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==Lecture 1 Techniques==  
==Lecture 1 Techniques==  
<i>What can these be used for?</i><br>
<i>What can these be used for?</i><br>
===Golgi staining===
===Golgi staining===
Also called the Black Reaction, Golgi staining stains a subset of cells within the brain, because staining all neurons and cellular processes would make anatomical analyses difficult and cumbersome. While the exact mechanisms behind the Golgi stain is not well understood, this technique labels axons, dendrites, and cell somas in black and brown along their entire length. Hence, neural ciruits can be visualized, tracked, and mapped. Golgi stains are made by injection of potassiumdichromate and silver nitrate; the brown-black color of neurons stems from the microcrystallization of silver chromate.
Also called the Black Reaction, Golgi staining stains a subset of cells within the brain, because staining all neurons and cellular processes would make anatomical analyses difficult and cumbersome. While the exact mechanisms behind the Golgi stain is not well understood, this technique labels axons, dendrites, and cell somas in black and brown along their entire length. Hence, neural ciruits can be visualized, tracked, and mapped. Golgi stains are made by injection of potassiumdichromate and silver nitrate; the brown-black color of neurons stems from the microcrystallization of silver chromate.
Line 30: Line 29:


===Electrical stimulation===
===Electrical stimulation===


===fMRI===
===fMRI===


 
==Lecture 2 Model Systems==
 
<i>What are the advantages of each?</i><br>
==Lecture 2==
===Frog visual system===
<i>Model Systems Introduced:  What are the advantages of each?</i><br>
===Vertebrate spinal cord===
Frog visual system<br>
===C. elegans sensory and motor neurons===
Vertebrate spinal cord<br>
===Drosophila embryo===
C. elegans sensory and motor neurons<br>
===Cell culture===
Drosophila embryo<br>
===Grasshopper===
Cell culture<br>
===Xenopus axons in culture===
Grasshopper<br>
Xenopus axons in culture<br>
<br>
<br>
<i>Techniques and Tools Introduced:  What can these be used for?</i><br>
==Lecture 2 Techniques==
Biochemistry<br>
<i>What can these be used for?</i><br>
Genetics: mutation and over expression<br>
===Biochemistry===
Co-culture on a 3D collagen gel matrix<br>
===Genetics: mutation and over expression===
Antibody staining<br>
===Co-culture on a 3D collagen gel matrix===
Cloning genes and expressing them in cell culture<br>
===Antibody staining===
Forward genetic screen<br>
===Cloning genes and expressing them in cell culture===
Dye injection<br>
===Forward genetic screen===
Poo assay<br>
===Dye injection===
Explant overlay assay<br>
===Poo assay===
Incubating slices in media with chemical cues<br>
===Explant overlay assay===
Mammalian pyramidal neurons<br>
===Incubating slices in media with chemical cues===
===Mammalian pyramidal neurons===
<br>
<br>
==Lecture 3==
==Lecture 3 Model Systems==
<i>Model Systems Introduced:  What are the advantages of each?</i><br>
<i>What are the advantages of each?</i><br>
Drosophila olfactory system<br>
===Drosophila olfactory system===
Three-eye frogs<br>
===Three-eye frogs===
<br>
<br>
<i>Techniques and Tools Introduced:  What can these be used for?</i><br>
==Lecture 3 Techniques==
In vitro stripe assay<br>
<i>What can these be used for?</i><br>
2D gel electrophoresis<br>
===In vitro stripe assay===
Transplantation<br>
===2D gel electrophoresis===
Radiolabel injection<br>
===Transplantation===
 
===Radiolabel injection===
 


===TTX===
===TTX===
Tetrodotoxin.  A toxin from the puffer fish that blocks [[voltage gated sodium channels]].<br>
Tetrodotoxin.  A toxin from the puffer fish that blocks [[voltage gated sodium channels]].<br>


===TEA===
===TEA===
Tetraethylammonium.  A compound which selectively blocks [[voltage gated potassium channels]].<br>
Tetraethylammonium.  A compound which selectively blocks [[voltage gated potassium channels]].<br>


===Differential Display===
===Differential Display===
A technique used to determine the differences in [[expression]] of mRNA between two cells under different conditions or between two different cell, using [[mRNA probes]].  This technique is rapidly being replaced by [[expression profiles]] using [[microarray]]s.<br>
A technique used to determine the differences in [[expression]] of mRNA between two cells under different conditions or between two different cell, using [[mRNA probes]].  This technique is rapidly being replaced by [[expression profiles]] using [[microarray]]s.<br>


===In-situ hybridization===
===In-situ hybridization===
In-situ uses mRNA probes (also called oligos) that anneal to the mRNA strand of interest in fixed animal tissue. Because the probes are usually fluorescently-tagged, this technique allows visualization of mRNA in cells/tissue, providing quantitative data on the amount of genetic information being expressed.
In-situ uses mRNA probes (also called oligos) that anneal to the mRNA strand of interest in fixed animal tissue. Because the probes are usually fluorescently-tagged, this technique allows visualization of mRNA in cells/tissue, providing quantitative data on the amount of genetic information being expressed.


Knockout mice<br>
===Knockout mice===
Monocular enucleation<br>
===Monocular enucleation===


==Paper 1==
==Paper 1 Model Systems==
<i>Model Systems Introduced:  What are the advantages of each?</i><br>
<i>What are the advantages of each?</i><br>
Chick optic tectum<br>
===Chick optic tectum===
Mouse superior colliculus<br>
===Mouse superior colliculus===
Mouse retina<br>
===Mouse retina===
<br>
<br>
<i>Techniques and Tools Introduced:  What can these be used for?</i><br>
<i>Techniques and Tools Introduced:  What can these be used for?</i><br>
In-situ hybridization – sense controls<br>
===In-situ hybridization – sense controls===
HEK293 cells<br>
===HEK293 cells===


===SF9 cells===
===SF9 cells===
An insect cell line (from a kind of caterpillar) used for the production of recombinant protein.<br>
An insect cell line (from a kind of caterpillar) used for the production of recombinant protein.<br>


===Baculovirus system===
===Baculovirus system===
Baculovirus is a natural pathogen of the caterpillars producing the SF9 cell line.  In the lab, genes are encoded into a baculovirus vector which is then used to infect SF9 cells.<br>
Baculovirus is a natural pathogen of the caterpillars producing the SF9 cell line.  In the lab, genes are encoded into a baculovirus vector which is then used to infect SF9 cells.<br>


===Affinity-purified protein===
===Affinity-purified protein===
A protein purified by passing a solution of protein through a column where the protein becomes associated with a matrix of immobilized [[ligand]] somehow attatched to the column.  In most cases the protein must be tagged, or appended to a functional motif called a [[fusion tag]].  Common fusion tag-ligand pairs include:  [[Histidine tag]] (6 or more extra Histidines) and the "ligands" Chelated Nickel or Cobalt, [[Maltose Binding Protein]] and its ligand [[dextrin]], [[Glutathione S-transferase]] and its ligand [[reduced glutathione]], and [[Green Fluorescent Protein]] and Anti-GFP [[antibody]].<br>
A protein purified by passing a solution of protein through a column where the protein becomes associated with a matrix of immobilized [[ligand]] somehow attatched to the column.  In most cases the protein must be tagged, or appended to a functional motif called a [[fusion tag]].  Common fusion tag-ligand pairs include:  [[Histidine tag]] (6 or more extra Histidines) and the "ligands" Chelated Nickel or Cobalt, [[Maltose Binding Protein]] and its ligand [[dextrin]], [[Glutathione S-transferase]] and its ligand [[reduced glutathione]], and [[Green Fluorescent Protein]] and Anti-GFP [[antibody]].
 


Mock infection<br>
===Mock infection===
Blocking with antibodies or proteins<br>
===Blocking with antibodies or proteins===
Western Blot, α-tubulin<br>
===Western Blot, α-tubulin===
Retina explant assay<br>
===Retina explant assay===
Electroporation into ventricular zone<br>
===Electroporation into ventricular zone===
Dominant-negative<br>
===Dominant-negative===
In ovo electroporation<br>
===In ovo electroporation===


===DAPI staining===
===DAPI staining===
Fluorescently labels cell nuclei by binding to DNA.<br>
Fluorescently labels cell nuclei by binding to DNA.


AP (alkaline phosphatase)<br>
===AP (alkaline phosphatase)===
Tagged proteins<br>
===Tagged proteins===
Protein overexpression<br>
===Protein overexpression===


===sFRP2===
===sFRP2===
secreted frizzled related protein 2 is an [[antagonist]] of the [[Wnt]] ligand in [[Wnt-Frizzled]] mediated [[cell signalling]].<br>
secreted frizzled related protein 2 is an [[antagonist]] of the [[Wnt]] ligand in [[Wnt-Frizzled]] mediated [[cell signalling]].
 


===DiI===
===DiI===

Revision as of 02:54, 14 October 2006

WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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Lecture 1 Model Systems

What are the advantages of each?

Mammalian visual system

Mammalian olfactory system

Spinal cord motor neurons

Human brain


Lecture 1 Techniques

What can these be used for?

Golgi staining

Also called the Black Reaction, Golgi staining stains a subset of cells within the brain, because staining all neurons and cellular processes would make anatomical analyses difficult and cumbersome. While the exact mechanisms behind the Golgi stain is not well understood, this technique labels axons, dendrites, and cell somas in black and brown along their entire length. Hence, neural ciruits can be visualized, tracked, and mapped. Golgi stains are made by injection of potassiumdichromate and silver nitrate; the brown-black color of neurons stems from the microcrystallization of silver chromate.

