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Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control

Featured protocol,  Authors: Jérémie Barral
Jérémie BarralAffiliation: Center for Neural Science, New York University, New York, USA
For correspondence: barral@cns.nyu.edu
Bio-protocol author page: a4664
 and Alex D Reyes
Alex D ReyesAffiliation: Center for Neural Science, New York University, New York, USA
Bio-protocol author page: a4665
date: 6/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2335.

Brief version appeared in Nat Neurosci, Dec 2016
We studied a network of cortical neurons in culture and developed an innovative optical device to stimulate optogenetically a large neuronal population with both spatial and temporal precision. We first describe how to culture primary neurons expressing channelrhodopsin. We then detail the optogenetic setup based on the workings of a fast Digital Light Processing (DLP) projector. The setup is able to stimulate tens to hundreds neurons with independent trains of light pulses that evoked action potentials with high temporal resolution. During photostimulation, network activity was monitored using patch-clamp recordings of up to 4 neurons. The experiment is ideally suited to study recurrent network dynamics or biological processes such as plasticity or homeostasis in a network of neurons when a sub-population is activated by distinct stimuli whose characteristics (correlation, rate, and, size) were finely controlled.

Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice

Featured protocol,  Authors: Arnau Busquets-Garcia
Arnau Busquets-GarciaAffiliation 1: INSERM, U1215 NeuroCentre Magendie, Bordeaux, France
Affiliation 2: University of Bordeaux, Bordeaux, France
For correspondence: arnau.busquets-garcia@inserm.fr
Bio-protocol author page: a4732
Edgar Soria-Gómez
Edgar Soria-GómezAffiliation 1: INSERM, U1215 NeuroCentre Magendie, Bordeaux, France
Affiliation 2: University of Bordeaux, Bordeaux, France
Bio-protocol author page: a1734
Guillaume Ferreira
Guillaume FerreiraAffiliation 1: University of Bordeaux, Bordeaux, France
Affiliation 2: INRA, Nutrition et Neurobiologie Intégrée, UMR, Bordeaux, France
Bio-protocol author page: a4733
 and Giovanni Marsicano
Giovanni MarsicanoAffiliation 1: INSERM, U1215 NeuroCentre Magendie, Bordeaux, France
Affiliation 2: University of Bordeaux, Bordeaux, France
Bio-protocol author page: a1733
date: 6/20/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2358.

Brief version appeared in Mol Psychiatry, Feb 2017
Several paradigms for rodent models of the cognitive and negative endophenotypes found in schizophrenic patients have been proposed. However, significant efforts are needed in order to study the pathophysiology of schizophrenia-related positive symptoms. Recently, it has been shown that these positive symptoms can be studied in rats by using representation-mediated learning. This learning measure the accuracy of mental representations of reality, also called ‘reality testing’. Alterations in ‘reality testing’ performance can be an indication of an impairment in perception which is a clear hallmark of positive psychotic-like states. Thus, we describe here a mouse task adapted from previous findings based on a sensory preconditioning task. With this task, associations made between different neutral stimuli (e.g., an odor and a taste) and subsequent selective devaluation of one of these stimuli have allowed us to study mental sensory representations. Thus, the interest of this task is that it can be used to model positive psychotic-like states in mice, as recently described.

Stereotaxic Surgery for Suprachiasmatic Nucleus Lesions in Mice

Featured protocol,  Authors: Kimiko Shimizu
Kimiko ShimizuAffiliation: Department of Biological Sciences, School of Science, the University of Tokyo, Tokyo, 113-0033 Japan
For correspondence: shimizuk@bs.s.u-tokyo.ac.jp
Bio-protocol author page: a4698
 and Yoshitaka Fukada
Yoshitaka FukadaAffiliation: Department of Biological Sciences, School of Science, the University of Tokyo, Tokyo, 113-0033 Japan
Bio-protocol author page: a4699
date: 6/20/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2346.

