The glianeuron index in the submolecular layers of the motor cortex in the cat.код для вставкиСкачать
THE GLIA/NEURON INDEX I N THE SUBMOLECULAR LAYERS O F THE MOTOR CORTEX I N THE CAT1 K. R. BRIZZEE AND L. A. JACOBS Anatomy Department, University of U t a h College of Medicine, Salt Lake City, U t a h THREE FIGURES INTRODUCTION It has been shown in recent quantitative histological studies on the cerebral cortex (Hawkins and Olszewski, '57) that the glia index (number of glia divided by number of neurons) in the whale is significantly higher than in man. The authors interpreted these findings as indicating that the glia index is correlated with brain weight rather than the position of a given species in the phylogenetic scale as previously suggested by Friede ( '54). If the glia index actually reflects only brain weight rather than phylogenetic development, one might expect the index to increase directly as brain weight increases in ontogenetic development. On the other hand, if the increase in the index does not coincide with the increase in brain weight throughout ontogenetic development, factors other than brain weiglit alone may be suspected to influence the glia index. It is the purpose of the present investigation to study the glia index in a series of cats ranging from kittens to adults in order to determine what relationships exist between the index and brain weight in growing and maturing animals. This investigation was supported in part by research grants, B-146 ( C 5 ) I and B-2016 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, and the University of Utah Research Fund. 97 98 K. R . BRIZZEE AND L. A. JACOBS MATEKIALS AND METHODS We have limited our studies to animals weighing from $$ to 3 kg (about 60 days of age to adulthood) because the brain is still growing rapidly in normal kittens of an s - k g weight, and the number of cells with nuclei intermediate in appearance between neuroglia and small neurons is relatively small in kittens weighing 1/2 kg and in older cats. I n earlier stages the number of such cells is somewhat larger, particularly in layers two and three, and might possibly introduce an appreciable error in such determinations. W e have used body weight as the criterion of the stage of development because most of the cats and kittens were obtained from the local animal pound and few records of their exact ages were available. Five apparently normal, healthy animals at each weight level were selected for study. The brains in all animals were fixed by perfusion with a saline-gum acacia mixture in Bouin's fluid (Peters and Flexner, '50). Tissues were embedded in paraffin, sectioned at 25 and stained with the buffered tliionin method (Windle et al., '43) at a pH of 4.5. The cortical site under investigation in this work was restricted insofar as possible to the region of the posterior sigmoid gyrus mdiich, according to Ward and Clark ('35) and Garol ('42) should constitute the motor area for the thigh. Only the submolecular cortex of the crown of the gyrus was studied and the counts were restricted to an area within 15" on either side of the midline of the gyrus. Where brain weight lias been used as a basis for comparison of cell densities or evaluation of growth phenomena in previous investigations, it has been customary to use total brain weight. I n the present study we have adopted the procedure of sectioning each brain at the ponto-mesencephalic junction before weighing in order to eliminate as many regions of the brain not concerneld with the current problem as possible. It would, of coin-se, be desirable to m7eigh only the cerebral cortex for such determinations a s me have carriccl out, hat WP THE GLIA/NEURON INDEX 99 believe any method of dissecting out the cortex alone would result in appreciable errors due t o incorporation of variable amounts of white matter or portions of other structures closely associated with the cortex. The density counts were carried out with the use of a 3-mm circle drawn on a thin disc of photographic film placed in the ocular of the microscope. When projected onto a stage micrometer using the oil immersion objective, the circle measured 30.5 I-( in diameter. I n each section the circle was focused through a standard thickness of exactly 15 j.