prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What did the decision to base the water rates on usage reflect?

Read the following text and answer briefly.

Context 1: Time to clean house in Paso Robles Home
Front Page » Time to clean house in Paso Robles
September 5, 2010 Opinion By JIM REED
I’d like to give you an update on the issue of our civil servants cramming hundreds of millions of dollars in spending down our throats after the people of Paso Robles voted down the water rate increase last November  The rate increase is being hung up in the courts by the City Attorney  What was supposed to be a quick issue to get in front of a judge, has been drug out as long as possible by the City Attorney 
Even if the courts throw out the current rate increase, I expect that our civil servants will just change a couple of words in the rate increase notice and force the same old plan on us again 
There is a real problem with the people we have hired to work for us in Paso Robles  It seems that decisions are made based on some agenda, even if it is contrary to citizens’ wishes 
City Councilmen Ed Steinbeck, Nick Gilman and Mayor Duane Picanco, on August 19th, voted unanimously to hire the same law firm employed by the City of Bell  You may have heard the recent news story about the City of Bell’s corrupt city representatives 
This law firm allowed the elected officials and City employees to pillage the General Fund for their own benefit, contrary to the rights and interests of the citizens  We are already paying several City employees $12,000 per month with equally ridiculous benefits and pensions  What does this say about our elected representatives?
I believe most residents are like me  We elect people we believe have our best interest in mind  Over the last few years I have seen that nothing is farther from the truth  The people we have elected have lost track of the fact that “the City” exists to protect and deliver services to the citizens  To them it is some all-important ideal they strive to cultivate and improve according to their agenda  They have forgotten that they are elected to represent the citizens 
We have an election coming up in November  We have the opportunity to elect some responsible, principled people to represent us  If we elect more people from within this system, we will get more of the same type of government  We need to look at where the new candidates stand  Will they lawfully represent the citizens of the city? Or, are they happy with the way things are being run?
We have stood together in the past and have made real significant changes in important matters that are going to affect our lives for years to come  There are several thousand citizens that made their voice heard on the water issue, more than enough votes to make a change in our city government 
Please come out and vote for a democratic representative governing body for Paso Robles instead of the tyrannical leadership that exists now 
Jim Reed is a longtime resident of Paso Robles 
Subjects: Opinion Paso Robles Paso Robles City Council Vote	Related:
<- Previous Next ->	Endless Summer Nights at Edna Valley, event photos Trial postponed for Paso Robles woman accused of forgery The comments below represent the opinion of the writer and do not represent the views or policies of CalCoastNews com  (moderator@calcoastnews com Comment Guidelines )
2 whatisup says:	09/13/2010 at 9:27 pm
pasoobserver – Here is something to observe and get you going in the right direction:
California Government Code Section 65584
(a) (1) For the fourth and subsequent revisions of the
housing element pursuant to Section 65588, the department shall
determine the existing and projected need for housing for each region
pursuant to this article  For purposes of subdivision (a) of Section
65583, the share of a city or county of the regional housing need
shall include that share of the housing need of persons at all income
levels within the area significantly affected by the general plan of
(2) While it is the intent of the Legislature that cities,
counties, and cities and counties should undertake all necessary
actions to encourage, promote, and facilitate the development of
housing to accommodate the entire regional housing need, it is
recognized, however, that future housing production may not equal the
regional housing need established for planning purposes 
(b) The department, in consultation with each council of
governments, shall determine each region’s existing and projected
housing need pursuant to Section 65584 01 at least two years prior to
the scheduled revision required pursuant to Section 65588  The
appropriate council of governments, or for cities and counties
without a council of governments, the department, shall adopt a final
regional housing need plan that allocates a share of the regional
housing need to each city, county, or city and county at least one
year prior to the scheduled revision for the region required by
Section 65588  The allocation plan prepared by a council of
governments shall be prepared pursuant to Sections 65584 04 and
65584 05 with the advice of the department 
(c) Notwithstanding any other provision of law, the due dates for
the determinations of the department or for the council of
governments, respectively, regarding the regional housing need may be
extended by the department by not more than 60 days if the extension
will enable access to more recent critical population or housing
data from a pending or recent release of the United States Census
Bureau or the Department of Finance  If the due date for the
determination of the department or the council of governments is
extended for this reason, the department shall extend the
corresponding housing element revision deadline pursuant to Section
65588 by not more than 60 days 
(d) The regional housing needs allocation plan shall be consistent
with all of the following objectives:
(1) Increasing the housing supply and the mix of housing types,
tenure, and affordability in all cities and counties within the
region in an equitable manner, which shall result in each
jurisdiction receiving an allocation of units for low- and very low
(2) Promoting infill development and socioeconomic equity, the
protection of environmental and agricultural resources, and the
encouragement of efficient development patterns 
(3) Promoting an improved intraregional relationship between jobs
(4) Allocating a lower proportion of housing need to an income
category when a jurisdiction already has a disproportionately high
share of households in that income category, as compared to the
countywide distribution of households in that category from the most
recent decennial United States census 
(e) For purposes of this section, “household income levels” are as
determined by the department as of the most recent decennial census
pursuant to the following code sections:
(1) Very low incomes as defined by Section 50105 of the Health and
(2) Lower incomes, as defined by Section 50079 5 of the Health and
(3) Moderate incomes, as defined by Section 50093 of the Health
and Safety Code 
(4) Above moderate incomes are those exceeding the moderate-income
level of Section 50093 of the Health and Safety Code 
(f) Notwithstanding any other provision of law, determinations
made by the department, a council of governments, or a city or county
pursuant to this section or Section 65584 01, 65584 02, 65584 03,
65584 04, 65584 05, 65584 06, 65584 07, or 65584 08 are exempt from
the California Environmental Quality Act (Division 13 (commencing
with Section 21000) of the Public Resources Code) 
pasoobserver says:	09/13/2010 at 6:52 pm
To whatisup —- First of all, I reviewed AB 602 Assembly Bill  Thanks  I am sorry to inform you but AB 602 is not the LAW as you so stated in your blog  I contacted the Deputy Chief Council’s office in Sacramento handling AB 602 to confirm your misstatement of facts  You know,in the English language, It shouldn’t be so difficult to answer some simple questions with a “YES” or “NO” answer  Yet, you are reluctant to do so, but you go on and on with a thesis along with some rhetoric  I never talked about a court suit over the “water issue”, I asked YOU, not about waiting for a court decision  Maybe, you did with some other people  Also, I was not ranting about the wineries usage of water  My response to you on your vague question about “there are people not paying their fair share for their use of water”  I related, are you talking about the wineries? I am well aware that most of the wineries are outside the city limits using the same aquifer  You took my question out of context , nice try! You are just being a popinjay and rhetorical  Also, you didn’t answer another question about “what is the unit cost of water” in Templeton? as compared to Paso Robles 
whatisup says:	09/13/2010 at 8:54 pm
I am on a well  I am sure you are capable of doing your own homework  I also am quite sure if you really contacted the Deputy Chief Counsel’s Office you have been set straight  What I gave you is a proposed small adjustment in the wide range of laws that make up the California Housing element  I assumed you could stumble onto the facts based on what I gave you  By the way, I believe you can review the Paso Robles Housing element plan on the City’s website or at the Library  The California Housing Element Laws that all cities and counties have to follow have been in place for almost 25 years  I realize you don’t actually have a clue how to look the laws up  Either educate yourself or keep making a fool of yourself, your choice  A simple Google search of California Housing Element Laws will get you going  Good Luck!
TO WHATISUP — I WOULD LIKE TO KNOW WHAT LAW YOU ARE REFERRING TO THAT SAYS “WE” THE PEOPLE HAVE TO SUBSIDIZE NEW DEVELOPMENT? AGAIN, FOR THE THIRD TIME, YOU FAILED TO ANSWER MY QUESTIONS POSED TO YOU IN MY PRIOR RESPONSES TO YOU ON SEPT 10TH &11TH  IS THERE A REASON WHY YOU DON’T WANT TO ANSWER THEM? YOU DO WHAT OUR ELECTED OFFICIALS DO SO WELL, AND THAT IS “IN ONE EAR AND OUT OF THE OTHER EAR” IT SEEMS TO ME THAT YOU ARE EITHER EMPLOYED BY THE CITY OR YOU HAVE OTHER DEALING WITH THE CITY, SO BE IT  IT APPEARS TO ME THAT YOU THINK THE CITY DOES EVERYTHING RIGHT  APPARENTLY, YOU PRESENT YOURSELF AS BEING VERY BIAS ON CITY DECISIONS  IT LIKE THEY CAN’T DO ANYTHING WRONG ACCORDING TO YOUR LOGIC  THEY KNOW WHAT IS BEST FOR THE CITIZENS OF PASO,THAT IS A GOOD EXAMPLE OF ARROGANCE ALONG WITH NARCISSISM 
WHAT PEOPLE ARE YOU TALKING ABOUT THAT DOESN’T PAY THEIR FAIR SHARE OF WATER? ARE YOU REFERRING TO THE WINERIES USING THE SAME AQUIFER?
I BELIEVE YOU RELATED THAT YOU RESIDE IN TEMPLETON, BUT YOU OWN PROPERTY IN PASO  BY THE WAY, WHAT IS THE COST PER UNIT OF WATER USAGE IN TEMPLETON COMPARED TO PASO? OF COURSE, TEMPLETON IS IN AN UNINCORPORATED AREA (COUNTY JURISDICTION) 
WELL, I GAVE YOU SOME SUGGESTIONS ON HOW TO PAY FOR THE NACIMIENTO WATER PIPELINE AND SEWER TREATMENT PLANT  ALSO, REMEMBER IT’S THE CITIZENS’ MONEY THAT IS BEING SPENT  WHAT IS MOST IMPORTANT OF ALL, IS LET THE CITIZENS OF PASO DECIDE WITH THEIR VOTE ON HOW TO FINANCE THIS HUGE CAPITAL IMPROVEMENT PROJECT EXPENDITURE  JUST BE IN COMPLIANCE WITH STATE PROPOSITION 218 AND STOP CIRCUMVENTING THE LAW 
