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Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn

Featured protocol,  Authors: De-Li Cao
De-Li CaoAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3992
Peng-Bo Jing
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
Bao-Chun Jiang
Bao-Chun JiangAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3994
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
date: 1/5/2017, 88 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2098.

Brief version appeared in J Clin Invest, Feb 2016
Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular disease models, such as pain and itch. Because of the specific features of SDHN, it is necessary to establish a reliable culture method that is suitable for testing neural response to various external stimuli in vitro.

Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations

Featured protocol,  Authors: Susana Mingote
Susana MingoteAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
Bio-protocol author page: a3960
Nao Chuhma
Nao ChuhmaAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
Bio-protocol author page: a3961
 and Stephen Rayport
Stephen RayportAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
For correspondence: stephen.rayport@columbia.edu
Bio-protocol author page: a3962
date: 1/5/2017, 99 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2090.

Brief version appeared in J Neurosci, Dec 2015
Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DATIREScre mice. The efficacy and specificity of ChR2 expression in dopamine neurons is verified by immunofluorescence for the dopamine-synthetic enzyme tyrosine hydroxylase. Then, slice patch-clamp recordings are made from neurons in regions recipient to dopamine neuron projections and the incidence and strength of excitatory connections determined. The summary of the incidence and strength of connections in all regions recipient to dopamine neuron projections constitute the functional connectome.

Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn

Authors: De-Li Cao
De-Li CaoAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3992
Peng-Bo Jing
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
Bao-Chun Jiang
Bao-Chun JiangAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3994
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
date: 1/5/2017, 88 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2098.

[Abstract] Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular ...

Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations

Authors: Susana Mingote
Susana MingoteAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
Bio-protocol author page: a3960
Nao Chuhma
Nao ChuhmaAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
Bio-protocol author page: a3961
 and Stephen Rayport
Stephen RayportAffiliation 1: Department of Psychiatry, Columbia University, New York, USA
Affiliation 2: Department of Molecular Therapeutics, NYS Psychiatric Institute, New York, USA
For correspondence: stephen.rayport@columbia.edu
Bio-protocol author page: a3962
date: 1/5/2017, 99 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2090.

[Abstract] Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional ...

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 203 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2046.

[Abstract] Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex ...

Delayed Spatial Win-shift Test on Radial Arm Maze

Authors: Simone N. De Luca
Simone N. De LucaAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3841
Luba Sominsky
Luba Sominsky Affiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3842
 and Sarah J. Spencer
Sarah J. SpencerAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
For correspondence: Sarah.Spencer@rmit.edu.au
Bio-protocol author page: a3843
date: 12/5/2016, 223 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2053.

[Abstract] The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way to ...

Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish

Authors: Subhra Prakash Hui
Subhra Prakash HuiAffiliation 1: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
Affiliation 2: Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, Australia
Bio-protocol author page: a3817
 and Sukla Ghosh
Sukla GhoshAffiliation: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
For correspondence: suklagh2010@gmail.com
Bio-protocol author page: a3818
date: 12/5/2016, 236 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2043.

[Abstract] Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and various ...

Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice

Authors: Zhi-Jun Zhang*
Zhi-Jun ZhangAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Department of Human Anatomy, Nantong University, Jiangsu, China
Bio-protocol author page: a3741
Peng-Bo Jing*
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
 (*contributed equally to this work) date: 11/20/2016, 282 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2020.

[Abstract] In order to explore the role of a specific gene/protein in the specific segment of spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, ...

An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties

Authors: Hyeok Jun Yun
Hyeok Jun YunAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Bio-protocol author page: a3721
 and Byung S. Kim
Byung S. KimAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Affiliation 3: Department of Neurology, Ajou University School of Medicine, Suwon, Korea
For correspondence: kimbg@ajou.ac.kr
Bio-protocol author page: a1956
date: 11/20/2016, 290 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2012.

[Abstract] Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned ...

In vitro Brainstem-spinal Cord Preparation from Newborn Rat

Authors: Jean-Patrick Le Gal
Jean-Patrick Le GalAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3699
Angelo Nicolosi
Angelo NicolosiAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3700
Laurent Juvin
Laurent JuvinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3701
 and Didier Morin
Didier MorinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
For correspondence: didier.morin@u-bordeaux.fr
Bio-protocol author page: a3702
date: 11/20/2016, 243 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2003.

[Abstract] The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). ...

Apparatus and General Methods for Exposing Rats to Audiogenic Stress

Author: Serge Campeau
Serge CampeauAffiliation: Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado, USA
For correspondence: Serge.Campeau@colorado.edu
Bio-protocol author page: a3678
date: 11/5/2016, 245 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1994.

[Abstract] Most organisms react innately to the sudden onset of environmental stimulation. Audiogenic or loud noise in rodents provides an effective threatening signal to study the central nervous circuits responsible for the elaboration of various responses typically elicited by threatening/stressful environmental stimulation. Audiogenic stress offers many advantages ...

Protocol for Primary Microglial Culture Preparation

Authors: Hong Lian
Hong LianAffiliation: Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, China
For correspondence: honglian@zju.edu.cn
Bio-protocol author page: a3663
Ethan Roy
Ethan RoyAffiliation: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA
Bio-protocol author page: a3664
 and Hui Zheng
Hui ZhengAffiliation: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA
Bio-protocol author page: a3665
date: 11/5/2016, 396 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1989.