~ beginning 4 to 7 p below the upper (cut) surface of the section and the number of the nuclei in the “cylinder” of tissue thus delimited was counted. The counts were made at progressively deeper levels in the submolecular cortex beginning at the outer border of layer I1 and ending at the inner border of layer V I in increments of one-ninth of the thickness of this part of the cortex. Proceeding in this manner a total of 20 samples were counted in each section. I n each animal a total of 10 sections was counted at intervals of every fifth section. Following this procedure, a total of 200 samples were counted in each animal or a total of 1,000 samples in each weight group (5 animals). Due t o the fact that many nuclei are bisected by the edge of the circle in any given section, it was necessary to adopt some criteria which would permit 8 constant evaluation of such border-line cells. Accordingly, we have employed the procedure of counting all cells in which clearly more than onehalf of the nucleus is included within the circle and have excluded all those in which less than half of the nucleus was included in the circle. Every other “half” nucleus was counted. It was, of course, also necessary to carry out such an evaluation of border-line nuclei in a similar manner at the upper and lower borders of the cylindrical mass of tissue examined in each count. This was done by carefully focusing to determine the mid-point of such questionable nuclei and determining whether or not the mid-point was within the cylindrical tissue mass examined. 100 K. R. BRIZZEE AND L. A. JACOBS RESULTS The results of the cell counts are summarized in table 1and figures 1-3. The glia density (table 1,fig. 1)increased from a mean value of 0.572 x 1O5/mnl3 in the youngest animals studied kg) to 0.631 in the 1-kg group. A more rapid increase was found between the 1-and 2-kg stages where a mean value of 0.972 was obtained, but only a slight increase was re- (x TABLE 1 Sakrnmary of mean cell densities ( 105/mm3j, i glia indices and brain weights (less medulla, pons and cerebellum) in series of BO kittens and cats weighing between % k g and 3 kg (about 60 days of age t o adulthood) WEIGHT NO.OF GROUP A N I X A L S BRAIN WEIGHT GLIA DENSITY 0.572 & 0.033 0.631 & 0.078 0.972 2 0.035 0.994 2 0.075 ’+!kg 5 13.22 & 1.2 1k g 5 2 kg 3 kg 5 5 19.63 k 1.04 18.72 t 0.76 18.85 f 1.8 GLIA INDEX NEURON DENSITY 0.G89 0.639 0.680 0.672 & 0.034 & 0.010 & 0.076 & 0.017 0.834 2 0.047 1.004 k 0.138 1.428 2 0.032 1.478 t 0.100 Four animals. 1.1- 1.0 T - .go- . mc 10 Neuroglla I 5 \,‘I I I 30- .I. I > t v) 2 w 5 .?O- _I 0 60’ %! I AI T ,/ .” -r Nebrons .50- .401GMS. 5bo lcIO0 I I 2000 3000 BODY WEIGHT Fig. 1 Alterations in mean neuroglial and neuron density in submolecular motor cortex in 4 groups of kittens and adult cats. THE GLIA/NEURON INDEX 101 corded after this stage, the maximal mean value being 0.994. Multiple “ t ” tests showed that the difference between the 1/-kg and the 1-kg and between the 2- and 3-kg groups was not significant ( p > I5- 1.4- 1.3- x X 1.2- G z 0 LL 3 1.1- w z \ 5 1.0-I a 0 LL 3 z“ 0.90.8 0.7 - 0.05) while the difference between the 1-and 2-kg groups was highly significant ( p < 0.01). The mean neuronal density (table 1 ; fig. 1) ‘dropped from a mean value of 0.689 x 105/mm3in 1/2-kg animals to 0.638 in the 1-kg group, and comparatively small changes occurred 102 I<. R. BRIZZEE AND L. A. JACOBS thereafter, the mean f o r the oldest animals being 0.672. From multiple ‘(t” tests it was shown that the differences in neuron density throughout the entire series were not statistically significant ( p > 0.05 in all groups). The glia index (table 1; fig. 2) increased from a mean value of 0.83 in the 1/-kg group to 1.42 in the 2-kg animals. I n the older animals, the value increased more slowly to 1.48. Multiple “ t ” tests indicated that the differences in the means of the glia indices between the 1/2- and 1-kg groups and between GMS. 22r cc ‘GMS. 500 1000 2 000 BODY WEIGHT 3000 Fig. 3 Changes in brain weight (exclusive of medulla, pons, and cerebellum) in 4 groups of kittens and cats. the 2- and 3-kg groups were not statistically significant ( p > 0.20) while between the 1-and 2-kg stages the differences were significant ( p 0.01). The most rapid increase in brain weight (exclusive of medulla, pons and cerebellum) was found between the 1/2-kg and 1-kg stages (table 1; fig. 3) ranging from a mean weight of 13.22 grams to 19.63 grams respectively. In older animals the brain weight was slightly lower than in the 1-kg group, but remained at about the same general level. From ‘4t”tests, it was shown that the difference in brain weight between the r~ THE GLIA/NEURON INDEX 103 %-kg and 1-kg groups was statistically significant ( p < 0.01) while in later stages the differences were not significant (p > 0.05 in both). DISCUSSION Our findings in this investigation support the views of Hawkins and