Brief version appeared in Nat Commun, Sep 2016
Site-specific lesions are invaluable methods for investigating the function of brain regions within the central nervous system and can be used to study neural mechanisms of behaviors. Precise stereotaxic surgery is required to lesion small regions of the brain such as the suprachiasmatic nucleus (SCN), which harbors the master circadian clock. In this protocol, we describe stereotaxic surgery optimized for bilateral lesion of the mouse SCN by loading electric current. Success of the SCN lesion is verified histologically and behaviorally by monitoring arrhythmic locomotor activity. The SCN-lesioned mouse allows for the evaluation of behavioral, biochemical, and physiological consequences of ablation of the master circadian clock.

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

Featured protocol,  Authors: Reka A. Haraszti
Reka A. HarasztiAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4671
Andrew Coles
Andrew ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
date: 6/20/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2338.

Brief version appeared in Mol Ther, Oct 2016
Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural trafficking ability gives extracellular vesicles the potential to be used as delivery vehicles for oligonucleotides, i.e., siRNAs. However, robust and scalable methods for loading of extracellular vesicles with oligonucleotide cargo are lacking. We describe a detailed protocol for the loading of hydrophobically modified siRNAs into extracellular vesicles upon simple co-incubation. We detail methods of the workflow from purification of extracellular vesicles to data analysis. This method may advance extracellular vesicles-based therapies for the treatment of a broad range of neurological disorders.

Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice

Featured protocol,  Authors: Cong Wang
Cong WangAffiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou, China
Bio-protocol author page: a4734
Hao Gao
Hao GaoAffiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou, China
Bio-protocol author page: a4735
 and Shengxiang Zhang
Shengxiang ZhangAffiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou, China
For correspondence: sxzhang@lzu.edu.cn
Bio-protocol author page: a4736
date: 6/20/2017, 76 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2360.

Brief version appeared in Sci Rep, Sep 2016
Transplantation of embryonic cortical tissue for repairing the damaged brain has provided a potential therapy for brain injury and diseases. The grafted tissue can successfully survive and participate in reestablishing the functional neural circuit of the host brain. Transplantation surgery can be combined with fluorescently labeled transgenic mice to evaluate the reconstruction of neuronal network (Falkner et al., 2016) and the repopulation of a subset of cortical cells. By using this approach, we have shown that infiltrating cells from host brain can restore the microglial population in the graft tissue (Wang et al., 2016). This protocol describes the detailed procedure of the transplantation surgery in mice, including establishing a lesion model in the host brain, preparing the embryonic cortical graft, and transplanting the embryonic cortical graft to adult brain.

Contusion Spinal Cord Injury Rat Model

Featured protocol,  Authors: Chuan-Wen Chiu
Chuan-Wen ChiuAffiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan
Bio-protocol author page: a4670
Henrich Cheng
Henrich ChengAffiliation 1: Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan
Affiliation 2: Center for Neural Regeneration, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan
For correspondence: hc_cheng@vghtpe.gov.tw
Bio-protocol author page: a4669
 and Shie-Liang Hsieh
Shie-Liang HsiehAffiliation 1: Genomics Research Center, Academia Sinica, Taipei, Taiwan
Affiliation 2: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
For correspondence: slhsieh@gate.sinica.edu.tw
Bio-protocol author page: a118
date: 6/20/2017, 61 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2337.

Brief version appeared in J Neuroinflammation, Jun 2016
Spinal cord injury (SCI) can lead to severe disability, paralysis, neurological deficits and even death. In humans, most spinal cord injuries are caused by transient compression or contusion of the spinal cord associated with motor vehicle accidents. Animal models of contusion mimic the typical SCI’s found in humans and these models are key to the discovery of progressive secondary tissue damage, demyelination, and apoptosis as well as pathophysiological mechanisms post SCI. Here we describe a method for the establishment of an efficient and reproducible contusion model of SCI in adult rat.

