Hypothyroidism affects preferentially the dendritic densities on the more superficial region of pyramidal neurons of the rat cerebral cortex

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Developmental Brain Research, 28 (1986) 259-262 Elsevier BRD 60154

Short Communications Hypothyroidism affects preferentially the dendritic densities on the more superficial region of pyramidal neurons of the rat cerebral cortex* ANTONIO RUIZ-MARCOS and SANTIAGO L. IPIIqA Unidad de Neuroanatomia, Instituto Cajal, C.S.1. C. and Departamento Matematicas Aplicadas, Facultad Ciencias Biologicas, Universidad Complutense, Madrid (Spain) (Accepted March 3rd, 1986) Key words: neonatal hypothyroidism - - pyramidal neuron - - cerebral cortex - - dendritic density The effect of neonatal hypothyroidism on the dendritic density of layer III pyramidal neurons of the rat cerebral cortex has been investigated using a special matric algorithm. The results obtained indicate that this disease affects preferentially the dendritic density on the upper region of these cells.

Quantitative studies m a d e about the effect that hypothyroidism has on the d e v e l o p m e n t of p y r a m i d a l neurons of the cerebral cortex have suggested that this pathological condition affects the dendritic density, or dendritic length p e r unit area, at different distances from the soma of these cells4. In o r d e r to corr o b o r a t e these previous results and to elucidate whether this disease affects the whole dendritic field of pyramidal neurons h o m o g e n e o u s l y or, on the contrary, it affects some parts of their dendritic arborization with preference to others, the dendritic densities of two groups of neurons belonging to control and hypothyroid animals were analyzed using a special algorithm. For this purpose, a total of 18 p y r a m i d a l neurons** chosen from the layer III of the visual a r e a of the cerebral cortex of 10 control (C) rats, 80 days old, and an equal n u m b e r of neurons of 10 rats of the same age, surgically t h y r o i d e c t o m i z e d (T) when they were 10 days old, were drawn using a c a m e r a lucida with a total magnification of 500 x . T h e occipital re-

gion of these brains was stained according to the rapid Golgi p r o c e d u r e and 200-/~m-thick sections were obtained with a microtome. G i v e n the difficulty to find complete i m p r e g n a t e d neurons, those entering the present study were selected by looking at the preparations with the lowest magnification (25 ×). Since it is not possible to notice the cell-specific configuration when looking at the neurons with such a magnification, this m e t h o d was considered as adequate to select the m a x i m u m n u m b e r of impregnated cells as it avoided the possible bias introduced by the observer. O n a second step of the neuron selection, those cells with m o r e than 10% of cut ends were eliminated. This last limitation m a d e , in practice, that only those neurons well i m p r e g n a t e d and fully i m m e r s e d in the section enter into the present study. The 3 spatial coordinates of the most i m p o r t a n t points of each dendrite of these neurons, i.e., origin of each dendrite at the soma, inflexion points or points where the dendrites change their direction, bi-

* Presented in part at the IX International ThyroidCongress, Sao Paolo, Brazil, 1985. ** The pyramidal neurons considered in this work were selected from the histological preparations made at our laboratory and used in a previous study concerning the effect of hypothyroidism in the maturation of the rat cerebral cortex6. As it was pointed out in that publication, the low value attained by the weight, pituitary GH content and TSH plasma circulating level of thyroidectomized rats reveals that these animals were severely hypothyroid at the particular age (80 days) studied here. Correspondence: A. Ruiz-Marcos, Unidad Neuroanatomia, Instituto Cajal, Velazquez 144, 28006 Madrid, Spain.

0165-3806/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)

260 furcation points and end points, were stored in the permanent magnetic m e m o r y of a P D P 11/40 computer according to the instructions of a special program named A D Q U I and using a sonic digitizer which measures directly the x,y coordinates of each of the selected points described above. The third coordinate (z) of these points was previously measured by means of a sensor (Millitron) attached to the fine focus of the microscope, and transferred to the computer by touching with the pen of the digitizer the corresponding place on a list of numbers previously printed on the digitizer board. E v e r y set of 3 coordinates was followed by a numerical code indicating the machine to which type of point (initial, inflexion, etc.) the 3 coordinates previously stored correspond. In this way, the neuron is stored as an array f o r m e d by a series of records, each record containing the values of the 3 coordinates of every selected point followed by a numerical code. Using another p r o g r a m n a m e d A C R O N (from A v e r a g e C o m p u t e r n e u R O N s ) the c o m p u t e r is able to reconstruct each individual neuron using the a b o v e - m e n t i o n e d array, projecting it on a virtual grid created in its core m e m o r y (see Fig. 1) and, following the complete arborization of the neuron in steps of one micron, to measure the actual dendritic length traversing each square of the grid. The p r o g r a m has been m a d e so that the side of each particular square

