selective expression would facilitate DAGLα-mediated 2-AG synthesis at synapses receiving particular excitatory inputs, such as the Schaffer collaterals. CB1 is expressed at high levels in inhibitory terminals of cholecystokinin-positive interneurons (Katona et al., 1999; Tsou et al., 1999), whereas the level is ∼20–30 times lower in excitatory terminals (Kawamura et al., 2006). Electrophysiological data demonstrate that threshold for depolarization-induced ERS is much lower at inhibitory synapses than at excitatory synapses in the hippocampus (Ohno-Shosaku et al., 2002). Although strong activation is required, ERS at hippocampal excitatory synapses might occur rather selectively at the activated synapse because of low CB1 expression in excitatory terminals and abundant expression of DAGLα, AMPA receptors (Baude et al., 1995), mGluR5 (Luján et al., 1996, 1997), Gq-protein (Tanaka et al., 2000), and PLCβ1 (our unpublished data) in postsynaptic spines (Fig. 10B). In contrast, ERS will be readily induced at local inhibitory synapses carrying high levels of CB1. The lack of dendritic expression of DAGLα further suggests that ERS at such inhibitory synapses is triggered by 2-AG synthesized and released from spines forming excitatory synapses.
In conclusion, DAGLα is targeted to postsynaptic spines in both cerebellar and hippocampal neurons, which should be important for efficient production of 2-AG depending on excitatory synaptic activity. The difference in its fine distribution around spines may be attributable to differences in the synaptic organization and in the density of presynaptic CB1 between the cerebellum and hippocampus. The localization of DAGLα may be regulated so that 2-AG can readily reach presynaptic CB1 and exert its swift retrograde modulation.
This investigation was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
- Correspondence should be addressed to Masahiko Watanabe, Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan. Email:

domain: literature


text:
Luthermuir WFC travelled to Balgownie knowing that this would be a hard afternoon against a very good passing Aberdeen Reserves.
With the game being filmed for some of the girls getting an opportunity to apply for a scholarship to the USA, Luthermuir knew this would be a tough 90 minutes ahead.
Aberdeen started the brighter and within three minutes was 1-0 up, a great ball split the Luthermuir defence open and as the girl raced in on goal she coolly slotted it home by Charles.
Aberdeen were pinning Luthermuir back and continued to pass the ball around but the defence stayed strong when they needed to be. Luthermuir created their first real chance when Hendry took a free kick that just sailed by everyone and past the post.
Aberdeen doubled their lead when a great ball down the right had the winger racing to the by-line, as she crossed to the back post the attacker rose above Mowatt and headed home for 2-0.
Luthermuir were struggling to get their foot on the ball and Aberdeen was pressing for more goals.
A great ball through the middle had Hendry and Middleton scrambling to clear but the Aberdeen attacker nicked it away and she fired it goal wards but Charles saved well.
Within two minutes it was 3-0 when a harmless cross was not cleared by Hendry or Middleton and the attacker was first to react and fire home with Charles helpless.
Luthermuir got on the score sheet when Hendry fired a free kick over the back line, Brough raced clear and, as the keeper advanced, she coolly lobbed from 10 yards for 3-1.
Luthermuir then started to press the home side and Murison was taken down just outside the box out on the right. Hendry fired over the free kick and Boyack just poked it wide.
As the half was drawing to a close a mix up in the back line had Luthermuir failing to clear the ball, w Charles then kicked it clear but it ricocheted off the attacker’s back and fell into the goal for 4-1.
Luthermuir started the second half looking to get back in the game but Aberdeen created the first chance. A great ball down the middle had the attacker getting clear and, as she fired it goalwards, Charles made a great save.
Over the next 10 minutes Aberdeen continued to dominate the game and passed the ball about well but failed to get past Charles who was in a determined mood to keep a clean sheet in the second half.
A great one two between the midfield created a chance 25 yards out but her shot was held well by Charles. On the 63rd minute, a corner to Aberdeen was whipped over and Charles punched it clear but the attacker collided with her and the game was stopped for five minutes for treatment to both players.
Charles was a little shaken but continued in goal and then managed to pull off a string of saves to keep the score line at 4-1.
On the 71st minute, the ball was going for a corner and, as Charles raced to the byeline to collect, the referee deemed this to be a pass back, Godsall reacted by having words with the referee who then issued a red card.The free kick 10 yards from the line was fired to the side of the goal.
With Aberdeen having a one man advantage, Luthermuir failed to get a hold of the ball and continued to defend deep in their own half andCharles made some good saves to prevent the score line being higher. The final chance of the game came when Lownie played the ball in to the middle of the park and Calder raced on to it and rifled the ball into the top corner from the edge of the box to make it 4-2.
Player of the match: Rosie Charles.
Match ball sponsor: Scots Corner, Luthermuir
Luthermuir - Charles, Murray, Godsall, Middleton, Hendry, Mowatt, Calder, Boyack, Brough, Murison, Lownie – Subs: Bruce

domain: sports


text:
Now showing items 1-3 of 3
Robots and Racism [Podcast]
Racism is a major problem in our society and it is an issue from which the HRI community cannot shy away from. Several studies showed that people transfer their racial biases onto robots. In this episode we will talk about ...
Robots and racism
Most robots currently being sold or developed are either stylized with white material or have a metallic appearance. In this research we used the shooter bias paradigm and several questionnaires to investigate if people ...
Persistence of the Uncanny Valley: the Influence of Repeated Interactions and a Robot’s Attitude on its Perception
(University of Canterbury. Human Interface Technology Laboratory, 2015)
The uncanny valley theory proposed by Mori has been heavily investigated in the recent years by researchers from various fields. However, the videos and images used in these studies did not permit any human interaction ...


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SpudGun 3 - Those Damned Junkie Deejays
Those Damned Junkie Deejays
Sarah Louisa - Vocals
Al - Guitar & Vocals
Anto - Guitar & Vocals
Poncho - Bass
Antonio - Drums
Those Damned Junkie Deejays are a Dublin based 5 piece funk-rock type thing. With naive plans for global domination they are relying on their wits and some pretty kick ass music. Those with a keen eye (and ear) might spot them giging around Dublin and beyond. Look out for their debut single due to be released real soon!!
In the mean time they have (foolishly) allowed self appointed music critic and general loudmouth Lester St. Vincent access to their Blog page so be sure to check out his gormless rantings.
Oh and don't forget to visit their website (www.junkiedeejays.com) where you can log on to their forum and give them abuse for not having updated it in a long time... but that should all change soon...
For a listen to a rare acoustic pre-production version of Something Temporarily visit the progressive productions website (www.progressiveprodutions.net) where you can download that track among other little gems.
Those Damned Junkie Deejays are currently in pre-production for the recording of their debut single tentatively titled "Guilty" and working on a shed load of new songs for your aural pleasure. Keep an eye on the blog for the latest news.

domain: artistic