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: Where is McPherson County located?

Read the following text and answer briefly.

Context 1: McPherson County (standard abbreviation: MP) is a county located in the U S  state of Kansas   As of the 2020 census, the county population was 30,223  The largest city and county seat is McPherson  The county is named for Civil War General James B  McPherson 

History

Early history

For many millennia, the Great Plains of North America was inhabited by nomadic Native Americans  From the 16th century to 18th century, the Kingdom of France claimed ownership of large parts of North America  In 1762, after the French and Indian War, France secretly ceded New France to Spain, per the Treaty of Fontainebleau  In 1802, Spain returned most of the land to France, but keeping title to about 7,500 square miles 

In 1803, most of the land for modern day Kansas was acquired by the United States from France as part of the 828,000 square mile Louisiana Purchase for 2 83 cents per acre  In 1848, after the Mexican–American War, the Treaty of Guadalupe Hidalgo with Spain brought into the United States all or part of land for ten future states, including southwest Kansas  In 1854, the Kansas Territory was organized, then in 1861 Kansas became the 34th U S  state 

19th century

From the 1820s to 1870s, the Santa Fe Trail passed through, what is now McPherson County  The trail entered the county, east of Canton, then south of Galva, then north of Inman, and west towards Lyons  In 1855, Charles O  Fuller established a ranch adjacent to the Running Turkey Creek Crossing about two miles south and one mile east of Galva  Fuller's Ranch provided accommodations for travelers on the Santa Fe Trail and was probably the first white settlement in McPherson County 

Peketon County was established in 1860, by the passage of a bill by S  N  Wood:  An act to establish Peketon County  Section 1  - That all that territory west of the sixth principal meridian and south of Township 16, in Kansas Territory, be and the same is hereby erected into a county, to be known by the name of Peketon County  On February 17, 1865, Peketon County was abolished, and McPherson County was made a part of Marion County, which extended from the west line of Chase County to the present western boundary of Kansas 

In 1868, Solomon Stephens and L  N  Holmberg were appointed Justices of the Peace—the first officers in what is now McPherson County  The next year (1869) occurred the first election for the township, now the county of McPherson  McPherson was regularly organized as a county in the spring of 1870, a mass meeting being held at Sweadal  Sweadal, the county seat thus selected, was located about one mile and a half southwest of the present site of Lindsborg  In September, however, the County Commissioners resolved to meet at the latter place, McPherson which had already been located some two years 

In April, 1873, a petition was filed for the county seat re-location  It was signed by 483 voters, and a special election was accordingly ordered for June 10  Upon that day, McPherson received 605 votes, New Gottland 325, King City 3 and Lindsborg 1; McPherson's majority over all, 276  In May the McPherson Town Company had offered, as an inducement for the location of the county seat at this point, the free use of rooms for ten years, and the donation of two squares of land on the town site  The offer was accepted the next month, the County Commissioners selecting blocks 56 and 65  Thus the county seat was established at McPherson and has remained since 

As early as 1875, city leaders of Marion held a meeting to consider a branch railroad from Florence  In 1878, Atchison, Topeka and Santa Fe Railway and parties from Marion County and McPherson County chartered the Marion and McPherson Railway Company   In 1879, a branch line was built from Florence to McPherson, in 1880 it was extended to Lyons, in 1881 it was extended to Ellinwood  The line was leased and operated by the Atchison, Topeka and Santa Fe Railway  The line from Florence to Marion, was abandoned in 1968  In 1992, the line from Marion to McPherson was sold to Central Kansas Railway  In 1993, after heavy flood damage, the line from Marion to McPherson was abandoned  The original branch line connected Florence, Marion, Canada, Hillsboro, Lehigh, Canton, Galva, McPherson, Conway, Windom, Little River, Mitchell, Lyons, Chase, then connected with the original AT&SF main line at Ellinwood 

In 1887, the Chicago, Kansas and Nebraska Railway extended its main line from Herington to Pratt   This main line connected Herington, Ramona, Tampa, Durham, Waldeck, Canton, Galva, McPherson, Groveland, Inman, Medora, Hutchinson, Whiteside, Partridge, Arlington,  Langdon, Turon, Preston, Natrona, Pratt   In 1888, this main line was extended to Liberal   Later, this line was extended to Tucumcari, New Mexico and Santa Rosa, New Mexico, where it made a connection with the Southern Pacific from El Paso, Texas   The Chicago, Kansas and Nebraska Railway was absorbed by the Chicago, Rock Island and Pacific Railway  This line is also called the "Golden State Route" 