Olseewski (’57) in part, in that the mean value for the glia index increased directly as the brain weight increased between the %-kg and 1-kg stages. Due to the great variability in the values for the glia indices in these groups, however, the difference in the means of the indices as indicated by the “t” test was not statistically significant. On the other hand, the difference between the means of the glia indices in the 1-and 2-kg groups was definitely significant with a p value very slightly greater than 0.01. Since there was no increase in brain weight after the I-kg stage, it appears that some factors in addition to brain weight must be of importance in the observed increase in the value of the glia index. To the knowledge of the authors there is no objective data available which would permit one to accurately delineate the period of “maturation” in the cat cortex. However, since normal animals which have attained a body weight of 2 kg can be considered young adults, it is probable that the complex processes of maturation are largely complete in animals which have reached this stage of development. The fact that the glia index undergoes very little increase after this stage, suggests that the increase observed in earlier stages may be related to increasing functional complexity of the cortex as well as brain weight. The mean value for the glia index obtained in the adult cat cortices in our studies (1.47) is on the average, slightly higher than those obtained by Friede (’54) for the horse, ox and swine and markedly higher than in the rabbit and other rodents but lower than either Friede or Hawkins and Olseewski (’57) found in the human cortex o r the latter authors found in the whale. If the glia index is related to brain weight alone, the values for the equine and bovine brains should be appreciably higher than in the cat. On the other hand, if the index 104 K. R. BRIZZEE AND L. A. JACOBS is determined only by functional complexity, the values for the horse shoulld be appreciably higher than for the ox but about equal to or less than that in the cat. The high values for the index found by Hawkins and Olszewski in the whale certainly indicate that the value of the index depends t o a large extent on brain weight. However, the fact that the glia index in the cat is higher than in the horse, ox or swine and increases during ontogenetic development after brain weight has become stable, indicates that the index must be related to functional complexity as well as brain weight. Further studies are in progress for the purpose of exploring these relationships in different areas of the cat cortex and in different mammalian species. SUMMARY The glia/neuron index in the submolecular layers of the motor cortex of a series of kittens and adult cats has been studied in order to determine what relationships exist between the index and brain weight in growing and maturing animals. Since no records of the exact ages of the animals were available, it was necessary to group them according to body weight. Five apparently normal, healthy animals at each weight level were selected for study. The glia index increased rapidly from a mean value of 0.83 in the youngest animals studied (1/2 kg) to 1.00 in the I-kg animals and 1.4 in the 2-kg group. I n the older animals ( 3 kg) the value increase'd more slowly to 1.48. The most rapid increase in brain weight (exclusive of medulla, pons and cerebellum) was found between the s - k g and 1-kg groups ranging from a mean weight of 13.2 gm to 19.6 gm, respectively. I n older animals the brain weight was slightly lower than in the 1-kg group, but remained a t about the same general level. These results suggest that the glia index is determined by functional complexity of the cortex as well as brain weight. Results are compared with those of other authors in different species of mammals. THE GLIA/NEURON INDEX 105 LITERATURE CITED FRIEDE,VON R. 1950 Der quantitative Anteil der Glia an der Cortexentwicklung. Acta Anatomica, 20: 290-296. GAROL, H. W. 1942 The motor cortex of the cat. J. Neuropath., Exp. Neurol., 1: 139-145. HAWKINS,A., AND J. OLSZEWSKI1957 Glia/nerve cell index for cortex of the whale. Science, 126: 76-77. PETERS,V., AND L. FLEXNE~ 1950 Biochemical and physiological differentiation during morphogenesis. Am. J. Anat., 86: 133-161. WINDLE,W. F., R. RHINESAND J. RANKIN 1943 A Nissl method using buffered solutions of thionin. Stain Tech., 18: 77-86. WARD,J. W., AND S. L. CLARK 1935 Specific responses elicitable from subdivisions of the motor cortex of the cerebrum of the cat. J. Comp. Neur., 6 3 : 49-64.