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can be adjusted according to the initial data given by the operator. In this way, each neuron is transformed into a numerical matrix; the actual size of the matrix used in the present study has been of 32 columns and 32 rows with an individual square side of 40 ~tm. The soma of the neurons was always placed at the center of each matrix. Once the individual matrices corresponding to a homogenous group of neurons (i.e., neurons belonging to a certain area of the brain of animals of the same age raised under the same condition) have been calculated, the c o m p u t e r proceeds further adding together all these matrices thus finding the matrix sum corresponding to the group of neurons studied. Dividing the elements of this matrix by the n u m b e r of neurons forming the group it is obtained the mean matrix; the quotient between each e l e m e n t of this last matrix and the area of each individual square gives as a final result the density matrix, normalized mean matrix or A C R O N . Thus the elements of the A C R O N represent the mean dendritic density of the group of neurons entering the study at a certain distance from their soma (Fig. 1). The comparison made using specific statistical tests described below, between each pair of homologous elements (i.e., elements with the same position inside the matrices) of the density matrices corresponding to neurons of a C group vs those corre-

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261 sponding to animals T at 10 days of age, could give us information about the region of the neuronal arborization on which t h y r o i d e c t o m y has affected significantly the total dendritic density. In a previous work 5 these differences were assessed by means of a multiple t-test, p e r f o r m e d b e t w e e n each pair of homologous elements. H o w e v e r , as this test requires a multiple comparison, according to the Bonferroni's inequalities 3 the too high value attained by the final significance level m a k e s such a type of t-test ineffective. To o v e r c o m e this difficulty, a series of new programs were i m p l e m e n t e d to p e r f o r m the following type of tests. On one hand, the overall difference between the m e a n dendritic densities of the two A C R O N s (C and T) of neurons was assessed by means of a t-test applied to the logarithmic transformed original data. This transformation was necessary in o r d e r to obtain a n o r m a l probability distribution in both samples and the h o m o g e n e i t y of variances which were tested using the K o l m o g o r o v Smirnov and variance ratio tests 9. The t-value obtained from this analysis was 4.381 (t0,05 = 1.96; df = 34). On the o t h e r hand, in o r d e r to find those positions inside the dendritic field where the m a x i m u m difference b e t w e e n the m e a n dendritic densities ( C - T ) occurs and those places where the differences e n c o u n t e r e d do not differ significantly from the m a x i m u m difference m o r e than the 0.05 level, the whole series of differences between all pairs of homologous matrix elements was analyzed by means of a one-way analysis of variance ( A N O V A ) followed by the Least Significant Difference (LSD) m e t h o d s applied to the logarithmic t r a n s f o r m e d data. To check w h e t h e r the error terms of these differences are i n d e p e n d e n t , an 'up and d o w n ' cycle test 9 was p e r f o r m e d (ts = 1.54 with 152 df; t0.05 = 1.96); in addition, the goodness of fit to a normal distribution and the h o m o g e n e i t y of variances were assessed by means of the K o l m o g o r o v - S m i r n o v and Bartlett tests 9. A s a result of this p r o c e d u r e the c o m p u t e r prints the corresponding F-value ( F = 3.296 with 152 and ~ df; F0.05 = 1.20) and a graphical pattern on which it appears a sign + on those places where these differences ( C - T ) are positive, a sign - - where such differences are negative and nothing where the value of the differences differs from the m a x i m u m difference b e y o n d the 0.05 level of significance. The letter X which appears on the center of this graph

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Fig. 2. Results from the comparison between the mean dendritic densities of the neuronal samples belonging to C and T animals. The unitary square used to obtain the dendritic density at each location of the dendritic field had 40-/~m sides. For meaning of symbols see text.

represents the position of the soma of the p y r a m i d a l cells studied with their apical shafts going upwards, perpendicular to the pia surface which is located parallel and just below the u p p e r I I I I line. Fig. 2 shows the pattern o b t a i n e d from this analysis. The position of signs + on this p a t t e r n indicates that thyroidectomy produces a m o r e conspicuous decrease of the dendritic density on the u p p e r part of the apical shafts than on the basal arborization of the

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Fig. 3. Results from the comparison between the mean dendritic densities of the same groups of neurons of Fig. 2 after rotating the cells 90° around an axis parallel to their apical shafts. The unitary square used to obtain this pattern had 40-/~m sides. For meaning of symbols see text.