20th century
The National Old Trails Road, also known as the Ocean-to-Ocean Highway, was established in 1912, and was routed through Windom, Conway, McPherson 

Geography

According to the U S  Census Bureau, the county has a total area of , of which  is land and  (0 3%) is water 

Adjacent counties
 Saline County (north)
 Dickinson County (northeast)
 Marion County (east)
 Harvey County (southeast)
 Reno County (southwest)
 Rice County (west)
 Ellsworth County (northwest)

Major highways
  Interstate 135
  U S  Route 56
  U S  Route 81
  K-4
  K-61
  K-153

Demographics

The McPherson Micropolitan Statistical Area includes all of McPherson County 

2000 census
As of the census of 2000, there were 29,554 people, 11,205 households, and 7,966 families residing in the county   The population density was 33 people per square mile (13/km2)   There were 11,830 housing units at an average density of 13 per square mile (5/km2)   The racial makeup of the county was 96 53% White, 0 81% Black or African American, 0 34% Native American, 0 32% Asian, 0 06% Pacific Islander, 0 79% from other races, and 1 16% from two or more races   1 94% of the population were Hispanic or Latino of any race  37 1% were of German, 12 9% Swedish, 12 1% American, 6 7% English and 6 3% Irish ancestry according to Census 2000 

There were 11,205 households, out of which 33 00% had children under the age of 18 living with them, 62 50% were married couples living together, 6 00% had a female householder with no husband present, and 28 90% were non-families  25 50% of all households were made up of individuals, and 11 80% had someone living alone who was 65 years of age or older   The average household size was 2 49 and the average family size was 2 99 

In the county, the population was spread out, with 25 40% under the age of 18, 10 30% from 18 to 24, 25 20% from 25 to 44, 21 80% from 45 to 64, and 17 30% who were 65 years of age or older   The median age was 38 years  For every 100 females there were 95 90 males   For every 100 females age 18 and over, there were 92 90 males 

The median income for a household in the county was $41,138, and the median income for a family was $48,243  Males had a median income of $33,530 versus $21,175 for females  The per capita income for the county was $18,921   About 4 20% of families and 6 60% of the population were below the poverty line, including 5 20% of those under age 18 and 8 10% of those age 65 or over 

Government

Presidential elections
McPherson county is often carried by Republican candidates  The last time a Democratic candidate has carried this county was in 1964 by Lyndon B  Johnson 

Laws
Following amendment to the Kansas Constitution in 1986, the county remained a prohibition, or "dry", county until 1996, when voters approved the sale of alcoholic liquor by the individual drink with a 30 percent food sales requirement 

Education

Colleges
 McPherson College in McPherson
 Bethany College in Lindsborg
 Central Christian College in McPherson

Unified school districts
 Smoky Valley USD 400
 McPherson USD 418
 Canton-Galva USD 419
 Moundridge USD 423
 Inman USD 448

School district office in neighboring county
 Goessel USD 411
 Little River-Windom USD 444

Museums
 Birger Sandzén Memorial Gallery in Lindsborg
 McCormick-Deering Days Museum in Inman
 McPherson Museum in McPherson
 Lindsborg Old Mill & Swedish Heritage Museum in Lindsborg
 Kansas Motorcycle Museum in Marquette

Communities

Cities

 Canton
 Galva
 Inman
 Lindsborg
 Marquette
 McPherson (county seat) 
 Moundridge
 Windom

Unincorporated communities
† means a Census-Designated Place (CDP) by the United States Census Bureau 
 Conway
 Elyria†
 Groveland
 Johnstown
 New Gottland
 Roxbury†

Ghost towns
 Alta Mills
 Battle Hill
 Christian
 Doles Park
 Elivon
 King City
 Sweadal

Townships
McPherson County is divided into twenty-five townships   The cities of Lindsborg and McPherson are considered governmentally independent and are excluded from the census figures for the townships   In the following table, the population center is the largest city (or cities) included in that township's population total, if it is of a significant size 

