BIO254:Toolbox OLD

Lecture 1 Model Systems

What are the advantages of each?

Mammalian visual system

The Mammalian visual system, of which the human visual system is an important member, is among the most complex in the animal world. The mammalian visual system is composed of several components, listed in the order which they act: the eye itself (consisting most importantly of the lens and the retina), the optic nerve, the optic chiasm, the left and right optic tracts, the lateral geninculate nucleus, the optic radiation and lastly the visual cortex itself. The light focused by the lens falls on photoreceptors in the retina, which, in humans, are bound to two different proteins called opsins: rodopsins (in rods) and cone opsins (in cones). The human, and some primates', visual system is different from most other mammals in that we have one extra type of cone opsin that allows us to distinguish colour. Most mammals are colourblind; colour evolved fairly recently in a sub-branch of the primate evolutionary tree. Despite this, the mammalian visual system (i.e. that of mammals other than the higher primates) is a powerful model for study as most of its components are analogous to those in humans. For further information on the specific components of the visual system, see lecture 8 below.

Mammalian olfactory system

Mammalian sense of smell depends on chemoreceptors. The olfactory sensors are sensory neurons embedded in a layer of epithelial tissue at the top of the nasal cavity. These neurons directly project their axons to the olfactory bulb of the brain (i.e. they are not relayed through the thalamus like other senses' axons). Their dendrites end in olfactory hairs on the surface of the nasal epithelium. Each functional olfactory receptor protein that is expressed is found in a limited number of sensory cells in the olfactory epithelium. All of the cells that express the same receptor protein project to the same regions in the olfactory bulb. A given odorant molecule may bind to one or to more than one receptor protein. Therefore each odorant molecule can excite a unique combination of cells in the olfactory bulb, so an olfactory system with even hundred of different receptor proteins can discriminate a large number of cells. Interestingly, the more odorant molecule that bind to receptors, the more action potentials are generated, and the greater the intensity of the perceived smell.

Spinal cord motor neurons

The spinal cord may be divided up into sections based on the nerve roots that extend from it. At each segment, rootlets come out of both the dorsal and ventral halves of the spinal cord. In fact, nerves go in through the dorsal side and then exit through the ventral face. The dorsal horns respond to sensory perception by receiving axons from the periphery via the dorsal root. The ventral horns contain motor neurons with axons that leave the cord via the ventral roots and travel to stimulate the muscles. The dorsal root ganglion contains a collection of cell bodies that have all the receptor neurons sending processes to the peripheral muscles. These are the motor neurons located in the spinal cord. Lower motor neurons are either alpha or gamma cells. Alpha cells comprise the principle motor neurons of the spinal cord and are a part of the main portion of the common reflex pathway. These neurons conduct rapid motor impulses; each alpha cell innervates about 200 muscle fibers. Gamma neurons, also part of the common pathway, are only half as numerous as alpha cells. They conduct slower motor impulses and their major function is to stretch muscle spindles.

Human brain

AS the most anterior part of the central nervous system, the human brain acts as the primary control center for the peripheral nervous system. Autonomic functions of the brain include controlling heartbeat, digestion, respiration, sensation, and movement. Higher order functions of the human brain include conscious activities like thinking and reasoning. The human brain is unique from other mammalian and vertebrate brains because it contains millions of billions of synaptic connectins, resulting in a complex and dense neural circuit. Neuroscience is the study of the brain and its functions; psychology is the study of the mind and behavior; and neurophysiology is the study of normal healthy brain activity. Advantages of using the human brain system for study is that results obtained will be the closest model system for determining the causes and pathways of mental illness in humans. Neuroimaging allow scientists to study the brain not only in detail, but in real time (functional neuroimaging). The human brain is most often compared with a computer: individual neurons are analogous to microchips, and specific areas of the brain are said to resemble graphic cards. However, no clear sequential (one-to-one command) set of instructions are consistently observed in the brain, and the study of the human brain directly cannot be replaced with computer models with great accuracy.

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

A recent method developed for detecting transposition and may genetically label conjugative plasmids that do not result in an apparently identifiable phenotype. Genetic labeling results in the transformation of the host organism with a plasmid containing a heterologous DNA fragment. This DNA labels genetic areas of interest which may then be visualized. The method is also beginning to be studied in vivo, where mice can be orally inoculated with genetically-labelled probiotic bacteria plasmids to study products of the digestive tract.

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

Coupled with the patch clamp technique, electrical stimulation is a form of manual current that may be delivered to the neuron under investigation in order to study its electrophysiological properties in a controlled manner. Usually only a few milliAmps are applied to the neuron to evoke passive responses. Greater current is introduced to evoke action potentials. Through this manner, the firing threshold for various types of neurons may be quantitatively determined with great accuracy. Furthermore, recent experiments have used this method to purposefully stimulate neocortical neurons to study the effects of prolonged activity (or "stimulation") on axonal growth.

fMRI

Functional magnetic resonance imaging is a technique used to visualize not only the neural anatomical images created by traditional MRI scans, but also overlaid images of event-related hemodynamic responses in the brain. The hemodynamic activation levels refer to the amount of blood oxygenation ocurring at a particular "voxel" of the image, which is a kind of three-dimensional pixel. This hemodynamic response is often referred to as BOLD (blood-oxygen level dependent) contrast. High BOLD contrast reflects a decreased amount of deoxygenated hemoglobin present in the brain. General changes in BOLD signal are highly correlated with changes in blood flow to different regions of the brain. Images of both anatomical and functional (BOLD) data are recorded every few seconds. Data can be analyzed in such a way as to contrast the activations associated with two separate paradigms, effectively subtracting the activation of one dataset from another and presenting the difference visually. This technique is generally applied to psychophysical ventures, quantifying the results of a multitude of psychological questions.

