Journal of Undergraduate Research
Volume 1, Issue 2 - February 2000

The Effect of Alcohol on the Inferior Olivary Nucleus of the Brainstem and Climbing Fibers of the Cerebellum

Adam Reig

ABSTRACT

Fetal Alcohol Syndrome is a major cause of birth defects throughout the world. This study focused on how alcohol affects climbing fibers and inferior olivary nucleus (ION) cells during rat brain development. There was a qualitative, climbing fiber aspect of the study, and a quantitative, ION portion of the study. It could not be determined by qualitative analysis if alcohol has an affect on climbing fibers. The quantitative aspect of the study is still underway and results should be conclusive as to whether or not alcohol has an affect on ION cells.

INTRODUCTION AND BACKGROUND

Fetal Alcohol Syndrome (FAS) is a condition that afflicts thousands of children around the world. The nervous system is severely affected by alcohol in the developing stage of animals. Some of the indications of FAS children include poor motor skills, imperfect balance, deformed face, and underdeveloped limbs. This particular study focused on the cerebellum and brain stem of rats. The cerebellum is responsible for the regulation and coordination of complex voluntary muscular movement as well as the maintenance of posture and balance, which explains how cerebellum damage causes symptoms of FAS. During brain development, there are cells located in the inferior olivary nucleus (ION) of the brainstem that serve as the origins of climbing fibers. These fibers migrate up to all the layers of the cerebellum and connect to the dendrites of Purkinje cells. Climbing fibers are excitatory bodies that send signals from the brainstem to Purkinje cells in the cerebellum. It was investigated how alcohol affects ION cells and climbing fiber migration during rat brain development.

In past experiments, Dr. Heaton and her colleagues have found that there is a 45% Purkinje cell loss in the cerebellum of rats exposed to alcohol on postnatal days four and five compared to the control groups. Napper and West (1995) have determined that postnatal alcohol exposure causes permanent neuronal loss in the inferior olivary nucleus. To determine whether alcohol has a direct effect on the ION, two postnatal days were compared. Since it is known that alcohol exposure on P4-5 causes a 45% loss in Purkinje cells, the rats were exposed on those days. Four groups of animals were formed; a control group and an alcohol group that were sacrificed on P6 and a control and an alcohol group that were sacrificed on P12. Postnatal day six and postnatal day 12 was utilized because Jansen and Brodal (1954) determined that with Purkinje cell destruction, cells in the ION were lost within 4-6 days following exposure. The use of P6 and P12 would determine if ION loss is direct or indirect.

If there is ION cell loss on P6, (i.e., comparable to the Purkinje cell loss) then the effect of alcohol on those neurons is direct. If there is no loss noted on P6, then either the cells aren't sensitive to alcohol during those exposure days, or P6 is too early to see a loss in ION number. If the latter is true, then the effect of alcohol on ION cells is probably indirect, and their loss is dependent of Purkinje cell loss. If we notice ION cell loss on P12 (with no loss on P6), then it would be apparent that there is a delayed death in cells, which could be attributed to Purkinje cell loss. If there is no loss seen at P6 or P12, then exposure at P4-5 is not a sensitive period for ION cells.

If there is a loss of climbing fibers observed, then we could determine whether their loss can be attributed to the loss of ION cells by analyzing P6 and P12 cerebella. If there is normal development of climbing fibers and no loss of ION seen at P6, then it is probable that their innervation will decline at a later time due to the loss of Purkinje cells. Not only can climbing fiber numbers be affected by losing their target cells, since cells without targets die rapidly, but also their growth patterns could be affected. Climbing fibers will also be analyzed at P12, if the outcome of the ION cells counts produce no loss from exposure on days P4-5. This would tell us about the longer-term effects of loss of the Purkinje cell targets on the climbing fiber innervation patterns.

MATERIALS AND METHODS

The method of exposure to alcohol by the rats was performed by means of inhalation. The rats were put into a tank that has apparatus that pumped ethanol fumes inside. The rats raised their blood alcohol levels (BALs) by breathing in the fumes.

Then, depending on the day (P6 or P12), the rat's brains were dissected out. The brains used for the ION portion of the study were fixed in Bouin's solution for 48 hours, then stored in 70% ethanol. The brains used for the climbing fiber aspect of the study were fixed for three hours in 4% paraformaldehyde, then stored in a 30% sucrose solution.