See also
 List of people from McPherson County, Kansas
 National Register of Historic Places listings in McPherson County, Kansas
 McPherson Valley Wetlands
 Maxwell Wildlife Refuge

References

Notes

Further reading

 Wheeler, Wayne Leland  "An Analysis of Social Change in a Swedish-Immigrant Community: The Case of Lindsborg, Kansas " (PhD dissertation, University of Missouri-Columbia; ProQuest Dissertations Publishing, 1959  5905657) 

County
 Through the Years: A Pictorial History of McPherson County; McPherson Sentinel' Heritage House Publishing Co; 1992 
 McPherson County First Courthouse Built About 1869 or 1870; Lindsborg News-Record; March 30, 1959 
 Pioneer Life and Lore of McPherson County, Kansas; Edna Nyquist; Democratic-Opinion Press; 1932 
 A History of the Church of the Brethren in Kansas (includes McPherson College history); Elmer LeRoy Craik; McPherson Daily; Republican Press; 397 pages; 1922 
 Portrait and Biographical Record of Dickinson, Saline, McPherson, and Marion Counties, Kansas; Chapman Bros; 614 pages; 1893 
 Standard Atlas of McPherson County, Kansas; Geo  A  Ogle & Co; 82 pages; 1921 
 Plat Book of McPherson County, Kansas; North West Publishing Co; 50 pages; 1903 
 Edwards' Atlas of McPherson County, Kansas; John P  Edwards; 51 pages; 1884 

Trails
 The Story of the Marking of the Santa Fe Trail by the Daughters of the American Revolution in Kansas and the State of Kansas; Almira Cordry; Crane Co; 164 pages; 1915  (Download 4MB PDF eBook)
 The National Old Trails Road To Southern California, Part 1 (LA to KC); Automobile Club Of Southern California; 64 pages; 1916  (Download 6 8MB PDF eBook)

Mennonite Settlements
 Impact of Mennonite settlement on the cultural landscape of Kansas; Brenda Martin; Kansas State University; 1985/1988  
 Mennonite settlement : the relationship between the physical and cultural environment; Susan Movle; University of Utah; 1975/1886 
 Status of Mennonite women in Kansas in their church and home relationships; Eva Harshbarger; Bluffton College; 1925/1945 

External links

County
 
 McPherson County - Directory of Public Officials
Historical
 , from Hatteberg's People'' on KAKE TV news
Maps
 McPherson County Maps: Current, Historic, KDOT
 Kansas Highway Maps: Current, Historic, KDOT
 Kansas Railroad Maps: Current, 1996, 1915, KDOT and Kansas Historical Society

 
Kansas counties
1867 establishments in Kansas

Context 3: Sir Simon William English  (born 30 December 1961) is a New Zealand former National Party politician who served as the 39th prime minister of New Zealand from 2016 to 2017  He had previously served as the 17th deputy prime minister of New Zealand and minister of finance from 2008 to 2016 under John Key and the Fifth National Government 

A farmer and public servant before entering politics, English was elected to the New Zealand Parliament in  as the National Party's candidate in the Wallace electorate  He was elevated to Cabinet in 1996 and in 1999 was made minister of finance, although he served for less than a year due to his party's loss at the 1999 general election  In October 2001, English replaced Jenny Shipley as the leader of the National Party (and consequently as Leader of the Opposition)  He led the party to its worst defeat at the 2002 general election, and as a consequence, in October 2003 he was replaced as leader by Don Brash 

In November 2006, after Brash's resignation, English became deputy leader under John Key  After National's victory at the 2008 general election, he became deputy prime minister and was also made minister of finance for the second time  Under English's direction New Zealand's economy maintained steady growth during National's three terms of government  He became a list-only MP after stepping down as an electorate MP at the 2014 general election 

John Key resigned as leader of the National Party and prime minister in December 2016  English won the resulting leadership election unopposed and was sworn in as prime minister on 12 December 2016  His tenure was only ten months, and included a three-month election campaign  In the 2017 general election, National won the largest number of seats but fell short of a majority  The parties holding the balance of power declined to support the existing government, and English was subsequently replaced as prime minister by Jacinda Ardern, leader of the Labour Party  English initially continued on as Leader of the Opposition, but resigned as leader of the National Party on 27 February 2018 and left parliament two weeks later 