Lecture 2 Model Systems

What are the advantages of each?

Frog visual system

Some of the most groundbreaking work in understanding vision in humans were completed using the visual system of frogs. The retina of frogs has a uniform distribution, while mammalian eyes--such as those of humans--contain areas of higher resolution, called the fovea. The frog, however, has no fovea and it only moves its eyes to compensate for its own motion (either intended or accidental). This makes it easier to compare the patterns of light reaching the frog's retina with the signals leaving along its optic nerve. In early experiments, scientists placed electrodes along the nerve fibers emanating from the eye. Then, they shone a light on to the retina and looked at the pattern of responses along the outgoing nerves. The first studies of the responses in the frog's visual system were conducted in the late 1930's by the physiologist Hartline. Hartline introduced the term "receptive field" to describe the region of the retina to which an individual output responded. The discover of ON, OFF, and ON-OFF cells were made in the frog visual system and allowed us to gain a stronger understanding of how visual systems sharpen the constrast of perceived images.

Vertebrate spinal cord

C. elegans sensory and motor neurons

Caenorhabditis elegans is a model system due to its short life cycle (about four days) and ease of maintenance. These nematodes are also transparent, which allow for visual assays (such as with fluorescent proteins). In the study of neurobiology, C. elegans is useful because it has a simple nervous system of 302 neurons. For each of these neurons, the morphology and the connectivity to other neurons is known through electron micrographs.

Most sensory neurons of C. elegans can be categorized into two groups. The first type consists of chemosensory neurons with channels that are exposed to the external environment. The second group consists of mechanosensory neurons that lack these channels to the environment.

The interneurons of C. elegans vary in their function and types of connections. Some receive synaptic input from only a few neurons, while others receive signals from many neurons.

C. elegans neurons have the interesting property in which the presynapses and postsynapses are not localized to only after or before the cell body, respectively. For example, the interneuron AVE has a postsynaptic region and presynaptic region which both follow after the cell body. Another unique property is that C. elegans neurons do not conduct action potentials.

Drosophila embryo

Embryonic development has been studied extensively in the Drosophila embryo, particularly the establishment of the dorsal-ventral and anterior-posterior axis, as well as segmentation in Drosophila. The body axes of the embryo are prefigured in the oocyte by maternal effect genes. These are prelocalized cytoplasmic determinants as well as localized extracellular signals (signals in the egg shell covering). Scientists have identified about 50 maternal mRNA types that build up localized determinants during oogenesis that are pre-determined before zygotic genome is turned on. More specifically, the dorsal-ventral axis is specified by an extracellular signal called spatzle. Molecules laid down in the ventral extracellular egg covering during oogenesis locally activate a ligand (spatzle). Spatzle quickly and locally binds its receptor, activating a signal transduction cascade that releases the transcription factor dorsal from a cytoplasmic inhibitor called cactus by degrading cactus. As a result, dorsal enters nuclei on the ventral side. dorsal protein is localized in the nucleus in a gradient on the ventral side of the blastoderm embryo. The gradient of activity of the dorsal transcription factor sets up several different domains of target gene expression. The gradient of activation of transcription factor (high ventral, low dorsal) and affinity of binding sites, determines how embryo pattern will develop based on signaling pathways. The action of the bicoid gene during oogenesis is required to set up conditions for development of anterior structures in the embryo. Bicoid mRNA, like dorsal, is a localized cytoplasmic determinant that is localized to the anterior pole of the oocyte during oogenesis (by motor proteins moving on microtubule tracks). After fertilization the mRNA is translated and bicoid protein diffuses out to form a gradient. It functions as a DNA binding protein that turns on transcription of the hunchback gene in the embryo. Hunchback, in turn, acts to pattern the embryo as a gap gene. The levels of bicoid protein sets the position of hunchback expression along the anterior-posterior axis since a threshold level of bicoid is required to turn on hunchback transcription. Hunchback transcription is blocked in the posterior region by the nanos protein. Nanos mRNA is localized posteriorly and similarly sends out a protein gradient that opposes the bicoid protein gradient as well as the maternal Hb mRNA that is distributed uniformly throughout the cell. The gap genes, which include hunchback, giant, kruppel, and knirps, are transcription factors for segmentation. There is a tapering of the proteins in each direction from where it is expressed to create gap gene domain expression overlap, creating combinations of more than one of their protein products. The peaks overall to foster complexity since across head to tail axis there are different amounts and different locations of transcription factors, and genes sensitive to these differ in triggering influences that lead to segmented expression of genes. Pair rule gene transcription is under gap protein control. There are different DNA control elements for different stripes. Combinations of transcription factors act on particular silencers/enhancers to control segmentation. The regulation of lateral segments require different combinations of transcription factors, such as Wnt and Hedgehog signals that organize the pattern of bristles within each Drosophila segment. Hedgehog and Wnt are both short range signals, but Hedgehod is a secreted protein and Wnt is a signaling pathway. Adjacent cells talking to each other for feedback to reinforce each other’s signals in positive feedback. If the Hedgehog signal fades then there’s no communication within the poles of the segments and Wnt causes bristles to form on all cells. Finally, homeotic genes (Hox genes) are single transcription factors that can affect where development occurs by conferring different fates upon repeating body segments, inducing limb growth, and organizing organ placement. This is based upon their select expression along the dorsal-ventral and anterior-posterior axis in accordance to combinatorial coding of the genes described above.