WOULD YOU OBJECT TO HAVING TO FINANCE SOME NEW BONDS ON YOUR PROPERTY TAX BILL AS A ” SPECIAL TAX” OR AN ASSESSMENT TAX” TO PAY FOR THE NACIMIENTO WATER PIPELINE AND SEWER TREATMENT PLANT? A PERCENTAGE OF PASO CITIZENS FINANCE LOCAL SCHOOL BONDS ON THEIR PROPERTY TAX BILL AND DON’T HAVE ANY KIDS GOING TO SCHOOL  HOW ABOUT THAT COMPARISON FOR YOU TO THINK ABOUT? WHAT SAY YOU?
I say less CapsLock, please 
whatisup says:	09/12/2010 at 11:41 pm
I have answered your questions  I have been quite detailed in my answers and I am sorry if you can’t deal with the detail  I guess it is your inconvenient truth  You do seem to like to deflect and go around in circles  Another example, now you are ranting about the wineries using the same aquaifier as the City  Let me be clear for you, I don’t like the amount of water the wineries are using  However, the wineries are in the County, not in the City and the City can’t do anything about it  They wineries are allowed to take the water they are taking even if it drops the City’s water levels in their wells  You need to complain to Sacramento  It sounds like you just don’t want to pay anything for the infrastructure because you really just don’t want it built 
Several of your observations of my opinions are bizarre considering I have stated several times I believe the Courts need to decide if Paso Robles has, or has not followed the rules as to funding the infrastucture  Obviously, as I have stated before, if the City loses the lawsuit the infrastructure will have to be paid out of the City’s General Fund until a new method of payment is voted on by the Citizens of Paso Robles  Pretty clear 
Your idea of charging based on a special assesment rather than the amount of water a property uses means that people who use little water, but live on a more expensive property will pay more than their share, based on their water usage  In addition, how do you deal with a rental unit where the renter is supposed to pay the water bill? Your idea is inherantly unfair, but my guess is it will favor you, so you don’t care if it is unfair and other people would pay part of your share  You also have decided that since I have alternative ideas to yours I must work for, or have business with the City of Paso Robles, another attempt to deflect from the issue  However, once again, I have never worked for the City or have ever done business with the City and don’t expect to ever do business with the City  I do own property in the City which is why I pay attention  Finally, it turns out there needs to be a fix to the housing element laws, the existance of which you are questioning  As I understand it the fix to the housing elemnt laws is because of some lawsuit  This should give you all the information you need to educate yourself on the California Housing Element laws that every city and county in California has to follow:
BILL ANALYSIS ————————————————————
|SENATE RULES COMMITTEE | AB 602|
|Office of Senate Floor Analyses | |
|1020 N Street, Suite 524 | |
|(916) 651-1520 Fax: (916) | |
|327-4478 | |
———————————————————— THIRD READING
Bill No: AB 602
Author: Feuer (D), et al
Amended: 8/20/10 in Senate
SENATE TRANSPORTATION & HOUSING COMM : 6-3, 6/29/10
AYES: Lowenthal, DeSaulnier, Kehoe, Pavley, Simitian, Wolk
NOES: Huff, Ashburn, Harman
ASSEMBLY FLOOR : Not relevant
SUBJECT : Statute of limitations on housing element
SOURCE : California Rural Legal Assistance Foundation
Housing California DIGEST : This bill states the intent of the Legislature
in enacting this bill to modify the courts opinion in Urban
Habitat Program v  City of Pleasanton (2008) 164
Cal App 4th 1561, with respect to the interpretation of
Section 65009 of the Government Code, and revises and
clarifies statute of limitations and remedies for specified
housing related challenges 
Senate Floor Amendments of 8/20/10 revise the statute of
limitations and remedies for specified housing-related
ANALYSIS : The Planning and Zoning Law requires cities
and counties to prepare and adopt a general plan, including
a housing element, to guide the future growth of a
community  Following a staggered statutory schedule,
cities and counties located within the territory of a
metropolitan planning organization (MPO) must revise their
housing elements every eight years, and cities and counties
in rural non-MPO regions must revise their housing elements
every five years  These five- and eight-year periods are
known as the housing element planning period 
Before each revision, each community is assigned its fair
share of housing for each income category through the
regional housing needs assessment (RHNA) process  A
housing element must identify and analyze existing and
projected housing needs, identify adequate sites with
appropriate zoning to meet its share of the RHNA, and
ensure that regulatory systems provide opportunities for,
and do not unduly constrain, housing development  The
reviews both draft and adopted housing elements to
determine whether or not they are in substantial compliance
with the law  The Planning and Zoning Law and the Subdivision Map Act
also includes a number of sections governing zoning and
entitlements specifically related to housing, including:
? The Housing Accountability Act, which requires a city or
county to make one or more specified findings in order to
disapprove a particular housing development 
? A provision requiring cities and counties, when adopting
an ordinance which limits the number of housing units
which may be constructed on an annual basis, to make
findings as to the public health, safety, and welfare
benefits that justify reducing the housing opportunities
of the region  ? Density bonus law, which requires cities and counties to
grant a developer a density bonus, incentives, and
concessions when the developer proposes to include
specified percentages of affordable housing within a
development  ? The Least Cost Zoning Law, which requires cities and AB 602
counties to designate and zone sufficient vacant land for
residential use with appropriate standards to meet
housing needs for all income categories and to contribute
to producing housing at the lowest possible cost 
? A requirement that, when determining whether to approve a
tentative subdivision map, a city or county shall apply
only those ordinances, policies, and standards in effect
as of the date the developer’s application is deemed
Prior to a recent court decision, it was understood that
current law allowed a party to challenge the adequacy of a
city’s or county’s housing element at any time during a
planning period, provided that the challenger brought the
action “in support of or to encourage or facilitate the
development of housing that would increase the community’s
supply of [affordable] housing ” The challenging party was
required first to serve the city or county with a notice
identifying the deficiencies in the housing element  After
60 days or the date on which the city or county took final
action in response to the notice, whichever occurred first,
the challenging party had one year to file the action in
court  This process and statute of limitations also
applied to actions brought pursuant to the housing-related
statutes listed above  In 2006 Urban Habitat Program brought suit to challenge the
City of Pleasanton’s housing policies, including the city’s
annual cap on housing permits and the city’s cap on the
aggregate number of permissible housing units, both of
which Urban Habitat claimed were insufficient to allow the
city to meet its RHNA obligation  In 2008, the First
District California Court of Appeals issued an unpublished
decision in the case of Urban Habitat Program v  City of
Pleasanton allowing the case to proceed with respect to
some causes of action, but ruling that the challenge to the
housing element itself was time-barred  The court stated:
Although the statute does not specify the time within
which [a deficiency] notice must be given, it is our
conclusion that the statute must be interpreted as
containing a time limit within which this requirement
must be met? In sum, a party bringing a challenge AB 602
governed by section 65009, subdivision (d), has 90
days from the date a legislative action is taken or
approval is given to notify the local land use
authority of any claimed deficiencies in such an
action or approval  Its claim then accrues 60 days
after it gives this notice 
In other words, instead of being able to initiate a
challenge to a deficient housing element at any time during
the planning period, housing advocates and other interested
parties may now only initiate such a challenge by
submitting a deficiency notice within 90 days of the
housing element’s adoption 
1 Removes from the current list of city or county actions
which may be challenged pursuant to Government Code 65009
notice and accrual provisions those actions related to
the Housing Accountability Act, the Subdivision Map Act,
and the application of a Density Bonus ordinance to a
particular project, all of which are project-specific
actions  The bill maintains the ability to use these
notice and accrual provisions to challenge the adequacy
of a city’s or county’s density bonus ordinance
2 Extends lengthening the time in which a deficiency notice
may be served to cover all remaining city or county
actions described in this section of law, as opposed to
just housing element challenges  In other words, the
amendments apply the longer timeframe to serve the
deficiency notice to actions relating to the Least Cost
Zoning Law, annual limits on housing permits, and the
adequacy of a density bonus ordinance, in addition to
housing element law  3 Provides that an entity challenging such an action in
support of affordable housing may serve the deficiency
notice up to five years after the city’s or county’s
action  After 60 days or the date on which the city or
county takes final action in response to the notice,
whichever occurs first, the challenging party has one
year to file an action in court, except that the lawsuit AB 602
may not be filed more than five years after the city’s or
county’s action  In other words, the entity must file
the lawsuit within one year of the expiration of the
deficiency notice or within five years of the city’s or
county’s action, whichever occurs first 
4 Provides that a housing element from a prior planning
period may not be challenged if the city or county has
adopted a revised housing element for the new planning
Government Code 65755   Current law requires a court, if it
finds any portion of a general plan, including a housing
element, out of compliance with the law, to include within
its order or judgment one or more of the following remedies
for any or all types of developments or any or all
geographic segments of the city or county until the city or
county has complied with the law:
? Suspend the authority of the city or county to
issue building permits 
grant zoning changes and/or variances 
grant subdivision map approvals 
