Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1974 Lipid Metabolism of Neurons and Neuroglia Steven Roger Cohen Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Medicine and Health Sciences Commons Recommended Citation Cohen, Steven Roger, "Lipid Metabolism of Neurons and Neuroglia" (1974). Dissertations. 1437. https://ecommons.luc.edu/luc_diss/1437 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1974 Steven Roger Cohen LIPID .tv;:EIJ:'l-\BOLISi-1. OF 1:-."EURONS Al\TD NEUROGLil\ By STEVEN ROGER COI-illN A Dissertation Submitted to the Faculty of the Graduate School of rJoyola. University of Chicag-o in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy .June 1974 LOYOLA UJ.\ilVEESD ·y Tv1ED1CAL CENTER AC:h~O/JLEDGEMENTS 'I'he guidance of Dr. JosE: ph Bernsohn helped the work to proceed smoothly and, during those frustrating periods a researcher, especially a novice, sometimes experiences, his wisdom allov,red the work to proceed$ Dr. Bernsohn' s combination of wisdom a.nd patience \•.ras all that a graduate student could ask for~ Harold Norgello gave superb technical assistance in many of the experiments and his laboratory style vlill long serve as a standard of excellence for my 0'.\'D laboratory performance. I am very grateful to Dr. Michael Guarnieri, of the Department of Neurology at Johns Hopkins University, who gave many helpful suggestions during the writing of this disser·tation. f-.1y gratitude to Lena Tyson for typing the manuscript. And finally, to G.L. and K.G.P. for being there. TABLE OF CONTENTS Acknowledgements II Table of Contents III List of Figures IV List of Tables VI I. Introduction A. Background 1 B. Brain cell preparations 2 c. Lipids of neurons and glia 8 II. Materials and Methods 15 A. Cell isolation procedure 15 1. Neuronal-soma, astroglia and myelin 15 preparations. 2. Oligodendrocyte preparation 18 B. Lipid methodology 19 c. Incubations and Injections 24 III. Results and Discussion 31 A. Characterization of the cellular fractions 31 B. Incubation of cells versus slices 38 c. In vitro incorporation of {l-14c) linoleic and linolenic acid into neuronal soma and astroglia of rat brain and fatty acid compo-48 sit ion 1. Phospholipids 48 2. Fatty acids - incorporation 56 3. Fatty acids - composition 59 D. In vitro incorporation of fatty acids into neuronal soma, astroglia and oligodendroglia of calf brain 62 14 E. In vivo incorporation of {l- c) linolenate into neuronal soma, astroglia and oligodendroglia of rat brain 71 IV. Conclusions 111 v. References 116 VI. Biographical Sketch 123 iii List of Figures Figure Number Page 1. Methods of preparing brain cells. 4 2. Thin layer chromatograph of rat brain phos- 22 pholipids. 3. Gas-liquid chromatograph of rat brain phos- 25 phoglyceride fatty acids. 4. Neuronal soma isolated from rat brain. 32 5. Neuronal soma isolated from calf brain. 34 6. Astroglia isolated from rat brain. 35 7. Oligodendrocytes isolated from calf brain 36 white matter. a. Oligodendrocytes isolated from a mixture of 37 calf brain white matter and rat brain. 14 9. Incorporation of {l- c) Acetate into total 39 lipids of isolated neuronal soma and astro­ glia from developing rat brain. 14 10. Incorporation of (l- c) Acetate into lipid 42 classes of isolated neuronal soma and astro­ glia. 11. Two dimensional thin layer chromatography of 45 ethanolamine phosphoglyceride from rat brain c~lls incubated with (l-14c) acetate. 12. Incorporation of (l-14c) linoleic acid into 54 lipid classes of neuronal soma and astroglia. 13. Incorporation of (l-14c) linolenic acid into 55 lipid classes of neuronal soma and astroglia. iv Figure NUmber Page 14 14. Incorporation of ( 1- c) linolenate into 74 phospholipids of rat brain fractions of ten intracerebral injections. 14 15. Incorporation of ( l- c.) linolenate into 86 ethanolamine and choline phosphoglycerides of rat brain after intracerebral injection. 14 16. Incorporation of (l- c) linolenate into 96 phosphoglyceride fatty acids of rat brain fatty acids after intracerebral injection. v List of Tables Table Number Page 14 1. Incorporation of {1- c) acetate into 41 neurons and astrocytes of developing rat brain. 2. comparison of slice and cell incubated 44 incorporation of {l-14c) acetate into ethanolamine and choline phosphoglycerides of isolated neurons and astrocytes. 14 3. Incorporation of {1- c) acetate into 47 lipid fractions of rat brain under various incubation conditions. 4. Incorporation of {1-14c) linolenate into 49 phospholipid fractions of rat brain under various incubation conditions. 5. Incorporation of various precursors into 50 lipid fractions of neuronal soma and astro- cytes from developing rat brain. 6. Ratios of neuronal to astroglial lipid 51 specific activities after incubation of de­ veloping rat brain slices with labelled precursors. 