Cell culture

Small amounts of undifferentiated or single cells (normally from excised animal tissue) are placed in an artificial environment. The nutrient medium depends on the experiment being conducted, but usually the medium favors cellular growth and differentiation. By using cell cultures it is possible to pin down a cause and effect relationship between the carefully controlled culture and the development of the maturing cells. Cell cultures can be manipulated by adding chemicals, nutrients, etc. to the cellular environment to test a hypothesis or achieve desired characteristic results. Favorable qualities of cells can be precisely controlled, so that each cell is identical for the particular quality being sought, allowing for repetition within experimental methods. In the case of neuroscience, axon growth, protein secretion, receptor up/down-regulation, and neurotransmitter release can all be studied and manipulated within culture to test the effects of a wide variety of cellular environments.

Grasshopper

Xenopus axons in culture

Lecture 2 Techniques

What can these be used for?

Biochemistry

Genetics: mutation and over expression

A genetic mutation is a permanent change in the DNA sequence that makes up a gene. Mutations can affect a single DNA building block or even a large segment of an entire chromosome. Mutations may be induced in an egg or sperm cell or after fertilization; these changes are termed new (de novo) mutations, and may be experimentally beneficial for studying genetic diseases or for creating transgenic animal models that mimic aspects of human disease.

The protein encoded by a particular gene may be expressed in an increased quantity ("over-expression") such that the phenotype of the organism can be significantly altered. Two commonly used techniques to create gene over-expression are to either increase the number of the copies of the gene, or, to increase the binding strength of the promotor.

Co-culture on a 3D collagen gel matrix

Antibody Staining

Antibody staining, also known as immunostaining, is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. Now however, immunostaining encompasses a broad range of techniques used in histology, cell biology, and molecular biology that utilise antibody-based staining methods.

Cloning genes and expressing them in cell culture

Forward genetic screen

Genetic screens test and identify organisms with a specific phenotype. A forward genetic screen searches for new genes or mutant alleles, which rarely occur in nature. Hence, scientists perform a forward genetic screen by exposing the individual to a mutagen in order to induce mutations in their chromosome(s). Mutagens such as random DNA insertions by transformation or active transposons can also be used to generate new mutants.

The Poo Assay

The Poo Assay is used to assess growth cone turning responses to gradients of extracellular guidance factors. It is named after its originator, Mu-Ming Poo, who used it to demonstrate the attractive turning of a growth cone towards a gradient of netrin-1 and the repulsive turning of a growth cone away from a gradient of semaphorin 3A. Isolated growth cones are cultured in a cell-free environment in vitro and then are exposed to gradients of a potential signaling molecule. Within an hour turning of the growth cone is evident and the angle of turning can be used to gauge the strength of the molecule’s signal. Turning should not be observed when the culture medium is supplemented with an antibody against the signaling molecule of interest.

Explant overlay assay

The explant overlay assay, known more commonly as the slice overlay assay, is an in vitro assay in which neuronal explants are cultured over cortical slices. The principal use of the explant overlay assay is to characterize extracellular signaling molecules that regulate neuronal differentiation and patterning. The two methods used for this purpose before the innovation of the explant overlay assay had significant shortcomings. An in vitro assay using neuronal explants cultured on an artificial substrate was problematic because the substrate was no substitute for the actual in vivo environment in which neuronal outgrowth takes place. The limitation of the second method, an in vivo assay that involved transplanting and monitoring labeled neurons, was that the chemical environment could not be manipulated like in an in vitro assay. The explant overlay assay is able to resolve both problems, making it the most effective method for studying neuronal guidance molecules and mechanisms. Franck Polleux developed the explant overlay assay in 1998 to show that the initial growth of cortical axons toward the white matter is regulated by a semaphorin signal that is expressed in the marginal zone.

Incubating slices in media with chemical cues

Mammalian pyramidal neurons

Pyramidal cells are the primary projection neurons in the cerebral cortex and the hippocampus of the central nervous system (CNS, brain). Pyramidal cells have a pyramid-shaped cell a long and branching dendritic tree. An axon that carries nerve impulses emerges from one end of the cell. The axon may have local collateral branches but also project outside their region. These cells are multipolar neurons with a single apical dendrite and compose up to 80% of the neurons in the mammalian cortex. Pyramidal cells are excitatory neurons and release glutamate as their neurotransmitter.

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" [1])

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.

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

Paper 1 Model Systems

What are the advantages of each?

Chick optic tectum

Mouse superior colliculus

Mouse retina

Paper 1 Techniques

What can these be used for?

HEK293 cells

HEK 293 cells are an epithelial cell line originally derived from embryonic human kidney. As an experimentally transformed cell line, HEK cells are not a particularly good model for normal cells, cancer cells, or any other kind of cell that is a fundamental object of research. However, they are extremely easy to work with, being straightforward to culture and to transfect, and so can be used in experiments in which the behavior of the cell itself is not of interest. Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analyzing the expressed protein; essentially, the cell is used simply as a test tube with a membrane.