? Mandate the approval of building permits for
residential housing that meet specified criteria 
? Mandate the approval of final subdivision maps for
housing projects that meet specified criteria 
? Mandate the approval of tentative subdivision maps
for residential housing projects that meet specified
This bill clarifies that in any action or proceeding
brought pursuant to the notice and accrual provisions of
Government Code Section 65009 described above, neither the
court remedies described above nor any injunction against
the development of a housing project shall abrogate,
impair, or otherwise interfere with the full exercise of
the rights and protections granted to an applicant for a
tentative map or a vesting tentative map under specified
provisions of the Subdivision Map Act or to a developer
under a specified provision relating to development AB 602
Under current law, HCD operates a number of grant programs
to which cities and counties may apply  In many cases, the
law requires a city or county to have an HCD-approved
housing element in order to be eligible for funding  This bill provides that if a third-party challenges the
adequacy of a housing element in court and the court finds
that the housing element substantially complies with all of
the requirements of housing element law, the element shall
be deemed to be in compliance for purposes of state housing
The statutory language interpreted by the court and at
issue in this bill was added to statute by AB 998 (Waters),
Chapter 1138, Statutes of 1983, a bill sponsored by the
League of California Cities and the California Building
Industry Association  AB 998 created a short statute of
limitations period for land use decisions generally but
provided a specific exception to protect the ability to
challenge deficient housing elements  The Senate Housing
and Land Use Committee and the Senate Third Reading
analysis of the bill stated that the bill:
Specifies that for challenges in support of low- and
moderate-income housing requirements, the petitioner
shall notice local government 60 days prior to filing
action  The [one-year] statute of limitations then
begins on the first day the legislative body fails to
In the intervening 25 years prior to the Urban Habitat
ruling, housing advocates filed and successfully settled at
least ten cases in which the 60-day deficiency notice was
sent more than 90 days after adoption of the city’s or
county’s housing element  In none of these cases was the
timeliness on the advocates’ suit contested  Likewise, six
bills amended other portions of this statute during those
intervening years, and there was never any controversy
surrounding the lack of a deadline for housing advocates to
serve a deficiency notice nor any attempt to change the AB 602
statute in this regard  Current level of housing element compliance   According to
HCD’s website as of June 7, 2010, only 46 percent of cities
and counties have adopted an HCD-approved housing element
for the current planning period that began in 2005 for the
San Diego region, 2008 for the Southern California, Fresno,
Kern, and Sacramento regions, and the summer of 2009 for
the remaining areas of the state  Unlocking the private market   The purpose of housing
element law is to create opportunities for the private
housing market to function  Builders cannot build without
access to appropriately zoned land, and current land use
plans in many cities and counties in California fail to
provide sufficient opportunities to accommodate projected
population growth  The San Diego Association of
Governments’ Regional Comprehensive Plan describes this
typical California paradox in the following way:
Under current plans and policies, more than 90 percent
of [the San Diego region’s] remaining vacant land
designated for housing is planned for densities of
less than one home per acre, and most is in the rural
back country areas dependent upon scarce groundwater
supplies  And of the remaining vacant land planned for
housing in the 18 incorporated cities, only about
seven percent is planned for multifamily housing  When
taken together, the current land use plans of the 19
local jurisdictions do not accommodate the amount of
growth anticipated in our region  SANDAG’s population
forecast, which reflects the current adopted local
land use plans in the region, projects that while
population will increase by 37 percent by 2030,
housing will grow by just 30 percent  The forecast
shows that if local plans are not changed, demand for
housing will continue to outpace the supply, just as
Housing element law addresses this problem directly by
requiring cities and counties to zone land at appropriate
densities to accommodate the projected housing needs of all
income groups and to remove constraints that prevent such
sites from being developed at the allowed densities  AB 602
Cities and counties, however, are not required to build
housing because that is the role of private developers 
The law holds cities and counties accountable only for that
which they control: zoning and land use entitlements 
Without the ability to enforce housing element law, the
market’s ability to meet housing demand may well remain
locked up 
FISCAL EFFECT : Appropriation: No Fiscal Com : No
SUPPORT : (Verified 8/23/10)
California Rural Legal Assistance Foundation (co-source)
Housing California (co-source)
Advocates for Affordable Homes in Fremont
California Coalition for Rural Housing
Community Housing Improvement Program
Community Housing Works
Eden Housing
Fair Housing of Marin
Grassroots Leadership Network of Marin
Kennedy Commission
Public Advocates, Inc
San Diego Housing Federation
Self-Help Enterprises
Sierra Club of California
American Planning Association, California Chapter
JA:nl 8/23/10 Senate Floor Analyses SUPPORT/OPPOSITION: SEE ABOVE
pasoobserver says:	09/11/2010 at 11:17 pm
To whatisup — Thank you for your response to my comments  However, you failed to answer some of my questions that I mentioned to you  It’s almost like dealing with some City officials  They just let the public vent at their bimonthly council meetings  In my opinion, it’s difficult to deal with narcissism and arrogance  Over the years, there has been some very good input to our elected officials on how to proceed on the Nacimiento water pipeline,but it fell on deaf ears  You wanted me to answer some of your questions,but you did not answer some of my questions  Again, are you willing to subsidize new development?,Yes?or No?, are you willing to pay for a commodity that you are not receiving? Yes?or No? and another question for you  Are you willing to pay over 300% on your water bills within the five (5) year plan that the City has proposed? Also, the water rates will be subject to later increases too  By the way, I do concur with the city’s plan of “you pay for the amount of water units you use”  (748 gal=one unit)  However, the higher water rates are not good for our senior citizens on fixed incomes and other struggling families in our community  My first suggestion years ago was desalination  The response was it was too expensive  Of course, now it is more expensive  I would suggest that our elected officials recall the existing bonds (The bonds can be recalled early)  The City council can explain to the citizens in detail with financing of new bonds at a lower interest rate as of now for the sewer plant and Nacimiento water pipeline and present their new proposal in compliance with Proposition 218  Let the citizens of Paso VOTE on the financing bonds for their approval  Most of the citizens,that I had spoken to were not happy with the way our City Council handled the Nacimiento water pipeline project  The citizens of Paso didn’t give our City Council a “BLANK CHECK” for $176 million to spend without voter approval  I would suggest that it be a “special tax” or “an assessment” be levied on our property taxes  A percentage of those bonds can be deducted on Federal Income taxes  As it is now, a” fee” on a capital funding project is not deductible  Of course, there are homeowners would not go for this suggestion due to our poor economy  My analogy mentioned above would be, you would get something back on a “special tax” or an “assessment” verses nothing on a “fee”  What say you?
whatisup says:	09/12/2010 at 9:02 am
Unfortunately the law says we have to subsidize new development in California  I don’t like it, but it is the law  I know paying using the property taxes was bandied about  The argument against it was it would mean some would be paying for water they aren’t using and others could be big water users, but pay a small special assessment on their property taxes  I think the decision that was made to base it on usage was out of fairness  It seems to me if people are using water and not paying their share of the costs it is not fair  The Senior issue is very difficult  If someone is retired for twenty years is it realistic to think prices don’t go up during the 20 years of retirement  Think what prices were in 1990 compared to today  Should Seniors never have to pay for capital improvements? Paso Robles also had very low water rates  Rates that are no longer possible given the circumstances  Desalination will happen eventually  California is out of water  If you want to pay $1,000,000 a gallon there is no more allotable water of any consequence in California  The expense will be tremendous — still have to build a desalination plant, still have to build a pipeline  I don’t know if the plant has to be built along the ocean or if the salt water could be piped over to Paso Robles  If it has to be built along the ocean, Paso Robles doesn’t own land on the ocean and, in any case, the environmentalists will keep it in courts for years as they have done so for other proposed desalination plants in Southern California  Eventually necessity will force desalination past the environmentalists, but not yet 
pasojim says:	09/13/2010 at 7:46 am
Whatisup – On one of your previous post you made the comment you haven’t heard any of the legal suggestions for the water issue, But you obviously have  That is a good thing  So we can move the discussion ahead 
Once, again this was handled incorrectly by our city custodians from the beginning  And now here we are  The public is not supporting this very expensive, very limited benefit project  As you said, until a plan is developed that the public can support, things don’t look good 
All this discussion about the water issue has only reinforced my opinion the issue hasn’t been about water, only how the plan should be paid for  Or more specifically, to what extent do we allow our elected custodians and our un-elected GOD tzar decide which laws they will follow and which laws they will ignore  When the City GOD tzar tell citizens at a council meeting if we don’t agree with the City’s plan, then we should just sue him, and when the City Attorney explains to a citizen at a City Council meeting that she does have to respond to their questions because she does NOT work for them  When the project is voted down by the citizens and the council brings it right back up, it is clear that our elected representatives are not doing their job providing direction to their employees and listening to and representing the CITIZENS 