The Repeated Flurothyl Seizure Model in Mice

Featured protocol,  Author: Russell J. Ferland
Russell J. FerlandAffiliation 1: Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, NY 12208, USA
Affiliation 2: Department of Neurology, Albany Medical College, Albany, NY 12208, USA
For correspondence: ferlanr@mail.amc.edu
Bio-protocol author page: a4587
date: 6/5/2017, 180 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2309.

Brief version appeared in J Neurosci, Jul 2016
Development of spontaneous seizures is the hallmark of human epilepsy. There is a critical need for new epilepsy models in order to elucidate mechanisms responsible for leading to the development of spontaneous seizures and for testing new anti-epileptic compounds. Moreover, rodent models of epilepsy have clearly demonstrated that there are two independent seizure systems in the brain: 1) the forebrain seizure network required for the expression of clonic seizures mediated by forebrain neurocircuitry, and 2) the brainstem seizure network necessary for the expression of brainstem or tonic seizures mediated by brainstem neurocircuitry. In seizure naïve animals, these two systems are separate, but developing models that can explore the intersection of the forebrain and brainstem seizure systems or for elucidating mechanisms responsible for bringing these two seizure systems together may aid in our understanding of: 1) how seizures can become more complex overtime, and 2) sudden unexpected death in epilepsy (SUDEP) since propagation of seizure discharge from the forebrain seizure system to the brainstem seizure system may have an important role in SUDEP because many cardiorespiratory systems are localized in the brainstem. The repeated flurothyl seizure model of epileptogenesis, as described here, may aid in providing insight into these important epilepsy issues in addition to understanding how spontaneous seizures develop.

Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures

Featured protocol,  Authors: Dorette Freyer
Dorette FreyerAffiliation: Department of Experimental Neurology, Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin, Germany
Bio-protocol author page: a4585
 and Christoph Harms
Christoph HarmsAffiliation 1: Department of Experimental Neurology, Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin, Germany
Affiliation 2: Berlin Institute of Health (BIH), Berlin, Germany
For correspondence: christoph.harms@charite.de
Bio-protocol author page: a4586
date: 6/5/2017, 183 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2308.

Brief version appeared in J Neurosci, Aug 2016
The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a very long protein half-life (Hsieh and Blumenthal, 1956; Koh and Cotman, 1992; Koh et al., 1995).

Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control

Authors: Jérémie Barral
Jérémie BarralAffiliation: Center for Neural Science, New York University, New York, USA
For correspondence: barral@cns.nyu.edu
Bio-protocol author page: a4664
 and Alex D Reyes
Alex D ReyesAffiliation: Center for Neural Science, New York University, New York, USA
Bio-protocol author page: a4665
date: 6/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2335.

[Abstract] We studied a network of cortical neurons in culture and developed an innovative optical device to stimulate optogenetically a large neuronal population with both spatial and temporal precision. We first describe how to culture primary neurons expressing channelrhodopsin. We then detail the optogenetic setup based on the workings of a fast Digital Light ...

Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice

Authors: Arnau Busquets-Garcia
Arnau Busquets-GarciaAffiliation 1: INSERM, U1215 NeuroCentre Magendie, Bordeaux, France
Affiliation 2: University of Bordeaux, Bordeaux, France
For correspondence: arnau.busquets-garcia@inserm.fr
Bio-protocol author page: a4732
Edgar Soria-Gómez
Edgar Soria-GómezAffiliation 1: INSERM, U1215 NeuroCentre Magendie, Bordeaux, France
Affiliation 2: University of Bordeaux, Bordeaux, France
Bio-protocol author page: a1734
Guillaume Ferreira
Guillaume FerreiraAffiliation 1: University of Bordeaux, Bordeaux, France
Affiliation 2: INRA, Nutrition et Neurobiologie Intégrée, UMR, Bordeaux, France
Bio-protocol author page: a4733
 and Giovanni Marsicano
Giovanni MarsicanoAffiliation 1: INSERM, U1215 NeuroCentre Magendie, Bordeaux, France
Affiliation 2: University of Bordeaux, Bordeaux, France
Bio-protocol author page: a1733
date: 6/20/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2358.