Examples of such experiments include:

• A study of the effects of a drug on sodium channels
• Testing of an inducible RNA interference system
• Testing of an isoform-selective protein kinase C agonist
• Investigation of the interaction between two proteins
• Analysis of a nuclear export signal in a protein

In the Schmitt et al (2006), HEK 293 cells were used in a preliminary test to determine whether Wnt3 can regulate the growth of RGC axons. Schmitt el al created HEK293 cells transfected with the wnt3 gene in order to have the Wnt3 protein expressed in membrane fractions of HEK293 cells (Wnt3 is highly hydrophobic and associates tightly with cell membranes). They found that Wnt3-transfected HEK293 cell membranes inhibited the growth of both dorsal and ventral mouse RGC axons at higher concentrations, and stimulated the growth of dorsal but not ventral RGC axons at lower concentrations (data was not shown).

SF9 cells

An insect cell line used for the production of recombinant protein. The Sf9 cell line is derived from pupal ovarian tissue of the Fall armyworm Spodoptera frugiperda. The Sf9 cell line is highly susceptible to infection with Autographa california nuclear polyhedrosis virus (AcNPV baculovirus), and can be used with all baculovirus expression vectors. Sf9 cells are commonly used to isolate and propagate recombinant baculoviral stocks and to produce recombinant proteins. In the Schmitt et al. paper, Sf9 cells were used to overexpress Wnt3 (using the Baculovirus system) to obtain sufficient and consistent amounts of Wnt3.

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

A control used in infection experiments. Two specimens are used: one that is infected with the virus or vector of interest and the other is treated the same way except with the virus. Sometimes, a non-virulent strain is used in the mock-in the mock-infection experiments.

Blocking with antibodies or proteins

Western Blot, α-tubulin

Retina explant assay

Electroporation into ventricular zone

Dominant-negative

A dominant-negative is a mutation whose gene product adversely affects the normal, wild-type gene product within the same cell. This usually occurs if the product can still interact with the same elements as the wild-type product, but block some aspect of its function.

Examples: 1. A mutation in a transcription factor that removes the activation domain, but still contains the DNA binding domain. This product can then block the wild-type transcription factor from binding the DNA site leading to reduced levels of gene activation. 2. A protein that is functional as a dimer. A mutation that removes the functional domain, but retains the dimerization domain would cause a dominate negative phenotype, because some fraction of protein dimers would be missing one of the functional domains.

The Shmitt et al Ryk dominant negative: Wnt3 knockout mice fail in early embryonic patterning because Wnt3 is important for early nervous system development. This makes it impossible to examine the function of the Wnt3 gradient on the medial-lateral axis of the mouse superior colliculus because knockout Wnt3 mice die at birth and axon termination zones form at postnatal day 8. To circumvent this difficulty, Schmitt et al generated a dominant-negative form of Ryk. This truncated Ryk protein only contained Ryk ectodomain (extracellular) and the transmembrane domain, missing the intracellular domain. This dominant negative Ryk allowed Schmitt et al to test in vivo whether blocking Wnt3-Ryk function will shift the termination zone of RGX neurons in the superior colliculus of mice medially.

In ovo electroporation

DAPI staining

Fluorescently labels cell nuclei by binding to DNA.

AP (alkaline phosphatase)

Tagged proteins

Protein overexpression

Selective gene amplification by a cell results in more templates for transcription, which is the basis of natural protein overexpression. The cell can make more of a certain gene product by increasing the number of copies of the appropriate gene and transcribing them all. This strategy takes advantage of the transcription mechanisms already in place within the cell and merely feeds them more material to transcribe. In the lab, the polymerase chain reaction (PCR) technique makes multiple copies of a DNA sequence by copying a short region of DNA many times in a test tube. It is a cyclic process in which a sequence of steps is repeated over and over: A DNA molecule with a target sequence to be copied is heated to denature it. When the mixture cools, short, artificially synthesized primers bond to the single-stranded DNA. Then dNTPs (four deoxyribonucleotide triphosphates dATP, dGTP, dCTP, and dTTP) and DNA polymerase are added to synthesize two new strands of DNA. One goal of recombinant DNA technology is to produce many copies (clones) of a particular gene either for the purposes of analysis or to produce its protein product in quantity. Scientists normally use bacteria as hosts because they are easily grown and manipulated. Bacteria also contain plasmids, small circular chromosomes, which can be manipulated to carry recombinant DNA into the cell. But bacterium is not ideal for studying and expressing eukaryotic genes because they lack the splicing machinery to excise introns from the initial RNA transcript of eukaryotic genes. Many eukaryotic proteins are extensively modified after translation, so scientists use vectors rather than bacteria to carry the new DNA into host cells. Vectors already have a built-in origin of replication. The new DNA has to become part of a segment of DNA that contains an origin of replication (i.e. join a replication unit) in order to be replicated in the host cell as it divides. Plasmids are often used as vectors because they are small, naturally occurring in bacteria, often have only a single recognition site for a given restriction enzyme, and allows for the insertion of DNA at only one location. When the plasmid is cut with a restriction enzyme, it is transformed into a linear molecule with sticky ends that can pair with the sticky ends of another DNA fragment cut with the same restriction enzyme. Viruses can also be used as vectors to insert large numbers of base pairs into a genome. Even if the genes that cause the host cell to die and lyse are gone, the virus can still attach to a host cell and inject its DNA, which in our case is the new DNA to be expressed. Finally, expression vectors allow foreign genes to be expressed in host cells and can turns cells into protein factories, contributing to protein overexpression. Expression vectors contain the sequences for promotion, termination, and ribosome binding, which are necessary for protein synthesis in a foreign cell. The protein to be overexpressed is inserted at the restriction site, the bacteria of choice is transfected with the expression vector, and the protein is synthesized because of its locale in the DNA. Inducible promoters that respond to a specific signal, thus initiating protein synthesis, can be inserted into the expression vector so that the production of the target protein can be controlled.

