Laboratory Protocols

Under a dissecting microscope, in a 35-mm culture dish containing grasshopper saline (150 mM NaCl, 10 mM KCl, 4mM CaCl2, 2mM MgSO4, 5mM TES, and 140 mM sucrose, pH 7.1), embryos are removed from their egg cases by puncturing the opposite end of the egg that contains the embryo to relieve pressure (the dark pigment at one end of the egg indicates the presence of an embryo) some yolk will be expelled from the egg. 2. Using micro scissors, the very tip of the end of the egg containing the embryo...

Growing and Working with Peripheral Neurons

Department of Neurobiology and Anatomy Drexel University College of Medicine Philadelphia, Pennsylvania 19129 D. Preparation of Dissociated Cultures F. Preparation of Explant Cultures G. Reducing Nonneuronal Contamination III. Cultures A. Short-Term Chick Dorsal Root Ganglia Culture B. Short-Term Rat Sympathetic Culture C. Long-Term Rat Sympathetic Culture References Cultures of vertebrate peripheral neurons have been used to address a variety of issues related to the cell biology of the...

Bsa

AA 20x prestock solution of SeO2 is prepared at 0.75 mg ml and then diluted in PBS. Indianapolis, IN), and (c) 10 by volume of a supplement mixture we call N9 (Table II). This supplement mixture was developed from the basic N2 supplement (Bottenstein and Sato, 1979), but several component concentrations have been changed, and we have added tri-iodothyronine, estradiol, and corticosterone (Akuzawa and Wakabayashi, 1985 Ferreira and Caceres, 1991). The final concentrations of N9 components (all...

Drosophila as a Genetic Model System for Molecular Neurobiology

The entire genome of Drosophila has been sequenced (Adams et al., 2000), and EST cDNAs are available from multiple libraries representing different stages of development (BDGP, http www.fruitfly.org ). The availability of both genomic sequence and cDNA clones has increased the efficiency of forward and reverse genetic approaches (for review, see Adams and Sekelsky, 2002). Mutant screens for novel genes remain a primary tool of Drosophila neurobiologists (for examples of reviews, see Broadie,...

Contents

Comparing the Properties of Neuronal Culture Systems A Shopping Guide for the Cell Biologist Peter J. Hollenbeck and James R. Bamburg II. Approaching Experimental Questions Using Neurons 2 III. Choosing a Neuronal Culture System 8 IV. Conclusions 14 References 15 2. Growing and Working with Peripheral Neurons Yan He and Peter W. Baas 3. Dissection and Culturing of Chick Ciliary Ganglion Neurons A System Well Suited to Synaptic Study IV. Variations on Dissection, Dissociation, and Culturing...

Dissection and Culturing of Chick Ciliary Ganglion Neurons A System Well Suited to Synaptic Study

Department of Biochemistry and Molecular Biology and Program in Molecular, Cellular, and Integrative Neurosciences Colorado State University Fort Collins, Colorado 80523 A. Neuroanatomy and Development of the Ciliary Ganglion B. Applications of Cultured Cells and Isolated Calyx Terminals II. Materials B. Materials for Dissociation and Culturing A. Dissection Isolation of Ciliary Ganglion B. Dissociation of the Ganglion C. Culturing of Ciliary Neurons IV. Variations on Dissection, Dissociation,...

Functional Analysis Techniques in the Drosophila Nervous System

Many mutants modifying the nervous system will alter the communication between neurons and their target cells. To rigorously determine how a particular gene product affects the activity of an excitable cell in vivo, several methods directly monitoring neuronal physiological function are used in Drosophila and described in this section. Elevation of intracellular calcium is the trigger for the exocytosis of synaptic vesicles, and the released transmitter typically leads to a postsynaptic...

Avian Purkinje Neuronal Cultures Extrinsic Control of Morphology by Cell Type and Glutamate

Balcar,* Ornella Tolhurst,* Ron P. Weinberger,* and Jenny A. Meany* *Developmental Neurobiology Group Children's Medical Research Institute Westmead, New South Wales 2145 Australia Institute for Biomedical Research University of Sydney Sydney, New South Wales 2600 Australia Oncology Research Unit Children's Hospital at Westmead Westmead, New South Wales 2145 Australia I. Introduction II. Methods and Systems E. Glutamate Uptake in Purkinje Neurons III. Applications...

Biochemistry of the Drosophila Nervous System

Although genetics is the well-known strength of Drosophila, biochemical studies are feasible in this system and represent a vital research avenue. The quantities of neuronal brain extract required for many biochemical studies can be readily obtained from flies. Initially, fly head extracts were used to study the biochemical, pharmacological, and electrical properties of various ion channels previously identified and characterized in vertebrate systems (Schmidt-Nielson et al., 1977 Pauron et...