The subject of the original post was the need to elect different representation  I think with all the conversation made on this post, as well as the post on Cal Coast about the hiring of the new legal firm you were involved in, Supports my original opinion 
Context 2: JoVE | Peer Reviewed Scientific Video Journal - Methods and Protocols
A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Downs syndrome  Octavio Garcia, Maria Torres, Pablo Helguera, Pinar Coskun, Jorge Busciglio 
PUBLISHED: 07-02-2010	Downs syndrome (DS) is the most common genetic cause of mental retardation  Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology  However, the mechanisms involved in DS spine alterations are not known  In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons  We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion  In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology 
Analysis of Dendritic Spine Morphology in Cultured CNS Neurons Authors: Deepak P  Srivastava, Kevin M  Woolfrey, Peter Penzes  Published: 07-13-2011 JoVE Neuroscience
Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses  These structures are rich in actin and have been shown to be highly dynamic  In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain  Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number  Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers  Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders  In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages  Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells  Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments  These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo 
Play ButtonIsolation and Culture of Mouse Cortical AstrocytesAuthors: Sebastian Schildge, Christian Bohrer, Kristina Beck, Christian Schachtrup  Institutions: University of Freiburg , University of Freiburg  Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics  In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail  This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups  The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture  Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes  Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers  This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology Neuroscience, Issue 71, Neurobiology, Cellular Biology, Medicine, Molecular Biology, Anatomy, Physiology, brain, mouse, astrocyte culture, astrocyte, fibroblast, fibrinogen, chondroitin sulfate proteoglycan, neuronal regeneration, cell culture, animal model50079Play ButtonImaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination MicroscopyAuthors: Marijn Schouten, Giulia M  R  De Luca, Diana K  Alatriste González, Babette E  de Jong, Wendy Timmermans, Hui Xiong, Harm Krugers, Erik M  M  Manders, Carlos P  Fitzsimons  Institutions: University of Amsterdam, University of Amsterdam Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain  Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity  The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit  Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy  However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm 
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines  These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen  Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures  Possible applications of SIM overlap with those of confocal microscopy  However, the two techniques present different applicability  SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light  In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM  The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal  After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software Neuroscience, Issue 87, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite51276Play ButtonSetting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated TransportAuthors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky  Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259 The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue  Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer  Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture  RBEC were isolated from 5-week-old rat cortices  The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion  Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue  The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix  The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium  They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells  RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders  The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average  The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0 26 ± 0 11 x 10-3 cm/min  Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer  In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),51278Play ButtonInducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing RatesAuthors: Alison X  Xie, Kelli Lauderdale, Thomas Murphy, Timothy L  Myers, Todd A  Fiacco  Institutions: University of California Riverside, University of California Riverside, University of California Riverside Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter  However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention  Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices  Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy  In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs  Applications of the technique for study of astrocytes are discussed  Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse51458Play ButtonInhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA ReceptorsAuthors: Laura E  Brown, Celine Fuchs, Martin W  Nicholson, F  Anne Stephenson, Alex M  Thomson, Jasmina N  Jovanovic  Institutions: University College London Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks  GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain  It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons  Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials  During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes  This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other  To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes  Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification  Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses  Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts  Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line52115Play ButtonTwo-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull PreparationAuthors: Xinzhu Yu, Yi Zuo  Institutions: University of California, Santa Cruz In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses  Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity  As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses  Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain  Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo  This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex  Therefore, images can be acquired immediately after surgery is performed  The experimental procedure can be performed repetitively over various time intervals ranging from hours to years  The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions Neuroscience, Issue 87, dendritic spine, mouse cortex, in vivo, two-photon microscopy, thinned-skull, imaging51520Play ButtonModeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered MiceAuthors: Robert S  McNeill, Ralf S  Schmid, Ryan E  Bash, Mark Vitucci, Kristen K  White, Andrea M  Werneke, Brian H  Constance, Byron Huff, C  Ryan Miller  Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development  In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture  Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes  The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro  Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo  Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma  Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time  Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft51763Play ButtonPaired Whole Cell Recordings in Organotypic Hippocampal SlicesAuthors: Chantelle Fourie, Marianna Kiraly, Daniel V  Madison, Johanna M  Montgomery  Institutions: University of Auckland, Stanford University Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron  When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased  This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue  A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation  Paired whole cell recordings are often perceived as too challenging to perform  While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed  The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings  While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation  In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity51958Play ButtonImaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium IndicatorsAuthors: Ruotian Jiang, Martin D  Haustein, Michael V  Sofroniew, Baljit S  Khakh  Institutions: University of California Los Angeles, University of California Los Angeles Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals  It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules  However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series  The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia  In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described  We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity  We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ  The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets  The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry Neuroscience, Issue 93, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging51972Play ButtonMethods to Assess Subcellular Compartments of Muscle in C  elegansAuthors: Christopher J  Gaffney, Joseph J  Bass, Thomas F  Barratt, Nathaniel J  Szewczyk  Institutions: University of Nottingham Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state  The loss of muscle mass and function with disease and age are significant public health burdens  We currently understand little about the genetic regulation of muscle health with disease or age  The nematode C  elegans is an established model for understanding the genomic regulation of biological processes of interest  This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species  Since C  elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo  Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo  These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance  Thus, C  elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function  Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism Developmental Biology, Issue 93, Physiology, C  elegans, muscle, mitochondria, sarcomeres, ageing52043Play ButtonImproved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term PotentiationAuthors: Agnès Villers, Laurence Ris



