Visualizing Different Mouse Brain Regions

Kristiana Lachiusa

University of Rochester, Department of Neuroscience

Introduction:

The cerebellum and hippocampus are two distinct regions. However both of these two regions have some of the more interesting morphological features. The cerebellum with its characteristically shaped sulci and the three layers that contain different cell types. The region of the hippocampus focused on in this study looked more at the region right on the edge of this area, the choroid plexus of the third ventricle. This area is where the CSF is made as well as have many tight junctions that act as the blood brain barrier. .




Samples and Preparation:

Due to the nature of the sample, many steps had to be done before placing the sample in the TEM.

*Fixation: Mouse was initially perfused with acrolein and a paraformaldehyde . Next the sample was post-fixed overnight in 2.5%glutaraldehyde with buffer. Next the tissue was fixed /stained in 1% osmium tetroxide and 1.5% potassium ferrocyanide and buffer.

*Dehydration: Following this the sample was dehydrated using progressively higher concentrations of ethanol. Initially 50% ethanol was added until and increased until 100% ethanol was reached.

*Infiltration: Next the transition chemical propylene oxide was used since it mixes more readily with the epoxy resin that the sample will be embedded in. After working up to just resin, the sample was embedded in fresh resin and placed in a 65 degree oven to allow for the epoxy resin to polymerize for 2 days.

*Ultra microtome/ light microscopy Cutting: Next the sample was cut using an ultra microtome into 1 micron sections placed on a glass slide and stained with toluidine blue and viewed under a light microscope. The rest of the sample was cut into 80nm slices and placed on carbon coated grids.

*Staining: The samples on the grid were stained with uranyl acetate followed by lead citrate.

*Imaging:  FEI Tecnai F20 TEM was used in both TEM and STEM modes.

 

Imaging and Results:


The above image was collected using the STEM HAADF detector. Using this technique one can see the white surrounding the cells. The stains used, including the osmium tetroxide, lead citrate, and uranyl acetate preferentially bind to lipoproteins that are often found in the membrane of cells, and as seen in this image in the myelin sheath. This white seen was hypothesized to be this color due to the stains that have higher elemental numbers and thus will eject more electrons giving the white color.

The above images that represent the x-ray spectral map acquired shows the different elements that were part of the staining process, as well as the carbon that acts as a control since the cut samples were placed on carbon coated grids, thus should be equally seen throughout the sample, as is seen above. These images show dark regions where the STEM image is also dark, and colored regions, showing where the x-rays that were reflected off that element came from. Thus this shows that the lipoproteins present in the membranes are where the stain attaches to on the sample.  By knowing where the stain is, the contrast seen in the images is able to be better understood. .



First I looked at general structures in brain tissue. This image shows a neuron making a synapse onto a dendritic spine. The smaller cell filled with synaptic vesicles is the pre-synaptic end of a neuron. The darker region between the two cells shows the post-synaptic density where the receptors are present on the dendrite. Within the dendrite one can also see a mitochondria.

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Cerebellum:

This region of the brain has very pronounced sulci. Within these there are three distinct layers: the molecular layer, pukinje cells layer, and the granule cell layer. These layers can be seen in the light microscope image below with the granular cell layer on the right side, the purkinje cells the large ones in the middle, and the molecular layer on the left side.


IM-4M 83X
In the below image this same region of the sample was imaged using the TEM. For clarity I colorized the purkinje cells purple,and to the left,the smaller cells are the granular cells. This higher resolution image allows the dense nucelus to be clearly seen. The other characteristic feature is the single large axon with lots of branching dendrites. Unfortunately due to where this sample was cut prevents this from being seen.

IM-2K 478X


Hippocampus:


The region imaged here was the choroid plexus. This region has a very interesting morphology. The below image shows the choroid plexus viewed with the light micrscope. Below you see one of the arms of the c-shaped loop that my sample showed.
IM-2K Spectrum
IM-2K 1720X

The above image shows some of the microvilli, mitochondria, a fragment of an epiplexus cell, and on the far right side a section of the third ventricle.
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The above image shows the edge of the basal lamina. The large structure in the middle is the nucleus of an epithelial cell. The structures on the right are the large golgi apparatus, and further to the right many mitochondria.

The different structures of the choroid plexus correspond to its function as the region that both makes cerebral spinal fluid, CSF, as well as being a barrier for molecules entering the brain. With the structure of its villi it is able to have increased surface area in order to have the most contact and have the ability to do both of these processes. The large number of mitochondria present surrounding the villi contribute to the ionic gradient that contributes to the BBB, as well as help provide metabolic potential for the secretion of CSF.



Conclusions:

The purpose of this study was to explore regions of the cerebellum and choroid plexus by using different microscopy techniques learned in OPT307. Allowing for further understanding of these sections of the brain and how their structures contribute to their function.  



Acknowledgements:

I would like to thank Emily Kelly for her help with obtaining the sample, Gayle Schneider and Karen Bentley for their help with the preparation, Rohit Puranik for being a great TA, and Brian McIntrye for all his help with imaging.

References and Further Reading:

Emerich, D. (2005). The choroid plexus in the rise, fall and repair of the brain. BioEssays, 27(3), 262-264. Retrieved fromhttp://onlinelibrary.wiley.com/doi/10.1002/bies.20193/pdf

Cornford, E., Varesi, J., Hyman, S., & Damian, R. (1997). Mitochondrial content of choroid plexus epithelium.Experimental Brain Research, 399-405. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9372289

Peters, A (2008) The Fine Structure of Synapses IBRO History of Neuroscience . Retrieved from http://www.ibro.info/Pub/Pub_Main_Display.asp?LC_Docs_ID=3313


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