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 is the score achieved by the authors for Track-2?

Read the following text and answer briefly.

Context 1: 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 
Context 2: 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



ground_truth: 85.61%.

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: Who is the county seat of McPherson County?

Read the following text and answer briefly.

Context 2: 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
Populated places established in 1867
Context 3: 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: McPherson.

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 3:  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.