Early life
English was born on 30 December 1961 at Lumsden Maternity Centre in Lumsden  He is the eleventh of twelve children of Mervyn English and Norah (née O'Brien) English  His parents purchased Rosedale, a mixed sheep and cropping farm in Dipton, Southland from Mervyn's uncle, Vincent English, a bachelor, in 1944  English was born in the maternity unit at Lumsden 

English attended St Thomas's School in Winton, then boarded at St  Patrick's College in Upper Hutt, where he became head boy  He played in the first XV of the school's rugby team  English went on to study commerce at the University of Otago, where he was a resident at Selwyn College, and then completed an honours degree in English literature at Victoria University of Wellington 

After finishing his studies, English returned to Dipton and farmed for a few years  From 1987 to 1989, he worked in Wellington as a policy analyst for the New Zealand Treasury, at a time when the free market policies favoured by Labour's finance minister Roger Douglas (known collectively as "Rogernomics") were being implemented 

English joined the National Party in 1980, while at Victoria University  He served for a period as chairman of the Southland branch of the Young Nationals, and became a member of the Wallace electorate committee  After moving to Wellington, he served for periods on the Island Bay and Miramar electorate committees, respectively 

Fourth National Government (1990–1999)

At the 1990 general election, English stood as the National candidate in Wallace, replacing the retiring Derek Angus, and was elected with a large majority  He would hold this seat, renamed Clutha-Southland in 1996, until 2014  He and three other newly elected National MPs (Tony Ryall, Nick Smith, and Roger Sowry) were soon identified as rising stars in New Zealand politics, and at various points were dubbed the "brat pack", the "gang of four", and the "young Turks"  In his first term in parliament, English chaired a select committee into social services  He was made a parliamentary under-secretary in 1993, serving under the Minister of Health 

First period in cabinet (1996–1999)
In early 1996, English was elevated to cabinet by Prime Minister Jim Bolger, becoming the Minister for Crown Health Enterprises and Associate Minister of Education (to Wyatt Creech)  He was 34 at the time, becoming the cabinet's youngest member  After the 1996 general election, the National Party was forced into a coalition with New Zealand First to retain government  In the resulting cabinet reshuffle, English emerged as Minister of Health  However, as a condition of the coalition agreement, NZ First's Neil Kirton (a first-term MP) was made Associate Minister of Health, effectively becoming English's deputy  This arrangement was described in the press as a "shotgun marriage", and there were frequent differences of opinion between the two ministers  After their relationship became unworkable, Kirton was sacked from the role in August 1997, with the agreement of NZ First leader Winston Peters 

As Minister of Health, English was responsible for continuing the reforms to the public health system that National had begun after the 1990 general election  The reforms were unpopular, and health was perceived as one of the government's weaknesses, with the health portfolio consequently being viewed as a challenge  English believed that the unpopularity of the reforms was in part due to a failure in messaging, and encouraged his National colleagues to avoid bureaucratic and money-focused language (such as references to "balance sheets" and "user charges") and instead talk about the improvements to services the government's reforms would bring  He also rejected the idea that public hospitals could be run as commercial enterprises, a view which some of his colleagues had previously promoted 

By early 1997, as dissatisfaction with Bolger's leadership began to grow, English was being touted as a potential successor, along with Jenny Shipley and Doug Graham  His age (35) was viewed as the main impediment to a successful leadership run  National's leadership troubles were resolved in December 1997, when Bolger resigned and Shipley was elected to the leadership unopposed  English had been a supporter of Bolger as leader, but Shipley reappointed him Minister of Health in her new cabinet 

English was promoted to Minister of Finance in a reshuffle in January 1999, a position which was at the time subordinate to the Treasurer, Bill Birch  After a few months, the pair switched positions as part of Birch's transition to retirement, with English assuming the senior portfolio  In early interviews, he emphasised his wish to be seen as a pragmatist rather than an ideologue, and said that the initiatives of some of his predecessors (Roger Douglas's "Rogernomics" and Ruth Richardson's "Ruthanasia") had focused on "fruitless, theoretical debates" when "people just want to see problems solved" 

Opposition (1999–2008)

After the National Party lost the 1999 election to Helen Clark's Labour Party, English continued on in the shadow cabinet as National's spokesperson for finance  He was elected deputy leader of the party in February 2001, following the resignation of Wyatt Creech, with Gerry Brownlee being his unsuccessful opponent 