Techniques to Dissect Cellular and Subcellular Function in the Drosophila Nervous System

Matthies and Kendal Broadie Department of Biological Sciences Vanderbilt University Nashville, Tennessee 37235 II. Background Sources of Information III. Drosophila as a Genetic Model System for Molecular Neurobiology A. Rapid Reverse Genetic Approaches for Initial Assessment of Gene Function B. Generation of Mutations in a Gene of Interest IV. Biochemistry of the Drosophila Nervous System V. Cell Biology Techniques in the Drosophila Nervous System B. Protein Expression Studies...

Neuron Replating

Is only available by collaborating with Curis (www.curis.com). IV. Maintenance of Sympathetic Neuronal Culture A. The day after plating, replace N2 medium with medium A. B. The next day, replace two-thirds of medium A with medium B. Repeat every other day. C. One week after plating, start feeding the culture with medium C. Each time, replace two-thirds of the medium with medium C. D. After 7-10 days in medium C, the cells will have well-developed dendrites. IV. Maintenance of Sympathetic...

Info

Fig. 6 Interaction of a motor neuron growth cone with an extrinsic cue. An individual motor neuron growth cone encounters the extrinsic cue tumor necrosis factor a, coupled covalently onto a small polystyrene bead (5 m in diameter). Upon intial filopodia contact, the growth cone displays changes in behavior and morphology over a time period of 30 min. Finally the growth cone collapses, which is indicated by a complete loss of morphology and cessation of advance. Fig. 6 Interaction of a motor...

Development of the Tibial1 Ti1 Pathway

Grasshopper Limb Bud Axon

Grasshopper Limb Development Grasshopper embryogenesis is divided into stages based on the percentage of development. Embryos develop roughly 5 per day with the earliest born neurons in the peripheral nervous system (PNS) and central nervous system (CNS) appearing at approximately 30 of development. The Ti1 pathway is established from 30 to 35 of embryonic development. During this time, the developing limb bud also undergoes molecular and morphological changes. At 30 of development, during...

Analysis of Axon Guidance Mechanisms in Grasshopper Procedures

Staging Grasshopper Embryos from 29 to 35 of Development Our staging protocol is essentially from Bentley et al. (1979) with some changes that reflect the difference in species used (Schistocerca americana vs Schistocerca gregaria). The shape of the limb bud changes substantially from 29 to 35 of embryonic development. At 29 , the distal tip of the limb bud is only slightly pointed and the overall shape of the limb bud is rounded and unsegmented. At this stage, Ti1 cell bodies are...

The Culture of Chick Forebrain Neurons

Chicken Embryonic Stem Cells

Heidemann, Matthew Reynolds, Kha Ngo, and Phillip Lamoureux Department of Physiology Michigan State University East Lansing, Michigan 48824 II. Growth and Development Characteristics A. Chick Forebrain Neurons Develop into Typical Pyramidal Neurons in Low-Density Culture B. Chick Forebrain Neurons Show Elongation Behaviors Similar to Hippocampal Neurons C. Chick Forebrain Neurons Differ in Important Ways from Hippocampal Neurons III. Isolation of Single Chick Forebrain Neurons A....

Cell Biology Techniques in the Drosophila Nervous System

Among the forward genetic systems, methods for the analysis of neurons in situ are the most advanced in Drosophila. Immunocytochemical methods are developed for all stages of the life cycle and many antibodies are available see Table II . In C. elegans, creating transgenic animals expressing marked proteins is much faster, but regulating the expression levels is fairly difficult. In flies, however, transgenic protein expression is a standard method and many variations of controlling expression...

Working with Xenopus Spinal Neurons in Live Cell Culture

Gomez, Dan Harrigan, John Henley, and Estuardo Robles Department of Anatomy Cell and Molecular Biology Training Program University of Wisconsin Medical School Madison, Wisconsin 53706 Department of Molecular and Cell Biology University of California Berkeley, California 94720-3200 Neuroscience Training Program University of Wisconsin Medical School Madison, Wisconsin 53706 I. Introduction II. Neuronal Labeling A. Injection Sample Preparation III. Culturing Xenopus Spinal Neurons IV....

Growing and Working with Spinal Motor Neurons

Department of Pharmaceutical Sciences University of Montana Missoula, Montana 59812 II. Incubation of Fertilized Chicken Eggs III. Preparation of Chick Embryo IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or Ventral Halves VI. Plating Motor Neurons or Spinal Cord Neurons VII. Motor Neuron Enrichment by Density Gradient Centrifugation VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures...