Toolbox/Lecture 3

Lecture 3 Model Systems
What are the advantages of each?

Drosophila olfactory system
The Drosophila olfactory system is a great model system for understanding how precise connections are made, what are the genes important for the formation of precise connections, and how formation of these precise connections are relevant for encoding olfactory information. Olfactory sensory neurons project their axons to discrete circular centers called glomeruli. At these glomeruli they connect with the dendrites of second order neurons, projection neurons. The projection neurons then send axons to the mushroom body calyx and the lateral horn for higher processing of olfactory information. The power of genetics has allowed scientists to label projection neurons. Since the advent of MARCM (Mosaic Analysis with a Repressible Cell Marker) one can label a subset of these projection neurons. One can even label a single projection neuron. Using MARCM, studies have shown that lineage and birth timing of projection neurons is correlated with their glomerular projections. MARCM has also been used to study the branching patterns of individual classes of projection neurons and the genes involved in the precise projections to single glomeruli (e.g. Sema1a, N-cadherin, Dscam).

Three-eye frogs
"An extra eye primordium was implanted into the forebrain region of embryonic Rana pipiens. During development both normal and supernumerary optic tracts terminated within a single, previously uninnervated tectal lobe. Autoradiographic tracing of either the normal or supernumerary eye's projection revealed distinct, eye-specific bands of radioactivity running rostrocaudally through the dually innervated tectum. Interactions among axons of retinal ganglion cells, possibly mediated through tectal neurons, must be invoked to explain this stereotyped disruption of the normally continuous retinal termination pattern." ("Eye-specific termination bands in tecta of three-eyed frogs" ) Frogs do not have binocular vision because the outputs of the left and right eye do not converge. All retinal ganglion cells (RGCs; the cells that relay information from eye to the next level of information processing) from the left eye project their axons to the optic tectum on the right side. All RGCs from the right eye project their axons to the optic tectum on the left size. Because the left and right eyes are completely segregated there is no competition during development and no stripe formation is seen. However, when you transplant a third eye, you induce competition among axons projecting to the optic tectum. The competion between RGC axons from the transplanted and non-transplanted eyes to the same optic tectum gives rise stripes.

Lecture 3 Techniques
What can these be used for?

In vitro stripe assay
Creating a stripe assay involves affixing various substrates of interest into thin (~50 micrometers width) stripes onto a tissue-culture dish (thus, "in vitro"). One can then apply another substance to the culture dish and observe the effects of combination of both substances on the dish. For instance, one might wish to understand the molecular differences between anterior and posterior tectum to explain retinal axon patterning (this was done by Walter et al. in 1987, pg 13 of lecture 3 notes). To do this using the stripe assay, one would extract the membranes from anterior or posterior tectum and place them in alternating stripes, using flourescent labels to distinguish the two types of tissue. Then, temporal or nasal axons are allowed to grow on the stripes. Observing the results of such a test reveals that temporal retinal axons do indeed recognize the position-specific properties of the tectal cell membranes, because the temporal axons are attracted by the anterior membranes and repelled by the posterior tectal membranes. Thus, the in vitro stripe assay is a useful tool for understanding in vivo processes.

2D gel electrophoresis
A 2D gel electrophoresis is a process whereby proteins may be compared visually. The "gel" refers to a matrix of a specifically chosen polymer used to separate the molecules of analysis. "Electrophoresis" is the term that describes the electro-motive force that is used to push the molecules along the gel matrix. Molecules are applied to wells at one end of the matrix, and an electric current is applied, causing the molecules to move in a certain direction (depending on their electric charge, towards the anode if negative and towards the cathode if positive. Visualization of the progress of the molecules is made possible by dyes. The example in lecture three comes from Drescher et al. (1995): the gel electrophoresis is used to comopare proteins from anterior and posterior tectal membrane (thus, "2D"). The ligand Ephrin for the Eph receptor tyrosine kinase was found to be present in posterior, but not anterior tectal membrane. The Ephrin mRNA was revealed to be expressed in a gradient from posterior to anterior tectum.

A two-dimensional electrophoresis combines isoelectric focusing with SDS-PAGE, thus separating proteins with very high resolution. First, a sample of protein mixture is “isoelectrically focused”. When a mixture of small multicharged polymers (having many pI values) called polyampholytes undergo electrophoresis in a single-gel lane, they create a pH gradient in the gel. Thus, when the protein sample is subjected to electrophoresis in a gel with such a pH gradient, the proteins will each move to a position in the gel at which pH=pI, where pI is the isoelectric point of the protein, where its net charge and electrophoretic mobility are zero. This gel is then placed horizontally on an SDS-polyacrylamide slab. When the proteins are again subjected to electrophoresis (now in a vertical, rather than horizontal, direction, they will be now be separated by mass, creating a 2-D separation. (Berg, Tymoczko, Stryer, Biochem 2001, 5th Ed.) If the protein molecules are negatively charged, the smaller molecules will travel closer to the positive electrode end of the gel matrix, and the larger molecules will remain relatively closer to negative end.

