Toolbox/Lecture 12

C. elegans olfactory system
According to lecture, C. Elegans olfaction consists of three sets of neurons: AWA, AWB, and AWC. AWA and AWC control attraction while AWB regulates repulsion. An interesting finding is that the C. Elegans olfactory system is spatially encoded; it doesn't matter what the signal is, it matters which neuron fires. For example, if an attractant receptor is expressed in the repulsive or AWB neurons, the animal will be repelled by the attractant.

Trans-neuronal labeling
Mice are engineered to express a transneuronal tracer. Only mitral cells connected to the glomerulus take up the tracer with subsequent uptake by cortical neurons. Visualization of cortical neurons provides insight into olfactory cortex organization.

G-CaMP
G-CaMP is a Ca2+ probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon Ca2+ binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes.

Drosophila brain warping
Every Drosophila brain is different. Thus, deriving information from many fly brains may be difficult. Averaging can produce a fairly fuzzy image, so researchers developed a technique in which they use Drosophila brain warping--also used in human fMRI studies--to form a clearer picture. It is a method whereby many images of many fly brains are "warped" to adhere to a more coherent image. The method used is called translation rotation scaling, or non-linear warping. Researchers can thus "register" many different brains onto a preselected standard brain. This method has been used in studying projection neuron axon pathways. The method allows investigators to compare structures across different individuals of the same species.

IRES
An internal ribosome entry site (IRES) is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence. Normally, in eukaryotes, translation can only be initiated at the 5' end of the mRNA molecule, since 5' cap recognition is required for the assembly of the initiation complex. IRES mimics the 5' cap structure, and is recognized by the 43S pre-initiation complex (the 40S ribosomal subunit plus eIF1A, eIF3, and eIF2-GTP-bound to the initiator tRNA, required for translation). IRES are located in the 5’ un-translated region (UTR) of RNA viruses and allow translation of the RNAs in a cap-independent manner. Some mammalian mRNAs have also been reported to have IRES, although their existence is still controversial. One hypothesis is that the IRES function in eukaryotic mRNAs as housekeeping genes involved in cellular survival.

When an IRES segment is located in between two reporter genes in eukaryotic mRNA molecules as bi-cistronic mRNA, it can drive translation of the downstream protein coding region independently of the 5'-cap structure bound to the 5' end of the mRNA molecule. As such, both proteins are produced in the cell. The first reporter protein located in the first cistron is synthesized by the cap-dependent initiation approach while translation initiation of the second protein is directed by the IRES segment located in the inter-cistronic spacer region between the two reporter protein coding regions. However, this so-called “method of bi-cistronic constructs” has several bottlenecks when used in vivo and can often be deceitful.

In Lecture 12, Professor Luo presented data from Mombaerts et al. (1996), which used IRES to enable the translation of the P2 receptor (an olfactory receptor gene which is expressed in a restricted subpopulation of olfactory sensory neurons) and the tau-lacZ fusion protein (the protein tau functions in microtubule binding, while lacZ is a common reporter gene) from one mRNA. Their strategy allowed them to visualize axons from olfactory sensory neurons expressing a given odorant receptor as they project to the olfactory bulb. The experiment showed that neurons expressing a given receptor, and therefore responsive to a given odorant, project with precision to 2 of the 1800 glomeruli within the olfactory bulb.