Leader of the Opposition
In October 2001, after months of speculation, Jenny Shipley resigned as leader of the National Party after being told she no longer had the support of the party caucus  English was elected as her replacement unopposed (with Roger Sowry as his deputy), and consequently became Leader of the Opposition  However, he did not openly organise against Shipley, and according to The Southland Times "there was almost an element of 'aw, shucks, I'll do it then' about Mr English's ascension" 

Aged 39 when he was elected, English became the second-youngest leader in the National Party's history, after Jim McLay (who was 38 when elected in 1984)  He also became only the third Southlander to lead a major New Zealand political party, after Joseph Ward and Adam Hamilton  However, English failed to improve the party's performance  In the 2002 election, National suffered its worst electoral defeat ever, gaining barely more than twenty percent of the vote  English described it as "the worst day of my political life"  Both party insiders and the general public were split as to how much to blame him for the loss, but most of the party believed that English would be able to rebuild National's support 

By late 2003, however, National's performance in opinion polls remained poor  The party had briefly increased its popularity in the year following the election, but by October its support had fallen to levels only slightly better than what it achieved in the last ballot  English also appeared in a boxing match for a charity against entertainer Ted Clarke  This did not boost his polling or that of the National party either, with suggestions that it devalued his image as a serious politician  Don Brash, former governor of the Reserve Bank and a relative newcomer to politics, began to build up support to replace English  On 28 October, Brash gained sufficient backing in Caucus to defeat English in a leadership contest 

Shadow cabinet roles and deputy leader
On 2 November 2003, when Brash changed responsibilities for certain MPs, English became National's spokesman for education, ranked at fifth place in the party's parliamentary hierarchy  He remained in parliament after the 2005 election  In his new shadow education portfolio, English performed strongly, and remained a party favourite despite his election defeat as leader in 2002, eventually being returned to the finance portfolio in August 2004 as deputy spokesman (while still retaining responsibility for education) 

In November 2006, Brash resigned as leader  English was considered as a potential replacement leader (running against John Key) or deputy leader (against incumbent Gerry Brownlee) in the ensuing leadership election  However, a contest was avoided when the MPs agreed a Key/English ticket would run unopposed in a display of party unity  English took over the deputy leadership and the finance portfolio in the Key shadow cabinet 

Fifth National Government (2008–2017)

Deputy Prime Minister and Minister of Finance (2008–2016)

At the 2008 election, English was re-elected by his electorate, winning by a margin of about 15,500 votes  He became Deputy Prime Minister of New Zealand and Minister of Finance in the fifth National Government, being sworn into office on 19 November 2008 and continued to serve in those roles until becoming Prime Minister on 12 December 2014  He was also made Minister of Infrastructure in National's first term of government and Minister responsible for Housing New Zealand Corporation and minister responsible for the New Zealand flag consideration process in its third 

He was comfortably re-elected in Clutha-Southland in the 2011 election but opted to run as a party-list candidate in 2014  

The pairing of John Key as leader of the National Party and English as his deputy has been compared to that of Bob Hawke and Paul Keating (in Australia) and Tony Blair and Gordon Brown (in the UK)



ground_truth: McPherson County is located in the U.S. state of Kansas.

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: 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  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 



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: In which electorate was Simon English elected to the New Zealand Parliament?

Read the following text and answer briefly.

Context 3: 

English acceded to the role of Finance Minister in the continuing wake of the financial crisis  In response to New Zealand's rising debt, English made budget deficit-reduction his main priority  His first budget outlined three focuses in New Zealand's financial recovery: "improving the business environment and removing roadblocks to growth; investment in productive infrastructure; and improving the way government works"  One of his first acts was creating the National Infrastructure Unit, charged with formulating a plan for infrastructure projects and investments  He commissioned a government-wide spending review, with an aim to reducing government expenditure—with the exceptions of a two-year stimulus package and long-term increases on infrastructure spending 

In April 2011, the Opposition criticised English for suggesting that New Zealand businesses could use New Zealand's low wages to help it compete with Australia  The National Government campaigned for re-election in 2011 on its economic record  The Government boasted growth for five consecutive quarters up to mid-2010, totalling 1 6% of real GDP 