ground_truth: Fairness.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What do dendritic spines contain?

Read the following text and answer briefly.

Context 2:  Institutions: University of Mons Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding  LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied  However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood  This could be partly due to the various experimental conditions used by different laboratories  Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity  It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection 
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation  This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices  It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena Neuroscience, Issue 76, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Surgery, Memory Disorders, Learning, Memory, Neurosciences, Neurophysiology, hippocampus, long-term potentiation, mice, acute slices, synaptic plasticity, in vitro, electrophysiology, animal model50483Play ButtonIn Vivo Modeling of the Morbid Human Genome using Danio rerioAuthors: Adrienne R  Niederriter, Erica E  Davis, Christelle Golzio, Edwin C  Oh, I-Chun Tsai, Nicholas Katsanis  Institutions: Duke University Medical Center, Duke University, Duke University Medical Center Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish  Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated 1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling  Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders  Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish  This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants  Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype  Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf)  This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol  We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others  Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model50338Play ButtonDirect Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)Authors: Samira Samtleben, Juliane Jaepel, Caroline Fecher, Thomas Andreska, Markus Rehberg, Robert Blum  Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich Visualization of calcium dynamics is important to understand the role of calcium in cell physiology  To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular  Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types  To date, TED was used in cell lines, glial cells, and neurons in vitro  TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES)  The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging  The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red  At the beginning of the procedure, cells are transduced with a lentivirus  Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging  Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM  The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen  After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope  Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging  Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity  Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0 Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging50317Play ButtonPreparation of Dissociated Mouse Cortical Neuron CulturesAuthors: Lutz G  W  Hilgenberg, Martin A  Smith  Institutions: University of California, Irvine (UCI) This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain  These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation  These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation  The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared  Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells  To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step  This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death  Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons Neuroscience, Issue 10, cellular, molecular, neurobiology, neuron, calcium/sodium imaging, primary cultures, mouse562Play ButtonAnalysis of Schwann-astrocyte Interactions Using In Vitro AssaysAuthors: Fardad T  Afshari, Jessica C  Kwok, James W  Fawcett  Institutions: University of Cambridge Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries  Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4  In addition they myelinate the demyelinated axons at the site of injury 5 
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7  This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally  Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13 
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour 
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts  As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide  This allows the behaviour of the two cellular populations to be analyzed at the boundary  Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries 
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15  This assay is commonly known as inverted coverslip assay  Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip 
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation  Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19 
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur Cellular Biology, Issue 47, Schwann cell, astrocyte, boundary, migration, repulsion2214Play ButtonQuantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse NumberAuthors: Dominic M  Ippolito, Cagla Eroglu  Institutions: Duke University, Duke University One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation  As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity  Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry  Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization  We define synapses as points of colocalization between the signals generated by these markers  The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c eroglu@cellbio duke edu) under the ImageJ analysis software platform  The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers  This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development Neuroscience, Issue 45, synapse, immunocytochemistry, brain, neuron, astrocyte2270Play ButtonPreparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid PathologyAuthors: Diana M  Mathis, Jennifer L  Furman, Christopher M  Norris  Institutions: University of Kentucky College of Public Health, University of Kentucky College of Medicine, University of Kentucky College of Medicine The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity  The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid  Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments  The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory  Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds  This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3  Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research  Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity  Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment  Here, we discuss the steps taken during the dissection procedure to minimize these problems  Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments  The possible impact of other methodological factors on synaptic function in these animal models (e g  recording solution components, stimulation parameters) are also discussed  While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models Neuroscience, Issue 49, aging, amyloid, hippocampal slice, synaptic plasticity, Ca2+, CA1, electrophysiology2330Play ButtonMesenteric Artery Contraction and Relaxation Studies Using Automated Wire MyographyAuthors: Lakeesha E  Bridges, Cicely L  Williams, Mildred A  Pointer, Emmanuel M  Awumey  Institutions: North Carolina Central University, Durham, North Carolina Central University, Durham, Wake Forest University School of Medicine Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance  These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body  The rat mesenteric artery has a diameter greater than 100 μm  The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2  The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented  Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires  Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3 The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors 
Medicine, Issue 55, cardiovascular, resistant arteries, contraction, relaxation, myography3119Play ButtonVisualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero ElectroporationAuthors: Emilie Pacary, Matilda A  Haas, Hendrik Wildner, Roberta Azzarelli, Donald M  Bell, Djoher Nora Abrous, François Guillemot  Institutions: MRC National Institute for Medical Research, National Institute for Medical Research, Université de Bordeaux In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5  To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8  Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions 
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development  Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells  In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines Neuroscience, Issue 65, Developmental Biology, Molecular Biology, Neuronal development, In utero electroporation, dendrite, spines, hippocampus, cerebral cortex, gain and loss of function4163Play ButtonImaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture SystemAuthors: Haruki Higashimori, Yongjie Yang  Institutions: Tufts University, Tufts Sackler School of Graduate Biomedical Sciences Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS)  This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2   Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology  Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia  What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i e , axon, dendrite, or soma)3  This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites  In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia  Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell 
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP)  In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels  In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels  In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth  The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Biophysics, Microfluidics, Microfluidic culture platform, Compartmented culture, Neuron to glia signaling, neurons, glia, cell culture4448Play ButtonFluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal NeuronsAuthors: Chan-Ying Zheng, Ronald S  Petralia, Ya-Xian Wang, Bechara Kachar  Institutions: National Institutes of Health, Bethesda FRAP has been used to quantify the mobility of GFP-tagged proteins  Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest  The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest  The mobility of the protein is then analyzed by measuring the fluorescence recovery rate  This technique could be used to characterize protein mobility and turnover rate 
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility2568Play ButtonPrimary Neuronal Cultures from the Brains of Late Stage Drosophila PupaeAuthors: Beatriz Sicaeros, Jorge M  Campusano, Diane K  O'Dowd  Institutions: University of California, Irvine (UCI) In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae  The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation  The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium  The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated  The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating  Images show live cultures at 2 days  Neurons continue to elaborate processes during the first week in culture  Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP  Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses  A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons  These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses 
Context 3: Paper Info

