Student Project: Chris Erickson

This is my summary of recent research in the field of Olfactory Development, based on the research article Analysis of article titled “Odorant receptors instruct functional circuitry in the mouse olfactory bulb.” Research conducted by Leonardo Belluscio, Claudia Lodovichi, Paul Feinstein, Peter Mombaerts, and Lawrence C. Katz. Link to paper

This project is for the course BIOL 475, Neurobiology, Dr. Grant Mastick, Biology Department, University of Nevada, Reno.

Link to course

Introduction:

ü Olfactory receptors are specialized neurons that can interact with many different odorant molecules.

ü When odorant molecules bind to them in the nose a signal is sent to the brain for processing.

ü Odorant receptors are remarkable structures that can identify thousands of odors that the human eye is unable to see.

ü Researchers set out to determine how the olfactory system was organized in the mouse brain.

ü Specifically to determine if the function of odor receptors influence the organization in olfactory bulb.

ü It was previously known that if you interrupt the function of a receptor it resulted in a change in the organization within the olfactory section of the brain.

ü It was also previously known that the olfactory bulb was made up of two sides the medial and lateral surfaces. It was further known that two of the same type of receptors send signals to identical locations one on each side.

ü It was unknown to researchers how these odor receptors influenced the overall structure of the olfactory bulb so they set out to determine that.

Rat Brain Side View Rat Brain Bottom View

Figure 1 Location of Rat olfactory bulb shown in red. Medial and lateral sides are shown on right hand side. Illustration modeled after Gary Matthews Neurobiology, Second edition, Black well press 2001: 418.

Experimental System:

ü Researchers used a “move it” experiment in order to observe the effect that moving a receptor would have on the organization of the olfactory system in the mouse.

ü The researchers extracted a rat receptor gene (rI7) and substituted it into the mouse receptor gene (M71) location by extracting and replacing the protein coding regions of each.

ü They also labeled the protein coding region for the new gene with green florescent protein (GFP).

ü They were able to label the rat receptor gene by tagging a complementary strand of DNA with the GFP. This allowed them to directly view the effect that new receptor would have on organization.

Advantages:

ü Recent advances in DNA sequencing have made transgenic mice more available.

ü Easy to directly view the results using GFP and a Confocal microscope.

ü Relatively simple.

Disadvantages:

ü The techniques and experiments done are expensive.

ü These experiments take new state of the art equipment.

Figure 2. Receptor labeled with green florescent protein taken with confocal microscope. Modeled after Leonardo Belluscio Developmental Neural Plasticity Unit Home Page Link

Results:

Experiment 1:

In an initial experiment the researchers used imaging to determine if the newly formed group of receptors would respond to known odor stimuli. They used four known ligands Octanal, Heptanal, Citral, and Hexanal. They found that the response to Ocanal and Heptanal was the same as in a mouse with normal gene function. The Citral and Hexanal did not elicit a response. They determined that it was not due to a malfunction in the newly formed bulb. They determined this by using a positive control, Propanal, and saw a response.

Figure 3. The number of spikes measured in vivo to the given ligands.

Illustration shows the strong response by octanal and heptanal but

little response to citral and hexanal. Modeled after Belluscio et al,

Nature 419: 296-300, 2002.

Experiment 2:

The second experiment was conducted to determine if the known afferent signals from the receptor could organize the post synaptic circuitry that is needed to convey the signal to higher areas of the brain. The post synaptic dendrites are known as mitral and tuft cells. In order to view this they mated a mouse homozygous for the newly formed receptor and a mouse with a yellow florescent protein (YFP) that could bind to the new gene. They observed that all the mice showed mitral innervations on both the medial and lateral glomeruli.

To determine if the tufted cell had formed a connection they injected the mouse with rhodamine-labelled dextran. The injection was injected in the area that it would bind and it is known to only bind to the area wanted. They were able to view the injection and sawthat the tufted cells bound to the correct area. This combination of experiments showed that the new receptor (rI7) is sufficient to organize the two main types of cells that receive input.

Experiment 3:

The next experiment was to determine if the connections made in the previous experiment were functional. They visualized the lateral rI7-M7I receptor using GFP and targeted to the immediate location. They were then viewed when stimulated with a known odorant stimuli and seen to be functioning in the same way as in a normal mouse.

Experiment 4:

This experiment was done to see if the newly formed receptors could connect with the correct target after receiving a signal. A tracer injection which only bound to the new receptor locust was viewed. It was seen to target a remarkably restricted region in the olfactory bulb. This connection was the same an in a normal mouse. The newly formed circuit innervated the bulb in the exact opposite position as its partner circuit. As was stated in the intro it was already known that circuits form in exact positions on the lateral and medial sides of the bulb.

Figure 4. Diagram of the reciprocal connection that links the receptors in identical places on each

side of the olfactory bulb. The rI7—M7I sensory neurons form excitatory connections

with tufted cells which in turn project to the other side of the bulb where they form mirror

image connections.

Conclusion:

The conclusion that the came out of the researchers experiments was that despite the difference in environment the rI7 receptor directed the formation of functional glomeruli (clusters of receptors) recruited postsynaptic dendrites and imparted rI7 function on unrelated neurons. This implicates that postsynaptic neurons may not be obligated to innervate a predetermined glomeruli as previously thought. This is thought to be due to common molecular cues that span the olfactory bulb. The activity of the pair of glomeruli may then be refined later. They may acquire functional identity by the properties of the sensory axons that make up glomeruli near their dendrites.

Significance:

ü This research has furthered our understanding of the organization of the olfactory system.

ü It showed that neurons are not predetermined as was previously thought, it showed that stimuli can influence them.

ü May lead to further understanding of overall organization in the whole brain not just the olfactory system.

ü Research may also prove to be important in helping people with degenerative problems.

Future Directions:

ü Identifying the signals that may draw the postsynaptic cells to the olfactory bulb.

ü More information is needed in the area of olfactory organization.

ü Determining if stimuli can influence organization in a mature brain.

ü Determining if other sensory systems are organized in the same way.

References:

Primary Research Article:

“Odorant receptors instruct functional circuitry in the mouse olfactory bulb.” Research conducted by Leonardo Belluscio, Claudia Lodovichi, Paul Feinstein, Peter Mombaerts, and Lawrence C. Katz. Link to paper

Annotation: Used transgenic mice to show odorant receptors can influence the organization in the olfactory bulb.

Review Articles:

"Genetic tracing reveals a stereotyped sensory map in the olfactory cortex." Zou, Horowitz, Montmayeur, Snapper and Buck, 2001. Nature 414: 173-179. Link to abstract

"Olfactory Bulb Mitral-Tufted Cell Plasticity: Odorant-Specific Tuning Reflects Previous Odorant Exposure." Fletcher, Wilson, 2003. Journal of Neuroscience 17: 6946-6955. Link to abstract

"Target neuron prespecification in the olfactory map of Drosophila." Jefferis, Marin, Stockers and Luo, 2001. Nature 414: 204-208. Link to abstract

This page was constructed by Chris Erickson  cle83@hotmail.com  November 18, 2003