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.

Lecture 4

Bungarotoxin

Toxin harvested from the snake species Bungarus multicinctus that binds Acetylcholine receptors and therefore paralyzes its prey. Alpha bungarotoxin is used as a label for Acetylcholine receptors.

Agrin

A proteoglycan made by nerve and glia. Agrin is transported to the nerve terminal and synaptic cleft. Due to the phenotype of agrin knockout mice (dispersed acetylcholine receptors), agrin was believed to be the factor which organizes the aggregation of acetylcholine receptors into clusters. Later experiments in model systems in which agrin could not have been present due to the absence of the pre synaptic nerve (Homeobox 9 or HB9 knockouts) showed that Agrin was not necessary for clustering. It has since been elucidated that agrin stops the dispersion of acetylcholine receptors. Dispersion of acetylcholine receptors is caused by the receptor's own ligand, the neurotransmitter acetylcholine.

Proteoglycan

A class of glycoproteins which contain glycosaminoglycan chains

Glycan

The polysaccharides which form the carbohydrate moiety of glycoproteins.

MuSk

A receptor tyrosine kinase found in muscle necessary for aggregation of Acetylcholine receptors into clusters. MuSK co-localizes with Acetylcholine receptors. Its expression peaks during the formation of neuro-muscular junctions.

Rapsyn

A cytosolic protein necessary for proper Acetylcholine aggregation. During early stages of muscle development Rapsyn co-localizes with acetylcholine receptors.

ChAT

Choline Acetyl transferase. The enzyme responsible for the synthesis of Acetylcholine Acetyl-Coenzyme A and Choline.

Neuregulin

A protein which is a known ligand for the erbB type receptor tyrosine kinase.

Lecture 4 Model Systems

Neuromuscular junction

Electric ray

Zebrafish

Squid giant axon

Genetic mosaic animals

Lecture 4 Techniques

α-bungarotoxin

Protein extracts

anti-NF

YFP

CFP

Radiolabeled amino acids

Radiolabeled amino acids are made by replacing a carbon atom with 11C in a physiologic amino acid. This does not chemically change the molecule, but allows for detection through positive electron tomography. Or radiolabeled amino acids can be imaged after being infused into cells and incorporated into synthesized proteins while in culture or in vivo. Tracking where the amino acids travel and their activities serves to garner information about cellular function and can aid in imaging structures. Methionine is the most popular amino acid for PET when made into L-[methyl-11C]-methionine (MET), and it is extremely effective at diagnosing brain tumors.

Time lapse

GFP fusion protein

Double knock-out

EP screen

Lethal enhancer screen

Lecture 5

Enhancer Promoter Screen

A screen for over expression mutant phenotypes. The genotype is created through random insertion of a strong promoter into the genome.

Lethal Enhancer Screen

A screen for a second mutation that enhances a (lethal) phenotype of another mutation which by itself is not lethal.

Lecture 5 Model Systems

Electric eel

Lecture 5 Techniques

Aldicarb

Aldicarb (chemical name: 2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime) is an carbamate-class insecticide applied directly to the soil and is used to control mites, nematodes, and aphids. In the laboratory, aldicarb acts as an inhibitor of acetylcholinesterase (AChE), the enzyme present in the basal lamina of the post-synaptic cell of the neuromuscular junction (NMJ) which breaks down the neurotransmitter acetylcholine (ACh). ACh is the excitatory neurotransmitter for muscular contraction; therefore, inhibition of AChE using aldicarb results in prolonged activation of ACh receptors in the post-synaptic cell, causing paralysis and eventually death.

Aldicarb is a useful tool in genetic screens that search for mutants resistant or hypersensitive to the aldicarb-induced paralysis. These mutants will likely have mutated genes involved in the ACh signaling pathway at the NMJ. Mutants resistant to aldicarb will likely have an impairment of normal ACh signaling, while mutants hypersensitive to aldicarb will likely have an exaggeration of normal ACh signaling.

PCR

PCR, or the polymerase chain reaction, is an experimental technique devised by Kary Mullis in 1984 and is used to amplify a targeted DNA segment for further experimental analysis. PCR does not require knowledge of the target DNA sequence; however, knowledge of the DNA sequences flanking the target is necessary.

A PCR cycle proceeds as follows: 1) The parent DNA duplex is separated by heating the solution to 95°C for 15 seconds, exposing the bases on each strand. 2) Primers for the flanking DNA sequences anneal to the 3'-end of each parent DNA strand when the solution is cooled to 54°C. 3) A heat-stable DNA polymerase called Taq DNA polymerase (derived from the thermophilic bacterium Thermus aquaticus) synthesizes complementary DNA strands starting at the primers and using available nucleotides in solution when the solution is heated to 72°C. Repeated cycles of PCR allow for an exponential amplification of the target DNA sequence.

