An autoradiographic and morphological investigation of the postnatal development of the pineal body.

Cell proliferation in the pineal body of the hooded rat was studied with thymidine-H:' autoradiography. Animals were either injected at birth and allowed to survive for variable periods or were injected at different ages and allowed to survive for a fixed period. Cell proliferation was high in the neonate (one and six hours of age) and continued at a decreasing rate into adulthood. The final development of the pineal body was believed to be due to cellular hyperplasia in the young animal and cellular hypertrophy in the adult. The morphological evaluation of the autoradiograms indicated that the principal proliferating components were parenchymal cells. Earlier autoradiographic studies revealed the presence of labeled cells in many regions of the brain in rats after injection of thymidine-H3 during infancy (Altman and Das, '65, '66). These postnatally forming cells were interpreted to be precursors of either neurons with short axons or of neuroglia cells and other supporting elements. In these studies it was observed that a large proportion of cells in endocrine glands associated with the brain, namely the pituitary and pineal, were labeled. This paper is concerned with an evaluation of cell proliferation in the pineal body combined with identification of the cell types formed. Studies dealing with development of the pineal body have shown that in the mouse, mitotic activity drops from a neonatal maximum to a low level by two weeks (Dill and Walker, '66); data obtained by counting mitotic figures in the rat, showed that cell proliferation is rapid up to two weeks of age and then drops to a low frequency that continues into the adult period (Quay and Levine, '57). After two weeks of age the size of the pineal body increases up to about ten weeks; this change has been attributed primarily to parenchymal cell hypertrophy (Izawa, '25; Quay and Levine, '5 7). Autoradiographic material obtained from earlier studies seemed to indicate that proliferation in the rat pineal body continued for a longer time and at a higher rate than had been suggested by prior investigations. Accordingly this study was carried out in an attempt to resolve this question concerning cell proliferation in the pineal body using the technique of fineresolution thymidine-H3 autoradiography. MATERIALS AND METHODS In this study Long-Evans rats were used. The animals were given intraperitoneal injections of thymidine-H3, dissolved in isotonic saline at a dose of 10 pclgm body weight, (1.0 mc/ml; specific activity 6.7 ClmM). The younger animals were sacrificed by decapitation with immediate immersion of the heads in 10% neutral formalin; the older animals were sacrificed by cardiac perfusion with formalin. The brains were fixed in formalin for a minimum of one week and then embedded in Paraplast. Serial sections were cut at 6 p and three consecutive sections (or set of sections) were preserved out of every 10 to 30. Of the preserved sections, two sets were stained with Einarson's modification of gallocyanin-chromalum; one of these was coated in the dark with Kodak NTB-3 nuclear emulsion by the dipping technique, exposed at 5°C for 91 days and then developed. The procedures are described in more detail elsewhere (Altman, '64). In addition to the gallocyanin-chromalum stained control sections, sagittal sections were obtained from several adult animals and were stained with cresylecht-violet. Quantitative autoradiographic studies using thymidine-H3 and aimed at estimat_ ~ 1 Now at University of Hartford, 200 Bloomfield Avenue, 2 Now at Purdue University. Department of Biological Sciences. West Hartford Connecticut West Lafayette, Indiana 175 AM. J. ANAT., 126. 175-184 176 R. B. WALLACE, J. ALTMAN AND G. D. DAS ing rates of cell proliferation are predicated on the assumption that thymidine-HR injected in a single dose remains available for a limited period and that the cells that are in the S phase at the time of injection are the only ones that will utilize and retain the radiochemical (Hughes et al., '58; Cronkite et al., '59; Messier and Leblond, '60). The other assumption (Hughes, '59) is that DNA is metabolically stable and, therefore, thymidine-HJ is lost only when the cell multiplies and half of its DNA is transferred to its daughter cells. Hence, continued multiplication of already labeled cells will lead to an increase in number of labeled cells combined with dilution of the radiochemical and reduction in the number of silver grains over cell nuclei. The various possible relationships between number of labeled cells and concentration of label within the cells in populations with different rates and time course of multiplication have been indicated in an earlier study dealing with the general topic (Altman and Das, '56). A summary of the design of the experiment with respect to age of the animals and survival time after injection is presented in table 1. The variability in proportions of labeled cells in the replicated pineals is between 5 and 8%. The animals were maintained on an 8:oo AM to 8 : O O PM photoperiod with the injections administered, with the exception of the six hour group where injection was determined by the time of birth, at approximately the midway point in the cycle. There are