Strong growth resulted in a surplus of $473 million for the 2015/16 financial year, projected to rise to $8 5 billion by 2020/21  In his 2016 Economic and Fiscal Update address, English stated that reducing debt and tackling the costs of the 2016 Kaikōura earthquake were higher priorities than reducing rates of tax 

Allowances issue
In 2009, the media, including TVNZ and TV3 revealed that English was receiving about NZ$900 a week as part of a living allowance for ministers, to live in his own NZ$1 2 million Wellington home  At the time, English also received $276,200 in his annual salary as Deputy Prime Minister  It was also revealed other ministers with homes in the capital city were also claiming accommodation allowances  On 3 August 2009, Prime Minister John Key started a review of the housing allowances claimed by cabinet ministers  English subsequently paid back $12,000 and only claimed about $24,000 a year in living allowances  The Auditor-General's office said in September 2009 that they were making "preliminary enquiries" into parliamentary housing expenses in response to a letter of complaint from Progressive party leader Jim Anderton  Two days later English stated that he would no longer take up any housing allowance and had paid back all the allowance he had received since the November 2008 election 

Prime Minister (2016–2017)

John Key resigned on 12 December, and endorsed English as his successor in the resulting leadership election  Following the drop-out of both Judith Collins and Jonathan Coleman from the leadership election, English was sworn in as the 39th Prime Minister of New Zealand on 12 December 2016 

English appointed his first cabinet on 18 December  In a reshuffle, he appointed Steven Joyce to succeed him as Finance Minister, while most ministerial portfolios remained the same 

In February 2017, English did not attend Waitangi Day commemorations at the historic treaty grounds, reportedly in response to the Ngāpuhi iwi's decision to stop the Prime Minister from speaking at the marae  Ngāpuhi have protested the Government's negotiation of the Trans Pacific Partnership Agreement (TPPA), which the iwi believe infringes upon Māori sovereignty, and thus does not adhere to the Treaty of Waitangi  English had been invited to attend in an official capacity; his non-attendance was criticised by a Ngāpuhi elder and Opposition leader Andrew Little 

In his first overseas trip as Prime Minister, English travelled to Europe to discuss trade ties, including a prospective New Zealand–European Union free trade agreement  He first travelled to London on 13 January 2017 to meet British Prime Minister Theresa May  Discussing trade relations, English said the two nations were "natural partners" and would "continue to forge ties" after the UK's withdrawal from the EU  He also arranged to meet with London Mayor Sadiq Khan, Belgian Prime Minister Charles Michel and German Chancellor Angela Merkel   In a meeting with Merkel, English received crucial backing from Germany for a trade deal with the EU  On 16 January, English stated that his government would continue to promote TPPA, despite the United States' decision to withdraw from the agreement  He explained that Southeast Asian countries would now be treated as a priority in negotiations—he also asserted that the United States was ceding influence to China by its rejection of the trade pact 

At a press conference at the Beehive on 1 February 2017, English announced that the 2017 general election would be held on 23 September  The Prime Minister later confirmed that his party would approach ACT, United Future and the Māori Party if confidence and supply agreements were required to form a government following the election  In his second cabinet reshuffle on 24 April, English appointed Gerry Brownlee as his new Foreign Affairs Minister; he also promoted Nikki Kaye to the portfolio of Education Minister, and moved Mark Mitchell into the cabinet to become Defence Minister  The reshuffle was perceived as an election preparation 

On 13 February 2017, English welcomed Australian Prime Minister Malcolm Turnbull to Wellington  The two leaders reaffirmed their shared trade agenda, and discussed changes to the Australian citizenship pathway which will affect permanent residents originating from New Zealand 

On 19 June, it was reported that Todd Barclay, who succeeded English as MP for Clutha-Southland, had clandestinely recorded one of his employee's conversations the previous year, and that John Key's leaders' budget was used to pay a confidential settlement after the employee resigned  English admitted that he had been aware of the illegal recording and the settlement, and thus implicated in the scandal 

During the 2017 National campaign launch, English introduced a $379 million social investment package including digital learning academies for high school students, more resources for mathematics, and boosting support for teaching second languages in schools, and maintaining National Standards in the school curriculum  Prime Minister English also sought to defend National's financial management and economic track record and claimed that the opposition Labour Party would raise taxes  Early opinion polling had forecast a poor showing in the election for the Labour Party, but in early August 37-year-old Jacinda Ardern took over as Labour leader and seemingly energised younger voters 