E. coli

Ion replacement

Low transmitter release conditions

Caged calcium

Lecture 6

Shibire

The shibire mutation causes temperature-sensitive paralysis in adult Drosophila. The shibire gene encodes a homologue of rat dynamin protein. Dynamin is a GTPase that localizes to the neck of budding vesicles and is involved in vesicle scission from the parent membrane. When shibire mutants are exposed to temperatures exceeding a restrictive temperature, the function of the shibire protein is disrupted and endocytic vesicles can no longer be separated from parent membranes. As a result, endocytosis is blocked, membrane cycling is prevented and synaptic vesicles are depleted, especially in synaptic terminals. Because synaptic vesicles are depleted, when shibire mutants are exposed to temperatures exceeding a restrictive temperature they become paralyzed. The shibire protein can regain its function and the paralyzed phenotype can be reversed upon lowering to a permissive temperature.

Electron Microscopy

MK801

MK801 is an experimental drug that, like PCP, blocks the flow of calcium ions through the NMDA receptor channel. It binds to a site within the open channel pore and thus is considered a non-competitive antagonist of the NMDA receptor. It binds with a high affinity and its radiolabeled form has been used to label NMDA receptor populations in brain slices. Blockage of the NMDA receptor results in hallucinations similar to those seen in schizophrenia, leading to the hypothesis that schizophrenia may be the result of a defect in NMDA receptor function. MK801 has been studied as a potential treatment for diseases that are the result of excitotoxic neurodegeneration such as stroke and Alzheimer’s.

APV (AP-5)

Hippocampus to study plasticity mechanisms

Lecture 6 Model Systems

Xenopus oocytes

Mammalian hippocampus

Lecture 6 Techniques

Sequence comparison

Hydrophobicity plot

Hydrophobicity plots are used to determine the relative polarities of amino acids found in a protein sequence. The main use of these plots is to predict transmembrane regions of proteins, which are usually characterized by long sequences of hydrophobic residues. In order to generate a hydrophobic plot, each amino acid in a sequence is scored based on one of two scales: the Kyte-Doolittle scale and the Hopp-Woods scale. In the Kyte-Doolittle scale, highly hydrophobic regions achieve large positive values, and this scale is used predominantly to pick out potential transmembrane regions of the protein. The Hopp-Woods scale was developed to predict potential globular protein binding sites, which are usually characterized by many polar residues. This scale can be views as a hydrophilic index, where the Kyte-Doolittle represents a hydrophobic index.

Tetanic stimulation

Tetanic stimulation is a technique used in neurobiology to induce long term potentiation in post-synaptic neurons. It is performed by applying a sequence of high-frequency stimulations to artificially induce rapid EPSP temporal summative effects, mimicking increased neurotransmitter release and binding by postsynaptic receptors (i.e. large amounts of glutamate binding to AMPA receptors on the postsynaptic membrane). This results in greater postsynaptic depolarization if each successive pulse of tetanic stimulus reaches the postsynaptic cell before the previous EPSP can appreciably decay. The progressive and prolonged depolarization removes the magnesium blockage of the NMDA receptor, and subsequent stimuli promote an extremely rapid calcium influx through the NMDA receptor-coupled ion channel. Rapidly dramatically raising calcium’s intracellular concentration triggers a sequence of events ending in enhanced excitability to future stimuli. The calcium made available by the NMDA channel binds to chelator calmodulin and among other things activates the calcium/calmodulin dependent CaMKII. CaMKII increases the excitability of existing AMPA receptors and voltage-gated potassium channels by phosphorylating them and also initiates the MAP kinase cascade which adds new AMPA receptors to the postsynaptic membrane. The incoming calcium also binds adenyl-cyclase, indirectly raising the level of cAMP in the neuron. cAMP turns on PKA, an important protein kinase that phosphorylates voltage-dependent potassium channels and also calcium channels thus lowering their threshold for opening in response to future stimuli.

Protein synthesis blockers

Lecture 7

adenylate cyclase

An enzyme that takes AMP as substrate and synthesizes cyclic AMP (cAMP). cAMP is secondary messenger in many signal transduction cascades. There are nine different isoforms of adenylate cyclase that respond differently to the various kinases, elevated calcium levels, and to G protein subunits other than the stimulatory alpha subunit (by which all adenylate cylcases are activated.)

cAMP Phosphodiestrase

An enzyme that takes the second messenger cyclic AMP (cAMP) as a substrate and hydrolyses the phosphodiester bond, creating AMP. Important enzyme in shutting off signal transduction cascades.

Isoproternol

An agonist of the beta andrenergic receptor, a receptor for the catelchomines epinepherine (adrenaline), and norepinepherine (noradrenaline).

Beta andrenergic receptor

A receptor for the catelchomines epinepherine (adrenaline), and norepinepherine (noradrenaline). It is one type of G-protein coupled receptor or GPCR, in which ligand receptor binding activates a G protein which in turn activates an adenylate cyclase.