[Abstract] Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. ...
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Stereotaxic Injection of LPS into Mouse Substantia Nigra

Author: Huiming Gao
Huiming GaoAffiliation: National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
date: 4/20/2012, 10721 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.153.

[Abstract] Stereotaxic injection is an attractive approach for studying genetic, cellular and circuit functions in the brain. Injection of anatomical tracers, site-targeted lesions and gene delivery by recombinant adeno-associated viruses and lentiviruses in mice are powerful tools to study nervous system development ...

Immunofluorescence Staining on Mouse Embryonic Brain Sections

Author: Xuecai Ge
Xuecai GeAffiliation 1: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, USA
Affiliation 2: , Howard Hughes Medical Institute, Cambridge, USA
For correspondence: xuecaige@stanford.edu
Bio-protocol author page: a46
date: 6/5/2012, 10704 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.192.

[Abstract] This protocol comprises the entire process of immunofluorescence staining on mouse embryonic brains, starting from tissue preparation to mounting of the tissue sections....

c-Fos and Arc Immunohistochemistry on Rat Cerebellum

Author: Soyun Kim
Soyun KimAffiliation: Neuroscience Program, University of Southern California, Los Angeles, USA
For correspondence: soyunkimucsd@gmail.com
Bio-protocol author page: a45
date: 5/20/2012, 10196 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.191.

[Abstract] This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural ...

[Bio101] Microglia Cultures and Mixed Glial Culture

Author: Huiming Gao
Huiming GaoAffiliation: Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
date: 11/5/2011, 10067 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.149.

[Abstract] Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during ...

Mouse Cochlear Whole Mount Immunofluorescence

Authors: Omar Akil
Omar AkilAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
For correspondence: oakil@ohns.ucsf.edu
Bio-protocol author page: a238
 and Lawrence R. Lustig
Lawrence R. LustigAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
Bio-protocol author page: a239
date: 3/5/2013, 9944 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.332.

[Abstract] This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest ...

In utero Electroporation of Mouse Embryo Brains

Author: Xuecai Ge
Xuecai GeAffiliation 1: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, USA
Affiliation 2: , Howard Hughes Medical Institute, Cambridge, USA
For correspondence: xuecaige@stanford.edu
Bio-protocol author page: a46
date: 7/20/2012, 9449 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.231.

[Abstract] This is a non-invasive technique to introduce transgenes into developing brains. In this technique, DNA is injected into the lateral ventricle of the embryonic brains, and is incorporated into the cells through electroporation. Embryos then continue their development in normal conditions in vivo. The ...

A Protocol for Electrophoretic Mobility Shift Assay (EMSA) from Primary Neuron

Author: Jiali Li
Jiali LiAffiliation: Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, Piscataway, NJ, USA
For correspondence: jli@dls.rutgers.edu
Bio-protocol author page: a179
date: 12/5/2012, 9279 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.300.

[Abstract] The interaction of transcriptional or co-transcriptional factors with DNA is crucial for changes of neuronal gene expression during normal brain development as well as neurodegeneration. The electrophoretic mobility shift assay (EMSA) is a very powerful technique for studying changes of neuronal gene ...

Novel Object Recognition for Studying Memory in Mice

Authors: Tzyy-Nan Huang
Tzyy-Nan HuangAffiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
For correspondence: eugene02@gate.sinica.edu.tw
Bio-protocol author page: a1680
 and Yi-Ping Hsueh
Yi-Ping HsuehAffiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
For correspondence: yph@gate.sinica.edu.tw
Bio-protocol author page: a1681
date: 10/5/2014, 8998 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1249.

[Abstract] Memory tests are important indexes of the brain functions for rodents behavior assay. Many memory tasks require external forces (e.g. electric shocks) or intrinsic forces (e.g. hunger and thirsty) to trigger the responses. Under those conditions, rodents are under stresses, such as pain, tired, malnutrition ...

Optical Clearing Using SeeDB

Authors: Meng-Tsen Ke
Meng-Tsen KeAffiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan
Bio-protocol author page: a1144
Satoshi Fujimoto
Satoshi FujimotoAffiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan
Bio-protocol author page: a1145
 and Takeshi Imai
Takeshi ImaiAffiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan
For correspondence: imai@cdb.riken.jp
Bio-protocol author page: a1146
date: 2/5/2014, 8732 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1042.

[Abstract] We describe a water-based optical clearing agent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose (80.2% w/w) in water with ...

Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers

Author: Yang Geng
Yang GengAffiliation: Department of Pediatrics and Bioengineering, Stanford University School of Medicine, Stanford, USA
For correspondence: yanggeng@stanford.edu
Bio-protocol author page: a64
date: 7/20/2012, 7028 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.235.

[Abstract] Microfluidics chamber is an ideal tool to study local events that occurring in neuronal projections by perfectly compartmentalizing the cell soma from certain branches. It is very well suited for live cell imaging or immunohistochemistry staining. This protocol has been carefully modified in detail ...
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