At the 2017 general election, National won the largest share of the party vote (44 4%) and the largest number of seats (56) in the House Representatives  However, National lacked enough seats to govern alone due to two of the party's support partners, the Māori Party and United Future, losing their parliamentary seats  In response, English stated that the party would be entering into talks to form a coalition with New Zealand First  Following talks with the two largest parties, New Zealand First entered a coalition arrangement with the Labour Party  English was succeeded as prime minister by Jacinda Ardern on 26 October 

Opposition (2017–2018)

Leader of the Opposition
English was re-elected as National Party leader on 24 October 2017  At the time of his re-election, English announced his intention to stay on as leader until the next general election  On 13 February 2018, however, he stood down as National Party leader due to personal reasons, and instructed the party to put into motion the processes to elect a new leader  He also retired from Parliament  English's resignation followed weeks of speculation that he would step aside for a new leader  On 27 February, he was succeeded as party leader by Simon Bridges as the result of the leadership election held that day 

Post-premiership 
In 2018, English joined the board of Australian conglomerate, Wesfarmers  English serves in Chairmanships of Mount Cook Alpine Salmon, Impact Lab Ltd and Manawanui Support Ltd  He is also a director of The Instillery, Centre for Independent Studies and The Todd Corporation Limited, and is a member of the Impact Advisory Group of Macquarie Infrastructure and Real Assets 

Political and social views

English is regarded as more socially conservative than his predecessor, John Key  He has stated his opposition to voluntary euthanasia and physician-assisted suicide, same-sex civil unions, and the decriminalisation of prostitution  As Prime Minister he opposed any "liberalisation" of abortion law 

In 2004, English voted against a bill to establish civil unions for both same-sex and opposite-sex couples  In 2005, he voted for the Marriage (Gender Clarification) Amendment Bill, which would have amended the Marriage Act to define marriage as only between a man and a woman  English voted against the Marriage (Definition of Marriage) Amendment Bill, a bill that legalised same-sex marriage in New Zealand  However, in December 2016 he stated, "I'd probably vote differently now on the gay marriage issue  I don't think that gay marriage is a threat to anyone else's marriage" 

In 2009, English voted against the Misuse of Drugs (Medicinal Cannabis) Amendment Bill, a bill aimed at amending the Misuse of Drugs Act so that cannabis could be used for medical purposes 

Personal life 
English met his future wife, Mary Scanlon, at university  She was studying medicine at the time, and became a general practitioner  Both her parents were immigrants, her father being Samoan and her mother Italian, born on the island of Stromboli  They have six children: a daughter and five sons 

English is a practising Roman Catholic, but has stated that he considers his religious beliefs personal and thus separate from politics 

In June 2002, English took part in TV3's Fight For Life, a celebrity boxing fundraiser to raise money for the Yellow Ribbon anti-youth-suicide campaign, influenced by the death of a teenage nephew in 1997  He lost a split decision to former university colleague Ted Clarke 

Honours
In the 2018 Queen's Birthday Honours, English was appointed a Knight Companion of the New Zealand Order of Merit, for services of over 27 years to the State 

See also

List of New Zealand governments
Politics of New Zealand

References

External links

Profile at National Party 
Profile on Parliament nz
Releases and speeches at Beehive govt nz

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1961 births
21st-century New Zealand politicians
Candidates in the 2017 New Zealand general election
Deputy Prime Ministers of New Zealand
Leaders of the Opposition (New Zealand)
Living people
Members of the Cabinet of New Zealand
Members of the New Zealand House of Representatives
New Zealand farmers
New Zealand finance ministers
New Zealand list MPs
New Zealand MPs for South Island electorates
New Zealand National Party MPs
New Zealand National Party leaders
New Zealand Roman Catholics
New Zealand people of Irish descent
People educated at St  Patrick's College, Silverstream
People from Dipton, New Zealand
People from Lumsden, New Zealand
Prime Ministers of New Zealand
University of Otago alumni
Victoria University of Wellington alumni
Knights Companion of the New Zealand Order of Merit
New Zealand politicians awarded knighthoods



ground_truth: The Wallace electorate.