G-proteins

G protein is a heterotrimeric protein that exists in two forms, a GTP bound form, and a GDP bond form, and acts as an on/off switch in signal transduction cascades. The trimer is activated when it binds GTP. When activated the trimer separates into two subunits, one being the alpha subunit, the other being a heterodimer of the beta and gamma subunits. The alpha subunit activates its target adenylate cyclase, causing synthesis of the cAMP second messenger. Recent research suggests that the beta/gamma subunit also has downstream targets. G protein to GTP binding is mediated by an enzyme called the Guanine nucleotide Exchange Factor or GEF which exchanges GDP for GTP in the G-protein binding site. The alpha subunit has intrinsic GTPase action, and therefore can turn the entire trimer into the inactivated GDP-bound form, however hydrolysis of GTP to GDP by the alpha subunit is accelerated by the enzyme GAP or GTPase activating protein. When in the inactivated GDP bound form, G protein reforms the heterotrimer.

Sodium Fluoride and G protein activation

Sodium fluoride and aluminum fluoride are G protein activators.

Narcoleptic dogs

Narcolepsy is a primary sleep disorder, whose prominent symptom is excessive sleepiness. It was first identified by Jean-Babtiste in 1880. In the 1950s the narcoleptic syndrome was defines as consisting of four symptoms: (1) daytime sleepiness, (2) cataplexy,thbe reversible loss of muscle tone (3) sleep paralysis, and (4) hypnagogic hallucinations. After the discovery of REM sleep, it was discovered that patients with narcolepsy begin sleep with REM sleep, whereas normal sleep begins with non-REM sleep. Narcolepsy has been associated with a class II antigen of the major histocompatibility complex on chromosome 6 at the HLA-DR2 or HLA-DQW1 locus. HLA-DR2 is also associated with autoimmune diseases such as multiple sclerosis and rheumatoid arthiritis, raising the possibility that narcolepsyt has an immunological basis. Some dogs are narcoleptic, and their narcolepsies are similar in most respects to human narcolepsy, except for the mode of genetic transmission. In narcoleptic dogs, abnormalities have been found in cholinergic and monoaminergic synaptic transmission, important components of REM sleep regulation. Dogs with narcolepsy have more muscaniric M2 receptors in the pons, suggesting a defect in cholinergic sensitivity. Consistent with this, cholinergic antagonists inhibit and agonists exacerbate canine cataplexy. Norepinephrine function also seems abnormal in that the number of α-2 receptors in the locus ceruleus is larger than normal. Moreover, the density of dopamine D2 receptors is greater both in dogs and in humans with narcolepsy. Some of the selective serotonin reuptake inhibitors reduce cataplexy in dogs and humans, implicating serotonergic systems at least in cataplexy. A group of researchers at Stanford University led by Emmanuel Mignot, MD, PhD associate professor of psychiatry at Stanford University School of Medicine, used a technique called positional cloning to pinpoint the “narcolepsy gene” in dogs. In the August 6 issue of Cell (*Mignot, E., et al. "The Sleep Disorder Canine Narcolepsy Is Caused by a Mutation in the Hypocretin (Orexin) Receptor 2 Gene." Cell, August 6, 1999.), Mignot and his colleagues report locating two defective versions of the narcolepsy gene, one in Doberman pinschers, the other in Labrador retrievers. The gene, known as hypocretin receptor 2, codes for a protein that juts out from the surface of brain cells and that functions as an antenna, allowing the cell to receive messages - transmitted via small molecules called hypocretins - from other cells. The defective versions of the gene encode proteins that cannot recognize these messages, in effect cutting the cell off from essential directives, including perhaps messages that promote wakefulness. Mignot predicts the finding will not only help the roughly 135,000 Americans who suffer from narcolepsy, but in time it will shed light on two of the biggest mysteries in sleep research: how and why we sleep.

Fluorescent Proteins (e.g. GFP)

Green fluorescent protein, isolated from the jellyfish Aequorea victoria, fluoresces upon exposure to blue light. The structure was solved by James Remington and colleagues - the fluorescent group is contained within the B-barrel. Fluorescent proteins are available in many colors and are most often used as reporters of protein expression to better understand cellular signaling.

FRET Imaging

Fluorescence Resonance Energy Transfer (FRET) is the radiationless transfer of energy between two fluorescent proteins. The fluorescent donor is excited at a specific wavelength. This energy can then be transferred to the fluorescent acceptor through a dipole-dipole coupling mechanism. FRET imaging can be used to determine protein-protein interactions, protein-DNA interactions, and protein conformational changes.

For example, one part of a protein is tagged with CFP and another part is tagged with YFP. When the protein is in a certain conformation in which the two fluorophores are far apart, the CFP will be excited but will not transfer its energy to the YFP. The assay would result in the visualization of the CFP wavelength. If a conformational change takes place and allows the CFP and YFP to come close together, the energy transfer will take place. In this case, the assay would result in the visualization of YFP wavelength.

One limitation of FRET is the inherent background noise that results from the direct excitation of the acceptor fluorescent protein. To avoid this problem Bioluminescence Resonance Energy Transfer (BRET) is used in which a bioluminescent luciferase is used instead of CFP as the fluorescent donor. Another solution to determine protein-protein interactions is BiFC. This technique attaches one half of the YFP molecule to one protein and the other half to another protein. When the two halves come together, the complete YFP is now functional.

CaM Kinase II

CaM Kinase II is a calmodulin-dependent protein kinase that shows history-dependent activity, remembering previous calcium pulses through autophosphorylation. CaM kinase II forms a complex with 12 subunits, arranged in two hexamer rings. It is activated by calcium-bound calmodulin, which relieves the autoinhibitory interaction between CaM kinase subunits. Once activated, CaM kinase II proceeds to phosphorylate itself and remains partially active even after the lowering of calcium levels, thereby prolonging the duration of its kinase activity. Consequently, activated CaM kinase II responds non-linearly to calcium oscillations, as (in the absence of calcium) its activity falls more slowly the more it is phosphorylated.

