Sanil Modi
Biol-4910
Summer 2014
M-F Dr. De Vries, Christopher T. Fields The Effects Of Toll Like Receptor 2 Deletion on Social Behavior Neural Network
Introduction
During this past summer I had the opportunity to conduct research at the Neuroscience Institute at Georgia State University. The research I participated in was under Christopher T. Fields who is working on getting his doctorial degree. In these last few months I have worked on many exiting projects, learned how create experiments and analyze them. From the first day of lab I learned to work with many different lab instruments, software and mastered the structures of the mice brain. The instruments I started working with were a digital microscope and its software Stereo Investigator that took pictures at HD quality of mice brain. Shortly after came the analysis of the pictures we captured and the software used was ImageJ and Excel. In ImageJ you can measure different thresholds of the mice brain and get analysis which is imputed into excel and then the numbers from excel are put into a statistical software where graphs are made and you can check if your experiments had any change from the control. What I also learned was how mice brains are put on a slide. First you would use a cryostat, which slices the mice brain at the amount of thickness needed. While you are slicing the mice brains you are putting them onto a slide. Then they are taken from the slide put into a buffer solution, which lets you add to another side where you can stain the structures in the brain where you want the analysis completed.
In the last few months we worked on some experiments involving the Toll-like receptors (TLR2), which have an important role in the innate immune system (first response), they are able to indicate when there is a presence of infectious bacteria and if there is an aberrant process occurring. The toll-like receptors also have an effect in the development of the brain. So what we see when the mice don’t have toll-like receptors is that they have really high levels of anxiety or they are socially inept. From these results we have researched the role of vasopressin, which are known to control anxiety and social behavior. In this project there were many multiple parts which tell us the effect of the TLR2 receptors and one of the projects were staining for the Iba-1 which is a marker for activated microglia (cells of the Brain). The activated microglia is caused by vasopressin expression. We also worked on situ hybridization for mRNA expression of vasopressin in Wild Type (WT) and TLR2-/- mouse brains. Immunohistochemistry (IHC) tells us that a certain cell carries a certain protein and we are staining cells to see weather vasopressin is being released and the affects of the release. In the experiments preformed now we are seeing that the Iba-1 IHC staining and AVP IHC staining could be used to research a new mouse model for autism in the TLR2-/- mice.
Results
In the TLR2 and sex differences in SCN vasopressin expression we used 6 male, 6 female TLR2-/- mice and 6 male, 6 female WT counterparts. From the use of a microscope using Kohler Illumination at 10x objective with Stereo Investigator we took images of Vasopressin projection areas in the brain and analyzed by using ImageJ and thresholding options. Our results were that the TLR2-/- mice did have an increase in vasopressin immunoreactivity in the suprachiasmatic nucleus (SCN) meaning we do see a change in vasopressin release when the TLR2-/- have been knocked out. We also see a higher immunoreactivity in WT females relative to WT males meaning that the TLR2-/- females compared to males have a higher release vasopressin. We do see vasopressin immunoreactivity in the SCN of the nuclei but we do not see the vasopressin immunoreactivity in SCN or BNST projection areas. This means that an SCN projection area in the TLR2-/- mice have lessened vasopressin immunoreactivity. For example the DM, which is one of the projection areas don’t have any increase vasopressin and doesn’t appear to have any increase in AVP-ir either.