The brains were processed in two different manners. Immunocytochemistry was performed on the brains for the climbing fiber study as a means of qualitative analysis. The cerebella were frozen and cut coronally on a microtome at 40 microns. Then they were stained with a peripherin marker, mounted and analyzed utilizing fluorescence microscopy. After trying a few peripherin-specific markers, R34 was used with a fluorescent label so that pictures of the tissue could be taken utilizing a fluorescence microscope. This type of stain only marks a protein found in climbing fibers so that comparisons between control and alcohol groups could be made without obstruction from other neurons.

The other means of processing the brains was a quantitative method using paraffin sectioning to perform ION cell counts. The brains were dehydrated and embedded in paraffin, cut coronally at 15 microns thick, and then mounted slides. With this type of histology, the inferior olivary nucleus, which is located in the brainstem, was able to be seen using a common Hematoxylin and Eosin Nissl stain. The nucleoli of these ION cells were counted using a morphometric microscope that has a moving stage and a stereology program loaded into an attached computer at the Optical Microscopy Lab in the UF Brain Institute. The program is very unique in the fact that it lets the user outline an area of tissue to be counted and in an unbiased and random manner goes to specific regions of the specimen for cell counts. The program is the called the MicroComputer Imaging Device (MCID) 5.0 and it was developed by Imaging Research, Inc.

RESULTS AND CONCLUSIONS

The qualitative aspect of the study yielded results that might not be accurate by the means with which the tissue was analyzed. The P6 and P12 brains were effectively stained with the peripherin marker, then analyzed for differences blind. It could not be determined if the alcohol produced a loss in climbing fiber growth or restricted the fiber's innervation by simply looking for differences under the microscope or in pictures.

Figure 1. Sample picture of a P6 control section of the granular layer of the cerebellum magnified 600 times. The climbing fibers are the thin strips of green tissue contrasted against a black background.

Figure 2. Sample picture of a P6 alcohol section of the granular layer of the cerebellum magnified 600 times.

Not much difference between the two samples can be seen in the pictures. The P12 groups gave similar, undetectable results as the P6 groups. There are many factors that can be attributed to the lack of unobservable differences between the two groups. The R34 marker might not have penetrated the tissue layer evenly, leaving blocks of fibers, rather than a continuous orientation. Also, since the fibers do not follow the same exact path in different brains, it is hard to deduce differences between the two groups.

A follow-up procedure that could give unbiased quantitative results for the experiment could be investigation of the fibers utilizing deconvolution or confocal microscopes. These types of microscopes section the specimen and project a 3-D image onto a computer screen. These images can be magnified so that isolation of single climbing fibers can be performed. Analysis such as density of the fiber and accurate lengths and widths of the fibers could be compared between the alcohol and control groups for differences.

The quantitative ION counting portion of the experiment is still in the process of being performed. Use of the morphometric microscope requires a lot of time per animal counted. The results of this aspect should give solid evidence as to what kind of effects, if any, alcohol has on the inferior olivary nucleus. Current counts of the P6 IONs range from 9,500-20,500 cells per animal. Since cell counting is still being performed on these groups, it is not known which counts are for the control or alcohol animals.

Photo by John Elderkin

ACKNOWLEDGEMENTS

I would like to thank the following people for their help and support on this ongoing project: Dr. Marieta B. Heaton, Mike Paiva, Izzy, Parletta, Dr. Gerry Shaw, Minnie, Tim Vaught, and Dr. Roger Reep. Without your help, this project couldn't have been accomplished.

REFERENCES

Jansen, J. (1954). On the morphogenesis and morphology of the mammalian cerebellum. In Aspects of Cerebellar Anatomy (J. Jansen and A. Brodal, eds.), pp.13-81. Johan Grundt Tanum, Oslo.
Napper, R. and West J. (1995). Permanent Neuronal Cell Loss in the Inferior Olive of Adult Rats Exposed to Alcohol During the Brain Growth Spurt: A Stereological Investigation. Alcoholism: Clinical and Experimental Research, Vol. 19, No. 5, 1321-1326.

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