Title: Two-stage Pipeline for Multilingual Dialect Detection
Publish Date: Unkown
Author List: Ankit Vaidya (from Pune Institute of Computer Technology), Aditya Kane (from Pune Institute of Computer Technology)

Figure

Figure 1: Class distribution of dialects
Figure 2: System diagram for dialect classification The LID classifies the input into one of 3 languages The sample is then further classified into dialects by language specific models 
Figure 3: Confusion matrix of 9-way classification Note that rows are normalized according to the number of samples is that class 
Our complete results for Track-1 using the two-stage dialect detection pipeline Model-* denotes the language of the models used for the experiments 
Performance on Track-1 validation dataset of individual models used in the two-stage pipeline "Lg" stands for language of the model used 
Comparative results of two-way classification using the finetuned (F T ) predictions and predictions adapted from three-way classification models 

abstract

Dialect Identification is a crucial task for localizing various Large Language Models  This paper outlines our approach to the VarDial 2023 DSL-TL shared task  Here we have to identify three or two dialects from three languages each which results in a 9-way classification for Track-1 and 6-way classification for Track-2 respectively 
Our proposed approach consists of a two-stage system and outperforms other participants' systems and previous works in this domain  We achieve a score of 58 54% for Track-1 and 85 61% for Track-2  Our codebase is available publicly 1  

Introduction

Language has been the primary mode of communication for humans since the pre-historic ages  Studies have explored the evolution of language and outlined mathematical models that govern the intricacies of natural language   Inevitably, as humans established civilization in various parts of the world, this language was modified by, and for the group of people occupied by that particular geographical region 
This gave rise to multiple national dialects of the same language  The VarDial workshop (colocated with EACL 2023) explores various dialects and variations of the same language  We participated in the Discriminating Between Similar Languages -True Labels (DSL-TL) shared task  In this task, the participants were provided with data from three languages, with each language having three varieties 
This shared task consisted of two tracks -Track-1 featuring nine-way classification and Track-2 featuring six-way classification  The second track included two particular national dialects of each language (eg  American English and British English), and the first track had one general  We ranked 1 st in both of the tracks 
Moreover, we beat the next best submission by a margin of 4 5% in the first task and 5 6% in the second task We were the only team to surpass the organizer baseline scores  We present our winning solution in this paper  We used an end-to-end deep learning pipeline which consisted of a language identification model and three language-specific models, one for each language 
We converged upon the best combination by doing an elaborate analysis of various models available  Furthermore, in this work we also analyze the performance of the pipeline as a whole and also provide an ablation study  Lastly, we provide some future directions in this area of research 

Related Work

The present literature encompasses various aspects of dialect identification  We study this from three perspectives: large language models, language identification and dialect classification problems 

Large Language Models

The success of transformers and BERT based models was inevitable since the initial boom of the transformer  2017) model  In recent years, many other architectures like RoBERTa and ELECTRA have further pushed the state-of-the-art in this domain  Moreover, autoregressive models like GPT and GPT-2 have also shown their prowess 
Multilingual versions of RoBERTA, namely XLM-RoBERTa are also available  Lastly, language specific models like Spanish BERT (la Rosa y Eduardo G  Ponferrada y Manu Romero y Paulo Villegas y Pablo González de Prado Salas y María Grandury, 2022) and Portuguese BERT are available as well  Our winning solution makes use of these large language models trained on specific languages 

Language Identification Models

Many multilingual language identification models have been developed in order to classify the language of the input sentence beforehand  Even though the initial works used n-gram models and generative mixture models or even conditional random fields and other classical machine learning methods like naive bayes , modern methods have shifted to the use of deep learning for language identification  
Recent works have mainly focused on deep learning based language identification, where handling codemixed data is a big challenge in the domain  For our experiments, we use a version of XLM-RoBERTa finetuned on a language identification dataset 2   This model has near-perfect test accuracy of 99 6% 

Dialect Classification

Dialect classification has been previously solved using statistical methods like Gaussian Mixture Models and Frame Selection Decoding or Support Vector Machines (SVM)   It has been explored relatively sparsely, mostly in the case for local languages   Deep learning approaches have been explored in previous editions of the VarDial workshop shared tasks and otherwise  
Dialect classification was also explored previously as a part of other shared tasks   We want to stress that given the multilingual nature of the dataset, using the present methods directly was not an option  In our work, although we take inspiration from the previous works, we propose a novel system that surpasses the performance of the previous systems by a large margin 

Data

The dataset  We observed that the class PT-BR had the most number of samples (2,724) and the class EN had the least number of samples (349), and thus the imbalance ratio was almost 1:8  We have illustrated the data distribution in Figure   We tried to mitigate this imbalance using over-sampling and weighted sampling methods 
However, the improved data sampling method did not affect the performance 

System Description

This was a problem of multi-class classification having 9 classes for Track-1 and 6 classes for Track-2  The samples were belonging to 3 languages having 3 varieties each, so the classification pipeline was made in 2 stages  The Language Identification (LID) model which is the first stage classifies the sentence into 3 languages: English (EN), Spanish (ES) and Portuguese (PT) 
The LID is a pretrained XLM-RoBERTa that is fine-tuned for the task of language identification  It is able to classify the input sentence into 20 languages  We classify and separate the samples according to their language  The samples corresponding to the specific languages are then fed into the language specific models for dialect identification 
For dialect identification we have used models like BERT and RoBERTa with a linear layer connected to the pooler output of the models  Then fine-tuning is done on the models for dialect identification   using the samples corresponding to the specific languages  For the task of dialect identification we experimented with several pretrained models like XLM-RoBERTa, BERT, ELECTRA, GPT-2 and RoBERTa 
All models were fine-tuned for 20 epochs with a learning rate of 1e-6 and weight decay 1e-6 with a batch size of 8  The best performing model checkpoint was chosen according to the epoch-wise validation macro-F1 score  5 Experiments and Results