See BIO254:CaMKII

Lecture 7 - No New Model Systems!

Lecture 7 Techniques

Calcium channel inhibitors

Calcium imaging

Lecture 8

Eye Anatomy

The eye is a light-sensitive organ through which visual information about the external world is transmitted to the brain. The pupil, a black, circular opening centered in the front of the eyeball, controls the amount of light entering the eye, widening when surroundings are dark and constricting when they are bright. The lens of the eye focuses light from the external world onto the retina, a thin, multilayered region composed of photoreceptors (rods and cones) and interneurons that lines the back of the eyeball. The fovea, located in the retina, is the focus point of the lens and contains the highest density of photoreceptors (only cones), making it responsible for high-acuity vision. Retinal axons leave the eye through the optic disc, an area also known as the “blind spot” for its lack of photoreceptors, and merge into the optic nerve, which transmits visual information to the brain.

Isolated Retinal Rod Cell

Phototransduction

See BIO254:Phototransduction

Adaptation

See BIO254:Adaptation

Lecture 8 Model Systems

Vertebrate eye

Isolated retinal rod cell

Lecture 8 Techniques

Exciting specific areas of the retina

Shearing rod cells

Sucrose density gradient

Lecture 9 Model Systems

Cat retina

Lecture 9 Techniques

Multielectrode array

Lecture 10 Model Systems

Toads

The toad’s stereotyped prey capture response was studied in order to better understand how releasers, features of a stimulus that activate a fixed action pattern, are detected. For the toad, the fixed action pattern was orienting its head towards potential prey and the releaser was a cardboard cutout that resembled a worm. Three different types of stimuli were placed in front of the frog and moved in a horizontal plane. The toad’s orienting response for each stimulus was measured. When a rectangular cardboard stimulus was moved across the toad’s visual field in the direction of its long axis, the so-called “worm” configuration, a strong orienting response was elicited. When a rectangular cardboard stimulus was moved across the toad’s visual field in the direction of its short axis, the so-called “anti-worm” configuration, no response was elicited. When the stimulus was a square piece of cardboard, the toad moved toward it if the square was small and moved away from it if the square was large, the shift in orientation taking place at the point where the square stopped being viewed as prey by the toad and started being viewed as a predator. A type of tectal neuron called TH5(2) which demonstrated frequent impulses in response to the worm configuration, infrequent impulses in response to the anti-worm configuration, and impulses of decreasing frequency in response to square stimuli of increasing size is a strong candidate for the feature detector.

Lecture 10 Techniques

Infrared camera

High speed monitor for stimulation

Gal4 inhibition of neurotransmission

The Gal4/UAS system is used for targeted gene expression in Drosophila as well as other model organisms. Gal4 is a transcription factor that does not activate native Drosophila genes but activates the expression of genes that are under the control of UAS. Gal4 can be used neurobiologically to selectively inhibit the neurotransmission of a subset of neurons. To do this, a Gal4-encoding gene is placed between some gene, present in a certain subset of neurons, and the promoter for this gene. In this way, whenever the gene is expressed, Gal4 is expressed. Next, UAS is placed next to a transgene that is incorporated into the neuronal DNA and that codes for the shibire protein. Gal4 will bind to UAS, activating the expression of the transgene’s shibire protein product. When exposed to temperatures higher than a permissive temperature, the shibire protein is disrupted and results in the blockage of synaptic signaling. Thus by manipulating the temperature a subset of neurons can be turned on and off.

Paper 3 Model Systems

Drosophila eye

Paper 3 Techniques

FLP/FRT system

MARCM system

GAL4-UAS system

The GAL4-UAS system allows for targeted gene expression in vertebrates using the transcription factor GAL4 and the gene of interest attached to a promoter specific to GAL4, the Upstream Activating Sequence (UAS). The system was originally discovered in the yeast Saccharomyces cerevisiae.

The system works by creating two transgenic strains of the organism and crossing them to yield progeny with both transgenes. In one strain, an activator line is introduced by inserting the gene for GAL4 near a specific promoter with a known expression pattern (e.g., a promoter used in the development of Drosophila melanogaster legs), while, in the other strain, an effector line is created by fusing the UAS upstream of the gene of interest. When the two lines are crossed, the progeny with both transgenes will express the gene of interest according to the expression profile of the promoter near which the GAL4 gene was placed. It is by this control over the insertion of the GAL4 gene that targeted expression (e.g., ectopic expression or expression of mutated genes) can be achieved.

Heat shock

Confocal imaging

Frozen sectioning

Water immersion microscopy

Plastic sectioning

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16 April 2024

			10:20		Yarn Microfluidics - Roger Dirth‎‎ 12 changes history +442‎ [Rcostello‎ (12×)]
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08:11

3D Printing Overview‎ diffhist +422‎ Michele Caggioni talk contribs

15 April 2024

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			22:11		The paper that launched microfluidics - Xi Ning‎‎ 14 changes history +9,705‎ [Xning098‎ (14×)]
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			18:16		Multilayer Paper Microfluidics - Madyson Redder‎‎ 15 changes history +4,948‎ [Mredder‎ (15×)]
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