7. Incorporation of {l-14c) acetate into phos- 57 phoglyceride fatty acids of neuronal and glial preparations of developing rat brain. 8. Incorporation of {1- C) linolenate into - 58 phosphoglyceride fatty acids of neuronal and astroglial preparations from ten day old rat brain. 9. Distribution of phosphoglyceride fatty acids 60 in n~uronal soma and· astroglia of ten day old rat brain. vi Table Number Page 10. Distribution of phosphoglyceride fatty acids 61 in cells from developing calf brain. 14 11. Incorporation of (l- c) fatty acids into 6 2a isolated neuronal soma, astroglia and oligo­ dendroglia from calf brain. 14 12. Incorporation of (l- c) linolenate into 65 phosphoglyceride fatty acids of isolated cells from developing calf brain. 14 13. Incorporation of (l- c) linolenate into phos- 67 phoglyceride fatty acids of rat brain cells in­ cubated in synthetic culture medium. 14 14. Incorporation of (l- c) linolenate into phos- 68 phoglyceride fatty acids of calf brain cells in­ cubated in culture medium. 15. Incorporat1on. of ( 1-14c ) 1'1nolenate 1nto. sl1ces. 69 and homogenate~ of rat brain and liver. 16. Incorporation of (l-14c) linolenate into phospho-70 glyceride fatty acids of rat brain tissue incu­ bated in culture medium. 17-22. Incorporation of (l-14c) linolenate into lipids of rat brain after intracerebral injection - twenty-one day old rats. 17. Five minutes after injection • 78 18. Thirty minutes after injection. 79 19. One_hour after injection. 80 20. Two hours after injection. 81 21. Four hours after injection. 82 22. Thirty-six hours after injection. 83 23. Ten day old rats. 84 vii Table Number Page 14 24-30. Incorporation of {l- c) linolenate into phosphoglyceride fractions of rat brain cells after intracerebral injection - twenty-one day old rats. 24. Five minutes after injection. 87 25~. one half hour after injection. 88 26. One hour after injection. 89 21. Two hours after injection. 90 28. Four hours after injection. 91 29. Twelve hours after injection. 92 30. Thirty-six hours after injection. 93 14 31. Incorporation of (l- c} linolenate into 94 phosphoglyceride fractions of rat brain cells after intracerebral injection 10 day old rats 32-38. Incorporation of (1-14c) linolenate into phosphoglyceride fatty acids of rat brain cells after intracerebral injection - twenty-one day old rats. 32. Five minutes after injection. 97 33. Thirty minutes after injection. 99 34. One hour after injection. 100 35. Two hours after injection. 101 36. Four hours after injection. 102 37. Twelve hours after injection. 104 38. Thirty-six hours after injection. 105 viii Table Number Page 14 Incorporation of (l- c)linolenate into . 39-40. phosph6glyceride fatty acids of rat brain cells after intracerebral injection - 10 day old rats. 39. one hour after injection. 106 40. One-half and two hours after injection. 107 41. Percentage of radioactivity in the carboxyl 110' carbon of brain fatty acids after injection of (l-14c) linolenate. '. ix Introduction A. Background For almost one hundred years, it has been knovm that the alian nervous system is composed of a variety of cell types, ma.mrn each of which sends out complex branching processes that inter- twine with each other to form a mesh-like pattern of nervous tissue (for a discussion see Peters, et al, 1970~ Cragg, 1968). Thus any section of nervous tissue, even if it is from a well defined anatomical region is composed of a hetc~rogeneous mix- ture of cells and their processes., There are two major types of cells, the neurons which carry the actual nervous impulse, and the neurog·lia, of \Alhich there are two rna jor kinds, the astrocytes, whose func·tion is largely unknown, and the oligo- dendrocytes, the cells presumably responsible for the formation and maintenance of the myelin sheat:h (Bunge, 1968, 1970). Esti·- mates have been advanced concerning the number of glial cells in the brain and the figure varies from brain region to brain region and between histologists (Brizzee, et al, 1964, Jacobson, 1970). Any compositional or metabolic experiments on nervous tissue will result in data that reflects the net average of all the cells and cell processes in the tissue sample. Methods have been developed to isolate the variou::> functional units from nervous tl'ssue such as_ cell. soma ( see b e 1 ow ) and even par.st I of cells such as nwelin (Autilio, et al, 1964), axons (DeVries, et a 1, 1972) and synaptosomes (Whittaker, 1964, deRobertis, et al, 1961). (For discussion of these various methods see Appel, et aL 1972 1 Whittaker, 1969, deRobertis andArnaiz, 1968). 'rhis •,..;ork was undert.aken for the purpose of elucidating the roles of neurons, astrocytes and oligodendrocytes in the overall pattern of lipid metabolism of the brain.