262 neurons studied. In o r d e r to test that these results were not biased by the anisotropy of the dendritic structure, the same type of analysis was p e r f o r m e d after rotating the cells 90 ° around an axis parallel to the neuron apical shaft. Fig. 3 shows the results obtained from this second comparison, confirming that the effect of thyroidectomy is m o r e intensive on the upper region of the cells. To complete this previous study the relative effect induced by t h y r o i d e c t o m y on the dendritic density with respect to its control value was analyzed. F o r this purpose, the c o m p u t e r further found the relative differences between each pair of h o m o l o g o u s elements of the two mean matrices with respect to its corresponding control value ( ( C - T ) / C ) , giving the result of these calculations in the form of an alphanumerical pattern. Those positions of the dendritic field where the c o m p u t e r has found dendrites of C neurons and not of T cells are indicated on the pattern with the letter E, representing with the letter C those positions where it has e n c o u n t e r e d the opposite situation. The percentage of increase or decrease in the mean dendritic densities are indicated by numbers ranging from +9 to - 9 ( n u m b e r 9 indicating 90% of the control value). Fig. 4 shows the alphanumerical pattern obtained from this study; although this result does not imply any statistical level of significance it is noticeable that most of letters E are located on the upper part of the neurons, revealing that the cells belonging to T animals stopped growing in this region. All the results here obtained are in a g r e e m e n t with previous findings concerning the effect of thyroidec-

1 Berbel, P.J., Escobar del Rey, F., Morrelae de Escobar, G. and Ruiz-Macros, A., Effect of neonatal hypothyroidism on the microtubule density and arrengement in apical dendrites of pyramidal cells of the rat visual cortex, Neurosci. Lett., Suppl. 14 (1983) $26. 2 Berbel, P.J., Escobar del Rey, F., Morreale de Escobar, G. and Ruiz-Marcos, A., Differential effect of neonatal hypothyroidism on myelinated process in different layers of the cerebral cortex of the rat, Trab. Inst. Cajal, LXXV, Suppl. (1984) 54. 3 Cox, J.R. and Small, N.J.H., Testing multivariate normality, Biometrika, 65 (1978) 263-272. 4 Eayrs, J.T., Thyroid and developing brain: anatomical and behavioural effects. In M. Hamburgh and B.J. Barrington (Eds.), Hormones in Development, Appleton-CenturyCrofts, New York, 1971, pp. 345-355. 5 Ruiz-Marcos, A., Mathematical models of cortical struc-

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E E E E E E E E E E E E E E & 6 9 8 9 9 E 7 3 7 6 8 E 9 E E E E E 9 7 8 7 6 - 7 E E E E 9 6 4 3 4 4 - 2 2 9 E E E 7 ~ 4 - 3 3 1 1 3 8 7 E E E E 9 2 - 9 - 4 2 2 - 1 - 3 4 E E EEE9&-3-812-7-38EEE 6 3 3 - I I - 1 3 9 E E C - 9 2 1 1 4 4 4 94111-1-51 32-4-2-2-I-148E -2-312X-II-IE El-2-1-2112E 2 2 1 3 - 1 1 2 5 9 517-5-261E 6-39-995 C

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Fig. 4. Alphanumerical pattern showing the relative differences between each pair of homologous elements of the ACRONs corresponding to the two groups of neurons compared (C vs T). For meaning of symbols see text.

tomy on the d e v e l o p m e n t of different layers of the cerebral cortex, according to which the effect of this disease on the d e v e l o p m e n t of dendritic spines 7, myelinated profiles 2 and microtubule densities 1 increase with the distance to the cell b o d y of the neurons. This work was s u p p o r t e d by Grants 3360/1 and 661/154 to A / R . - M . from the Comision A s e s o r a de Investigacion Cientifica y Tecnica. W e are grateful to Mrs. F e r n a n d e z de Molina for typing the manuscript and to Mrs. G a b r i e l de Ruiz-Marcos for her assistance with the c o m p u t e r w o r k .

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tures and their application to the study of pathological situations. In S. Grisolia, C. Guerri, F. Samson, S. Norton and F. Reinoso-Suarez (Eds.), Ramon y Cajal's Contribution to the Neurosciences, Elsevier, Amsterdam, 1983, pp. 209-222. Ruiz-Marcos, A., Sanchez-Toscano, F., Escobar del Rey, F. and Morreale de Escobar, G., Severe hypothyroidism and the maturation of the rat cerebral cortex, Brain Res., 162 (1979) 315-329. Ruiz-Marcos, A., Sanchez-Toscano, F., Obregon, M.J., Escobar del Rey, F. and Morreale de Escobar, G., Thyroxine treatment and recovery of hypothyroidism-induced pyramidal cell damage, Brain Res., 239 (1982) 559-574. Snedecor, G.W. and Cochran, W.G., Statistical Methods, Iowa State University Press, Ames, IO, 1967, pp. 259-339. Sokal, R.R. and Rohlf, F.J., Biometry, Freeman, San Francisco, 1969, pp. 689,780-798.