Microglia are cells in the brain that are targeted when we do the Iba-1 staining. This experiment is currently being conducted so the results will be what we are looking for and haven’t received yet. If the microglia are activated in the TLR2-/- brains then we know that the AVP in the SCN meaning that there is activating vasopressin proteins in the reading and that increased microglia activation and TLR2-/- removal is correlated. So henceforth knocking out the TLR2-/- gene is our independent variable. That means we know that the TLR2-/- knockout causes a difference in SCN immunoreactivity and microglia activation. What we don’t know is weather SCN immunoreactivity is brought by an increase in microglia activation or if it cause the other way around. Also it can be that both are straight results of TLR2-/- deletion. In situ hybridization of vasopressin in WT and TLR2-/- we see that vasopressin protein stains IHC tell us that if your cells carry a specific protein. Furthermore, the cells can transcribe and translate and collectively store proteins that are not degrading over a long period of time. On the other side seeing a decline in the protein staining could me that the cells are translating and transcribing the protein less, or the cell releases huge quantities of stored protein but is transcribing and translating that protein at the same rate. This generally means that in situ hybridization you can detect the change in levels of mRNA transcribed in cells of intact tissue. It’s exactly like the immunohistochemistry for mRNA. So the area that has the most staining for the mRNA transcript means that area has transcribed and transscripted more. This means that if the transcripts have higher stain density in a particular area, then that specific area is expressing the vasopressin gene at a higher rate. Now if we see an increase in mRNA transcript areas where the vasopressin protein is located mainly in the SCN of the TLR2 -/- mice compared to the WT mice, the we can propose that the increase in protein levels may be due to the increase in the gene expression. This would mean that the gene expression is the cause and not the increase in translation or decrease in protein breakdown. Recently in our laboratory Nicole Peters found that the Iba-1 IHC stain and the AVP IHC stain could be said to investigate a new mouse model for autism: the TLR2-/- mouse. She found that mice that have their entire normal flora taken out of their gut have an increase in vasopressin immunoreactivity (AVP IHC Stain) in the SCN. This is compared to normal mice that is untouched and has all its normal gut bacteria. We also found very similar increases in the SCN in TLR2-/- mice. This means that the gut bacteria may affect social behaviors like autism. A project we are working on that uses microbiota composition analysis on Brattleboro rats. This analysis can tell us using the Iba-1 IHC stain and the AVP IHC stain to investigate a new mouse model that autism also displays alterations in gut microbiota. Autistic children usually have very bad stomach problems and what we are working on is how the bacteria in the gut affect social behaviors. This means that reason autistic children might be socially inept is because they have bacteria that is not normal compared to normal gut bacteria. When these experiments were conducted on mice you can see that the mice without normal bacteria have higher anxiety and social disorders.
Discussion
WT females have greater AVP-IR in SCN nuclei than WT males. This also occurs in the TLR2-/- where the females see more Vasopressin protein than males. In this there were no genotype effects observed in vasopressin between males and female. For the projection areas Females did have greater AVP-IR in the PVA than males. The DM of female’s trends has a higher density than that found in males of the WT mice. In TLR2-/- mice we can see a sex difference but it appears to be abolished and the DM in TLR2-/- the mice are less AVP-/- than that of WT mice. The TLR2-/- Knock Out appears to affect patterns of vasopressin expression, the amount that is stored and the amount released, which mainly happens in the SCN nuclei and projections. Vasopressin in the projections is usually diminished and is increased in the cell soma of SCN neurons. In the future I think we could try to find out how much vasopressin is released normally and how much is released when there is immunoreactivity. Therefore we could tell how much vasopressin is released during different times, which could affect how behavior is looked at. Because if an increase in vasopressin helps with anxiety then we can we can try to control the amounts secreted at a time.
In the Microglia cells we are trying to see if SCN immunoreactivity is caused by an increase in microglia activation or if SCN immunoreactivity causes microglia activation or both cause it. I think in the future months we can construct an experiment that only measures microglia activation independent of SCN activity and do the same with SCN activity. We could also check what type of bacteria causes immunoreactivity and how it affects the SCN. I think the best way to figure what stimulates what is by maybe creating a new experiment, which can tell us when the microglia is activated and when then SCN is activated. By this you can tell if the one is causing the other or both cause each other stimulation.
In situ hybridization of vasopressin we see that certain cells carry a specific protein and cells can transcribe and translate and collectively store proteins that can express proteins like vasopressin over time. The protein can be stored in the cell at huge amount of quantities until later use so we don’t know if the vasopressin is released all at once or over time. A project we can conduct is to measure the amount of vasopressin that a normal cell can transcribe and translate and try to measure how much should be coming out. By understanding the amount release we can measure at what levels vasopressin can help come over social and anxiety disorders.
According to what Nicole Peters found that the Iba-1 IHC stain and the AVP IHC stain could be said to investigate a new mouse model for autism: the TLR2-/- mouse. She found that mice that have their entire normal flora taken out of their gut have an increase in vasopressin immunoreactivity (AVP IHC Stain) in the SCN. We could create experiments where we try to measure which bacteria in the gut are sending signals to the SCN region. We could try to identify if those bacteria are corrupting the other bacteria in the gut and if that is true then we could figure out a way to introduce the correct bacteria and help autistic children lessen their gut problems. This could help solve social issues and anxiety problems in autistic children that we think is caused by irregular gut bacteria.