Tissue culture

Tissue cultures allow researchers to grow tissues and/or cells outside of the organism under investigation. Primary cell cultures usually have a finite life span in culture compared to cell lines which are abnormal or transformed cell lines. The availability of tissue cultures enable the study of cells in a controlled environment without the external influences found in the organisms' physiological environment. Advantages of such a technique include the ability to study specific cellular mechanisms alone, and the opportunity to manipulate cell lines to better understand developmental abnormalities.

Electron microscopy

Through the use of electrons to create an image of the object, electron microscopy provides higher magnification and superior resolving power than a light microscope by almost a magnitude of two million. Various electron microscopy techniques exist for exploring morphology and mechanisms: scanning electron microscopes give a 3D image of the sample; transmission electrion microscopes produce 2D images at impressive magnifications (up to 500 million times); and scanning tunneling microscopes determine the height of the sample surface.

Biolistic transfection (gene gun)

This technique injects cells with a heavy metal coated with plasmid DNA, and is capable of transforming almost all types of cells including their genetic information and cellular organelles. Gene guns are also effective in delivering DNA vaccines to mammals for therapy.

Genetic labeling

Patch clamp

The patch clamp method allowed detailed understanding of the action potential after it was invented by Kenneth Cole in the 1940s. This method enables us to measure the membrane potential, or voltage, at any level desired by the experimenter through use of a microelectrode placed inside the cell. The voltage clamp technique reveals how membrane potential influences ionic current flow across the membrane, and was instrumental in providing Hodgkin and Huxley with information leading to membrane ion gradients and the action potential.

Electrical stimulation

fMRI

Lecture 2 Model Systems

What are the advantages of each?

Frog visual system

Vertebrate spinal cord

C. elegans sensory and motor neurons

Drosophila embryo

Cell culture

Grasshopper

Xenopus axons in culture


Lecture 2 Techniques

What can these be used for?

Biochemistry

Genetics: mutation and over expression

Co-culture on a 3D collagen gel matrix

Antibody staining

Cloning genes and expressing them in cell culture

Forward genetic screen

Dye injection

Poo assay

Explant overlay assay

Incubating slices in media with chemical cues

Mammalian pyramidal neurons


Lecture 3 Model Systems

What are the advantages of each?

Drosophila olfactory system

Three-eye frogs


Lecture 3 Techniques

What can these be used for?

In vitro stripe assay

2D gel electrophoresis

Transplantation

Radiolabel injection

TTX

Tetrodotoxin. A toxin from the puffer fish that blocks voltage gated sodium channels.

TEA

Tetraethylammonium. A compound which selectively blocks voltage gated potassium channels.

Differential Display

A technique used to determine the differences in expression of mRNA between two cells under different conditions or between two different cell, using mRNA probes. This technique is rapidly being replaced by expression profiles using microarrays.

In-situ hybridization

In-situ uses mRNA probes (also called oligos) that anneal to the mRNA strand of interest in fixed animal tissue. Because the probes are usually fluorescently-tagged, this technique allows visualization of mRNA in cells/tissue, providing quantitative data on the amount of genetic information being expressed.

Knockout mice

Monocular enucleation

Paper 1 Model Systems

What are the advantages of each?

Chick optic tectum

Mouse superior colliculus

Mouse retina


Techniques and Tools Introduced: What can these be used for?

In-situ hybridization – sense controls

HEK293 cells

SF9 cells

An insect cell line (from a kind of caterpillar) used for the production of recombinant protein.

Baculovirus system

Baculovirus is a natural pathogen of the caterpillars producing the SF9 cell line. In the lab, genes are encoded into a baculovirus vector which is then used to infect SF9 cells.

Affinity-purified protein

A protein purified by passing a solution of protein through a column where the protein becomes associated with a matrix of immobilized ligand somehow attatched to the column. In most cases the protein must be tagged, or appended to a functional motif called a fusion tag. Common fusion tag-ligand pairs include: Histidine tag (6 or more extra Histidines) and the "ligands" Chelated Nickel or Cobalt, Maltose Binding Protein and its ligand dextrin, Glutathione S-transferase and its ligand reduced glutathione, and Green Fluorescent Protein and Anti-GFP antibody.

Mock infection

Blocking with antibodies or proteins

Western Blot, α-tubulin

Retina explant assay

Electroporation into ventricular zone

Dominant-negative

In ovo electroporation

DAPI staining

Fluorescently labels cell nuclei by binding to DNA.

AP (alkaline phosphatase)

Tagged proteins

Protein overexpression

sFRP2

secreted frizzled related protein 2 is an antagonist of the Wnt ligand in Wnt-Frizzled mediated cell signalling.

DiI

A lipophillic compound used to label cells. DiI has affinity for any cell membrane and is therefore not cell specific, but will only label the cell individually injected with DiI.


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25 April 2024

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