Experiments using Large Language Models

For the task of Dialect Identification we have tried various language specific models like XLM-RoBERTa, BERT, ELECTRA, RoBERTa and GPT- 2  The base variant of all these models were used and all the models were used through the Hugging-Face library  The pooler output of these models was passed through a linear layer and the models were fine-tuned 
First, we experimented with different models for Track-1  All the models were trained for 20 epochs with learning rate 1e-6, weight decay 1e-6 and a batch size of 8  We used XLM-RoBERTa as the baseline for all 3 languages  The best performing models for the English language were RoBERTa and BERT whereas GPT-2 was the worst performing 
Similarly the language specific versions of RoBERTa and BERT performed well for the Spanish and Portuguese respectively  Overall the worst performing model was GPT-2 across all 3 languages  The validation F1 scores are present in Table   The two best-performing models for every language were chosen for Track-2 
The same procedure as specified above was used and the F1 scores are present in Table   The train and validation F1 scores for 2-class classification are higher for all models as compared to the F1 score of the same models for 3-class classification  This was mainly due to the poor representation and accuracy of classification of the third class 
We observed symptoms of overfitting in all models after 12-15 epochs and the best validation F1 score was obtained in the range of 4-8 epochs 

LID experiments

The pipeline for dialect identification is divided into two parts as the sentences in the dataset belong to different languages  The stages are described in Section 4  The XLM-RoBERTa we have used for language classification has a test accuracy of 99 6% meaning it correctly classifies all input sentences and hence, can be considered as a perfect classifier 
For the final pipeline we experimented using the two best performing models for each language in Track-1 and Track-2  For both the tracks we experimented with all 8 (2 3 ) possible combinations of models and calculated the validation F1 score for the combined validation dataset which had sentences belonging to all languages 
The validation scores for Track-1 and Track-2 are shown in Table and Table respectively  For both the tracks, the three pipelines with the best validation F1 scores were chosen for submission 

Using 3-way classifier as a 2-way classifier

In Track-1, participants are expected to train a classifier which classifies amongst 9 classes, and in Track-2, participants are expected to train a classifier which classifies amongst 6 classes  These 6 classes are a proper subset of the 9 classes from Track-1  Thus, an intuitive baseline for Track-2 is to use the model finetuned for Track-1, whilst considering only the relevant classes for the latter task 
The classes EN , ES and P T , i e  the classes without any national dialect associated with them are not included in Track-2 as compared to Track-1  Thus, we calculate the predictions for the Track-2 validation dataset using the models for Track-1 and exclude the metrics for Track-1 specific classes to get the metrics for this "adapted" 2-way classification 
We show the results of this experiment in Table and observe that, as expected, the adapted 2-way classification performs worse compared to the explicitly finetuned variant 

Results for Track-1 and Track-2

We now present our experiments and their performance for both tracks  Our experiments for Track-1 are described in Table and our experiments for Track-2 are described in Table   The participants were allowed three submissions for evaluation on the test set, so we submitted predictions using the three systems which performed the best on the validation set 
As mentioned in Section 5 2, we performed 2 3 , i e  a total of 8 experiments using the two best models for each language  We observed that RoBERTa base on English, Spanish BERT base on Spanish and Portuguese BERT base performed the best on the testing set for Track-1  The same combination, with RoBERTa base for English, worked best for Track-2 
All of our submissions were the top submissions for each track, which surpassed the next best competitors by a margin of 4 5% and 5 6% for Track-1 and Track-2 respectively 

Ablation of best submissions

We hereby make some observations of our submissions and other experiments  To assist this, we plot the confusion matrices of our best submissions for Track-1 and Track-2 in Figures respectively  Note that these confusion matrices have their rows (i e  true labels axes) normalized according to the number of samples in the class 
Here are observations from our experiments: 1  BERT-based models outperform other models across all languages: We observe that BERT-based models outperform ELECTRA-based and GPT-2-based models, as shown in Table   We speculate this is because of the inherent architecture of BERT, which combines semantic learning with knowledge retention 
This combination of traits is particularly useful for this task  2  Common labels perform the worst across all languages: We observe that the common labels EN , ES and P T perform the worst, both in the individual as well as the two-stage setup  We hypothesize this is because of the absence of dialect specific words, or words that are specific to the geographical origin of the national dialect (for example, "Yankees" for EN-US and "Oxford" for EN-GB) 
3  English models work better than models of other languages: It can be noted from Figures 4 and 3 that the English models have the best performance across all classes  This can be attributed to two reasons: absence of national dialect specific words and lesser pretraining data in the case of Portuguese 
4  British English is most correctly classified class: We can observe that the Spanish or Portuguese models make equal number of mistakes in the case of either national dialect, in the case of Track-2 (see Figure )  However, in the case of English, the label EN − GB is correctly classified for more than 95% of the cases 
We speculate this is because British English involves slightly distinctive grammar and semantics, which help the model separate it from other classes  5  The proposed 2-step method is scalable for multiple language dialect classification: We can strongly assert that the novel 2-step deep learning method for multilingual dialect classification is a scalable method for the task due to two specific reasons: firstly, the multilingual models (like XLM-RoBERTa) might not have the vocabulary as well as the learning capabilities to learn the minute differences between individual dialects 
Secondly, this system can be quickly expanded for a new language by simply adding a language specific dialect classifier, provided the language identification model supports that particular language 

Conclusion

In this paper we propose a two-stage classification pipeline for dialect identification for multilingual corpora  We conduct thorough ablations on this setup and provide valuable insights  We foresee multiple future directions for this work  The first is to expand this work to many languages and dialects 
Secondly, it is a worthwhile research direction to distill this multi-model setup into a single model with multiple prediction heads  The obvious limitation of this system is the excessive memory consumption due to the usage of language specific models  For low resource languages this system is difficult to train and scale 
We hope that these problems will be addressed by researchers in future works 



ground_truth: They are rich in actin and have been shown to be highly dynamic.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What models were used for dialect identification?

Read the following text and answer briefly.

Context 4:  While the original TABUCOL algorithm includes a "tabu-list" mechanism to avoid cycling, it is not always sufficient, and requires some hyper-parameter tuning in order to obtain a good performance on a large variety of instances  To overcome this issue, we use a neighbourhood, but replace the "tabu-list" by the conflict optimizer scheme presented above 
PARTIALCOL inspired neighbourhood PARTIALCOL another local search algorithm solving the vertex coloring problem was introduced in 2008  This algorithm proposes a new local search scheme that allows partial coloring (thus allowing uncolored vertices)  The goal is to minimize the number of uncolored vertices 
Similarly to TABUCOL, PARTIALCOL starts with an initial solution, removes one color (unassigning its vertices), and performs local search iterations until no vertex is left uncolored  When coloring a vertex, the adjacent conflicting vertices are uncolored  Then, the algorithm repeats the process until all vertices are colored, or the stopping criterion is met 
This neighbourhood was also introduced alongside a tabu-search procedure  The tabu-search scheme is also replaced by a conflict-optimization scheme  Note that this neighbourhood was predominantly used by the other teams 

Finding Initial Solutions

Lasa team used two approaches to find initial solutions: 1  DSATUR is the classical graph coloring algorithm presented in Section 1  2  Orientation greedy is almost the only algorithm where the geometry of the segments is used  If segments are almost parallel, it is likely that they do not intersect (thus forming an independent set) 
This greedy algorithm first sorts the segments by orientation, ranging from − π 2 to π 2   For each segment in this order, the algorithm tries to color it using the first available color  If no color has been found, a new color is created for coloring the considered segment  This algorithm is efficient, produces interesting initial solutions and takes into account the specificities of the competition 

Solution Initialization

The gitastrophe team uses the traditional greedy algorithm of Welsh and Powell to obtain initial solutions: order the vertices in decreasing order of degree, and assign each vertex the minimum-label color not used by its neighbors  During the challenge Gitastrophe attempted to use different orderings for the greedy algorithm, such as sorting by the slope of the line segment associated with each vertex (as the orientation greedy initialization presented in Section 3), and also tried numerous other strategies 
Ultimately, after running the solution optimizer for approximately the same amount of time, all initializations resulted in an equal number of colors 

Modifications to the Conflict Optimizer

Taking inspiration from memetic algorithms, which alternate between an intensification and a diversification stage, the algorithm continually switched between a phase using the above conflict score, and one minimizing only the number of conflicts  Thus during the conflict-minimization phase, the random variables f (C j ) and w(u) are both fixed equal to 1 leading to a conflict score
Each phase lasted for 10 5 iterations  Adding the conflict-minimization phase gave minor improvements to some of the challenge instances 

Shadoks

In this section, we describe the choices used by the Shadoks team for the options described in Section 2 1  The Shadoks generally chose to eliminate the color with the smallest number of elements  However, if the multistart option is toggled on, then a random color is used each time  The conflict set S is stored in a queue 
The Shadoks tried other strategies, but found that the queue gives the best results  The weight function used is w(u) = 1 + q(u) p , mostly with p = 1 2  The effect of the parameter p is shown in Fig    Notice that in all figures, the number of colors shown is the average of ten executions of the code using different random seeds 
The algorithm uses σ = 0 15, easy vertices, q max = 59022, but does not use the BDFS nor any clique  If q(u) is larger than a threshold q max , the Shadoks set w(u) = ∞ so that the vertex u never reenters S  If at some point an uncolored vertex v is adjacent to some vertex u of infinite weight in every color class, then the conflict optimizer is restarted 
When restarting, the initial coloring is shuffled by moving some vertices from their initial color class to a new one  Looking at Fig  , the value of q max does not seem to have much influence as long as it is not too small  Throughout the challenge the Shadoks almost exclusively used q max = 2000 • (75000/m) 2 , where m is the number of vertices 
This value roughly ensures a restart every few hours  q max =0 5k q max =5k q max =50k q max =100k q max =250k The Shadoks use the function f as a Gaussian random variable of mean 1 and variance σ  A good default value is σ = 0 15  The effect of the variance is shown in Fig    Notice that setting σ = 0 gives much worse results 
Option (e) The goal of BDFS is to further optimize very good solutions that the conflict optimizer is not able to improve otherwise  Fig  shows the influence of BDFS  While on this figure, the advantages of BDFS cannot be noticed, its use near the end of the challenge improved about 30 solutions  The bounded depth-first search (BDFS) algorithm tries to improve the dequeuing process 
The goal is to prevent a vertex in conflict with some adjacent colored vertices from entering in the conflict set  At the first level, the algorithm searches for a recoloring of some adjacent vertices which allows us to directly recolor the conflict vertex  If no solution is found, the algorithm  In both figures the algorithm uses p = 1 2, easy vertices, q max = 59022, but does not use the BDFS nor any clique 
For σ ≥ 0 25, no solution better than 248 colors is found  could recolor some vertices at larger distances from the conflict vertex  To do so, a local search is performed by trying to recolor vertices at a bounded distance from the conflict vertex in the current partial solution  The BDFS algorithm has two parameters: adjacency bound a max and depth d 
In order to recolor a vertex v, BDFS gets the set C of color classes with at most a max neighbors of v  If a class in C has no neighbor of v, v is assigned to C  Otherwise, for each class C ∈ C, BDFS tries to recolor the vertices in C which are adjacent to v by recursively calling itself with depth d − 1 
At depth d = 0 the algorithm stops trying to color the vertices  During the challenge the Shadoks used BDFS with parameters a max = 3 and d = 3  The depth was increased to 5 (resp  7) when the number of vertices in the queue was 2 (resp  1)  Degeneracy order Given a target number of colors k, we call easy vertices a set of vertices Y such that, if the remainder of the vertices of G are colored using k colors, then we are guaranteed to be able to color all vertices of G with k colors 
This is obtained using the degeneracy order Y   To obtain Y we iteratively remove from the graph a vertex v that has at most k − 1 neighbors, appending v to the end of Y   We repeat until no other vertex can be added to Y   Notice that, once we color the remainder of the graph with at least k colors, we can use a greedy coloring for Y in order from last to first without increasing the number of colors used 
Removing the easy vertices reduces the total number of vertices, making the conflict optimizer more effective  The Shadoks always toggle this option on (the challenge instances contain from 0 to 23% easy vertices) 

Results

We provide the results of the experiments performed with the code from the three teams on two classes of instances  First, we present the results on some selected CG:SHOP 2022 instances  These instances are intersection graphs of line segments  Second, we execute the code on graphs that are not intersection graphs, namely the classic DIMACS graphs , comparing the results of our conflict optimizer implementations to previous solutions 
The source code for the three teams is available at: • Lasa: https://github com/librallu/dogs-color • Gitastrophe: https://github com/jacketsj/cgshop2022-gitastrophe • Shadoks: https://github com/gfonsecabr/shadoks-CGSHOP2022

CG:SHOP 2022 Instances

We selected 14 instances (out of 225) covering the different types of instances given in the CG:SHOP 2022 challenge  The results are presented in Table   For comparison, we executed the HEAD code on some instances using the default parameters  The table shows the smallest number of colors for which HEAD found a solution 
We ran HEAD for 1 hour of repetitions for each target number of colors on a single CPU core (the HEAD solver takes the target number of colors as a parameter and we increased this parameter one by one)  At the end of the challenge, 8 colorings computed by Lasa, 11 colorings computed by Gitastrophe, and 23 colorings computed by Shadoks over 225 instances have been proved optimal (their number of colors is equal to the size of a clique) 
In order to compare the efficiency of the algorithms, we executed the different implementations on the CG:SHOP instance vispecn13806  The edge density of this graph is 19%, the largest clique that we found has 177 vertices and the best coloring found during the challenge uses 218 colors  Notice that vispecn13806 is the same instance used in other Shadoks experiments in Section 5  Notice also that HEAD algorithm provides 283 colors after one hour compared to less than 240 colors for the conflict optimizers 
We ran the three implementations on three different servers and compared the results shown in Figure   For each implementation, the x coordinate is the running time in hours, while the y coordinate is the smallest number of colors found at that time 

Results on DIMACS Graphs

We tested the implementation of each team on the DIMACS instances to gauge the performance of the conflict optimizer on other classes of graphs  We compared our results to the best known bounds and to the state of the art coloring algorithms HEAD and QACOL   The time limit for Lasa's algorithms is 1 hour 
CWLS is Lasa's conflict optimizer with the neighbourhood presented in TABUCOL , while PWLS is the optimizer with the neighbourhood presented in PARTIALCOL   Gitastrophe algorithm ran 10 minutes after which the number of colors no longer decreases  Shadoks algorithm ran for 1 hour without the BDFS option (results with BDFS are worse) 
Results are presented in Table   We only kept the difficult DIMACS instances  For the other instances, all the results match the best known bounds  The DIMACS instances had comparatively few edges (on the order of thousands or millions); the largest intersection graphs considered in the CG:SHOP challenge had over 1 5 billion edges 
We notice that the conflict optimizer works extremely poorly on random graphs, but it is fast and appears to perform well on geometric graphs (r250 5, r1000 1c, r1000 5, dsjr500 1c and dsjr500 5), matching the best-known results   Interestingly, these geometric graphs are not intersection graphs as in the CG:SHOP challenge, but are generated based on a distance threshold 
On the DIMACS graphs, Lasa implementation shows better performance than the other implementations 



ground_truth: BERT, RoBERTa, ELECTRA, GPT-2, and XLM-RoBERTa.