[Abstract] Several paradigms for rodent models of the cognitive and negative endophenotypes found in schizophrenic patients have been proposed. However, significant efforts are needed in order to study the pathophysiology of schizophrenia-related positive symptoms. Recently, it has been shown that these positive symptoms can be studied in rats by using representation-mediated ...

Stereotaxic Surgery for Suprachiasmatic Nucleus Lesions in Mice

Authors: Kimiko Shimizu
Kimiko ShimizuAffiliation: Department of Biological Sciences, School of Science, the University of Tokyo, Tokyo, 113-0033 Japan
For correspondence: shimizuk@bs.s.u-tokyo.ac.jp
Bio-protocol author page: a4698
 and Yoshitaka Fukada
Yoshitaka FukadaAffiliation: Department of Biological Sciences, School of Science, the University of Tokyo, Tokyo, 113-0033 Japan
Bio-protocol author page: a4699
date: 6/20/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2346.

[Abstract] Site-specific lesions are invaluable methods for investigating the function of brain regions within the central nervous system and can be used to study neural mechanisms of behaviors. Precise stereotaxic surgery is required to lesion small regions of the brain such as the suprachiasmatic nucleus (SCN), which harbors the master circadian clock. In this ...

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

Authors: Reka A. Haraszti
Reka A. HarasztiAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4671
Andrew Coles
Andrew ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
date: 6/20/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2338.

[Abstract] Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural ...

Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice

Authors: Cong Wang
Cong WangAffiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou, China
Bio-protocol author page: a4734
Hao Gao
Hao GaoAffiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou, China
Bio-protocol author page: a4735
 and Shengxiang Zhang
Shengxiang ZhangAffiliation: Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou, China
For correspondence: sxzhang@lzu.edu.cn
Bio-protocol author page: a4736
date: 6/20/2017, 76 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2360.

[Abstract] Transplantation of embryonic cortical tissue for repairing the damaged brain has provided a potential therapy for brain injury and diseases. The grafted tissue can successfully survive and participate in reestablishing the functional neural circuit of the host brain. Transplantation surgery can be combined with fluorescently labeled transgenic mice ...

Contusion Spinal Cord Injury Rat Model

Authors: Chuan-Wen Chiu
Chuan-Wen ChiuAffiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan
Bio-protocol author page: a4670
Henrich Cheng
Henrich ChengAffiliation 1: Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan
Affiliation 2: Center for Neural Regeneration, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan
For correspondence: hc_cheng@vghtpe.gov.tw
Bio-protocol author page: a4669
 and Shie-Liang Hsieh
Shie-Liang HsiehAffiliation 1: Genomics Research Center, Academia Sinica, Taipei, Taiwan
Affiliation 2: Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
For correspondence: slhsieh@gate.sinica.edu.tw
Bio-protocol author page: a118
date: 6/20/2017, 61 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2337.

[Abstract] Spinal cord injury (SCI) can lead to severe disability, paralysis, neurological deficits and even death. In humans, most spinal cord injuries are caused by transient compression or contusion of the spinal cord associated with motor vehicle accidents. Animal models of contusion mimic the typical SCI’s found in humans and these models are key to the ...

The Repeated Flurothyl Seizure Model in Mice

Author: Russell J. Ferland
Russell J. FerlandAffiliation 1: Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, NY 12208, USA
Affiliation 2: Department of Neurology, Albany Medical College, Albany, NY 12208, USA
For correspondence: ferlanr@mail.amc.edu
Bio-protocol author page: a4587
date: 6/5/2017, 180 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2309.

[Abstract] Development of spontaneous seizures is the hallmark of human epilepsy. There is a critical need for new epilepsy models in order to elucidate mechanisms responsible for leading to the development of spontaneous seizures and for testing new anti-epileptic compounds. Moreover, rodent models of epilepsy have clearly demonstrated that there are two independent ...

Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures

Authors: Dorette Freyer
Dorette FreyerAffiliation: Department of Experimental Neurology, Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin, Germany
Bio-protocol author page: a4585
 and Christoph Harms
Christoph HarmsAffiliation 1: Department of Experimental Neurology, Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, Berlin, Germany
Affiliation 2: Berlin Institute of Health (BIH), Berlin, Germany
For correspondence: christoph.harms@charite.de
Bio-protocol author page: a4586
date: 6/5/2017, 183 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2308.

[Abstract] The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell ...

Locomotor Assay in Drosophila melanogaster

Authors: Qingqing Liu
Qingqing LiuAffiliation 1: State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing, China
Affiliation 2: , University of CAS, Beijing, China
Bio-protocol author page: a4514
Jingsong Tian
Jingsong TianAffiliation 1: State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing, China
Affiliation 2: , University of CAS, Beijing, China
Bio-protocol author page: a4515
Xing Yang
Xing YangAffiliation 1: State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing, China
Affiliation 2: Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, CAS, Shanghai, China
Bio-protocol author page: a4516
Yan Li
Yan LiAffiliation: State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing, China
For correspondence: liyan@sun5.ibp.ac.cn
Bio-protocol author page: a4517
 and Aike Guo
Aike GuoAffiliation 1: State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing, China
Affiliation 2: Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, CAS, Shanghai, China
For correspondence: akguo@ion.ac.cn
Bio-protocol author page: a4518
date: 5/20/2017, 198 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2283.

[Abstract] This protocol describes a simple locomotor assay in Drosophila melanogaster. In brief, the locomotor of each single fly in the culture dish is recorded by a web camera. The moving time, walking length, speed and the locomotor trails of the single fly could be quantitatively analyzed. ...

Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining

Authors: Chao Wang
Chao WangAffiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
For correspondence: wang1438@purdue.edu
Bio-protocol author page: a4505
Feng Yue
Feng YueAffiliation: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
Bio-protocol author page: a4506
 and Shihuan Kuang
Shihuan KuangAffiliation 1: Department of Animal Science, Purdue University, West Lafayette, Indiana, USA
Affiliation 2: Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA
For correspondence: skuang@purdue.edu
Bio-protocol author page: a2572
date: 5/20/2017, 284 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2279.

[Abstract] Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber diameter, the presence of degenerated and regenerated myofibers, and adipocytes and fibrotic cells. Muscle fibers in adult skeletal muscles of rodents are classified into 4 subtypes based on the expression of myosin heavy chain proteins: Myh7 (type I fiber), Myh2 (type IIA fiber), Myh1 (type IIX fiber), Myh4 (type IIB fiber). A panel of monoclonal antibodies can be used to specifically label these muscle fiber subtypes. These protocols are commonly used in the study of muscle development, growth and regeneration (for example: Wang et al., 2015; Nie et al., 2016; Yue et al., 2016; Wang et al., 2017)....
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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, 11730 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....

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, 11679 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 ...

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, 11663 views, 2 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 ...

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, 11547 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 ...

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, 11219 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, 11215 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 ...

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, 10782 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 ...

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, 10728 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 ...

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, 10136 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 ...

Organotypic Slice Culture of Embryonic Brain Sections

Author: Froylan Calderon de Anda
Froylan Calderon de AndaAffiliation: Center For Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany
For correspondence: froylan.calderon@zmnh.uni-hamburg.de
Bio-protocol author page: a227
date: 2/5/2013, 7975 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.327.

[Abstract] This technique will allow using brain slices to study several aspects of cortical development (i.e. neurogenesis), as well as neuronal differentiation (i.e. neuronal migration, axon and dendrite formation) in situ. This protocol is suitable for various embryonic stages (Calderon de Anda et al., 2010; ...
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