Transplantation
In humans, tranplanted organs are used to replace a failing or damaged organ with a working organ from a donor. In research, transplatation is useful for exploring interactions between individual organisms--for example, the unique responses of an organism's immune system or the three-eyed frog to study axonal competition during neuron growth. Several types of transplatations are done:

1) Allografts = transplanting organs or tissues from a genetically non-identical member within the same species; 2) Autografts = transplanting tissue from one area of one's body to another, usually with surplus tissue to replace damaged areas; 3) Xenografts = transplanting organs or tissues across species (example, pig's heart to human body); 4) Isografts = transplanting organs or tissues to a genetically identical member of the same species (such as a twin). This type of transplantation may overcome difficulties associated with organ rejection or triggering a recipient's immune system.

Radiolabel injection
Using radiolabeled injections, neurobiologists are able to observe cellular mechanisms and metabolisms in real-time, such as the influx and efflux of calcium within a cell. This technique is completed by making and attaching a radiolabeled tag to the compound of interest, then injecting this compound into the organism or cell system under study. Through neuroimaging techniques such as MRI, fMRI and PET, we are able to see the brain regions where certain chemicals are taken up and metabolized.

TTX
'''Tetrodotoxin. (Also: anhydrotetrodotoxin 4-epitetrodotoxin, tetrodonic acid)''' A toxin from the puffer fish that blocks voltage gated sodium channels. Although originally found in the puffer fish and a few other organisms, TTX is now known to be synthetized by certain bacteria such as Pseudoalteromonas tetraodonis, some species of Pseudomonas and Vibrio, as well as others. The toxin works by blocking action potentials being created by binding to the pores of the voltage gated sodium channels in the neuron cell membranes. Tetrodotoxin binds to what is known as site 1 of the voltage-gated sodium channel. Site 1 is located at the extracellular pore opening of the ion channel. The binding of any molecules to this site will temporarily disable the function of the ion channel. Saxitoxin and several of the conotoxins also bind the same site. In humans, two categories of sodium channels with respect to TTX have been found: the tetrodotoxin-sensitive voltage-gated sodium channel (TTX-s Na+ channel) and the tetrodotoxin-resistant voltage-gated sodium channel (TTX-r Na+ channel). Nerve cells contain many TTX-s Na+ channels and thus TTX is a valuable tool in inducing paralysis of neurons in culture.

TEA
Tetraethylammonium. A compound which selectively blocks voltage gated potassium channels. Unlike TTX, TEA is synthesized for the purpose of being used as a potassium channel blocker in neuropharmacological experiments. The K+ eflux is responsible for the trailing part of the action potential so stopping it has a definite effect on the shape of the action potential.

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
Knock-out mice are genetically engineered animals with one or more genes that are made inoperable through a gene knock-out. Knock-out animals are significant to research because they allow us to test and identify the function of an identified gene whose effect is partially or fully unknown. Knock-out techniques are usually performed in mice, which are genetically similar to humans; this procedure is also easier to perform in mice compared to rats, in which knock-outs have only been possible since 2003. A typical procedure for creating knock-out mice are as follows:

1) Isolate the gene to be knocked-out from a mice genome library. A similar DNA sequence to the gene of interest is synthesized, but is made with significant changes so that the gene is inoperable. 2) Isolate stem cells from a mouse morulla, which can be grown in vitro. 3) Combine the stems cells with the re-created DNA sequence. Some of the cells will be able to incorporate the new DNA into their genomic sequence. 4) Insert stem cells into mouse blastocyst cells, then implant into a mouse uterus to complete the pregnancy. 5) Newborn mice are chimeras, sometimes not fully knocked-out mice. These animals are then crossed with other chimeras to potentially produce an offspring that is a full knock-out transgenic mouse.

Monocular enucleation
Monocular enucleation is a useful experimental tool for analyzing the mechanisms of neural plasticity. The process involves depriving one eye of environmental input during an early postnatal period and then observing the structural changes that result in the brain. What results is an adaptive reaction in which the visual system compensates for the lost sensory capacity. Afferent neurons from the deprived eye are connecting to the lateral geniculate nucleus (LGN) and the superior colliculus (CS) are destroyed as a result of the degeneration of retino-geniculate and collicular synapses, which receive no stimulation from the deprived eye. Inserting a tracer into the LGN of ME mice reveals a thinning of the ocular dominance columns. This is because the retinogeniculate fibers coming from the remaining eye replace begin to replace these synapses. Ipsalateral representation of the remaining eye thus becomes extended in the left LGN and in the left visual area. This raises the interesting question of how the deprivation of input results in adaptive plastic change. This is the primary question that is experimentally explored with monoculear enucleated organisms. (Reviewed by Toldi et al 1996)