Bio154JM08/Toolbox/Lecture 6
This page is part of the BIO154/254 Experimental Toolbox!
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
Electron microscopy, invented in 1931, is a form of microscopy that uses electrons (as opposed to photons in a classical optical microscope) to create a visualization of very small objects. Before the invention of electron microscopy, microscopes were limited by the wavelength of light, which meant that the minimum distance objects could be from each other before the microscope blurred them together and was incapable of distinguishing them was too high for many applications of microscopy. Electrons have a much smaller wavelength than light and thus microscopy based on them is capable of distinguishing much more detail. Advantages to electron microscopy include this increased resolution (0.5nm vs around 200nm for light microscopes) and much higher maginifications (250,000x vs 1500x). All this comes at a price, however: electron microscopes are expensive both to build and maintain. Furthermore, qualities of electrons mean that samples can only be observed in a vacuum, meaning that they have to be specially prepared. A critical consequence of this is that all samples will be killed in the process, and artifacts might be created. Contrast this with light microscopy, which allows viewing of even moving samples. As electrons have no color, electron microscopy creates a "monochrome" image, one which is often stained with arbitrary colors to increase viewability. Cells are sliced for viewing under an electron microscope using a tool called a microtome.
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)
APV (also called AP-5) is a selective NMDA receptor (NMDAR) antagonist that competitively inhibits the active site of NMDAR. Its chemical name is R-2-amino-5-phosphonopentanoate. APV is generally very fast acting within in vitro preparations, and can block NMDA receptor action at a reasonably small concentration.
Hippocampus to study plasticity mechanisms
Lecture 6 Model Systems
Xenopus oocytes
Xenopus oocytes are an ideal system in which to perform gene and protein expression experiments, as well as knockdown studies, given their large size (~1 mm in diameter). Their size makes them easy to culture, as well as to inject RNA from other species into.
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.
