Programmed cell death enhances uniformity in rat cerebral hemispheres.

Programmed cell death (apoptosis) in rat cerebral hemispheres (CH) reportedly occurs around postnatal day 7 and kills 15-75% of all cells whose continuing presence would be of disadvantage for the organism: neurons erroneously connected, or supranumerary neurons that do not find targets and are not protected by neurotrophins. In the present paper we report that apoptosis (or a concomitant phenomenon) reduces also variability (coefficient of variation, CV) of CH parameters: weight, DNA content and protein content (presented here as a percent of the mean of each of these parameters). Postapoptotic brains have significantly lower CV of these parameters than the preapoptotic brains. Presumably, this trend toward uniformity offers selective advantages (for rat) and established itself during evolution. The mechanisms and the control of this phenomenon are at present largely unknown.

Quantitative study of DNA contents (cell numbers) in parts of the rat brain.

DNA contents (cell numbers) of rat brain parts were determined for cerebral hemispheres, cerebral cortex, cerebellum, and brain stems at postnatal ages 30, 60, 90, and 120 days. It was found that for all these parts these contents reach their maxima (maturation plateaux) at the same age (weaning; 30 days). Thus, cells in these brain parts attain mature numbers in concert. The significance and the possible mechanisms to achieve such close synchrony are discussed.

Neonatal brain parameters: outstanding tails of normal distribution.

Previous studies were concerned with distribution curves of values of brain parameters (weight, DNA or cell number, and protein) in a population of 720 neonatal rats. These values followed normal distribution curves, and the tails of such curves, with values in excess of two standard deviations (SD) above (right hand) or below (left hand) the mean (X), were statistically significant. In the present work a larger sample (1,948) of neonatal rats, with particular reference to those whose brain parameters exceeded three SD above or below X, respectively, was examined. Such occurrences are rare; they are somewhat skewed with respect to numbers expected in the tails of a normal distribution curve. For DNA the highest deviations from X were for 'maximal' brain, +3.62 SD (right tail); and for 'minimal' brain, -4.33 SD (left tail). Further study suggested that one of the factors increasing the maximal values might be a lower number in the litter, and one of the factors decreasing the minimal values was a lower maternal weight at conception. Thus the factors affecting maximal and minimal brains are not the same. The occurrence of such individuals with maximal or minimal values of neonatal brain parameters is of particular interest if these parameters are correlated with behavioural performance.

Differential effects of chronic ingestion of tritiated water on prenatal brain development.

Female rats were given tritiated drinking water (3 microCi/ml) from 30 days before mating up to and throughout pregnancy. At this low dose, the course and the outcome of pregnancy were normal. The differences between newborn body and cerebral weights of the treated rats and those of control animals were on the borderline of significance. In contrast, cerebral DNA and cerebral protein were highly significantly lower. In 30-35% of the treated population the DNA and protein values were more than 2 standard deviations (SD) below the mean values for the control population. Thus the number of the progeny of the mothers exposed to tritiated water that were considered to have "outstandingly low" levels of DNA and protein was 14-17 times greater than in the control group. The irradiated population also had 3-5% of severely affected individuals with cerebral DNA and protein values more than 2 SD below the mean of the experimental population. However, even in this irradiated population, a certain number of individuals did not show the effects of radiation as indicated by DNA and protein values that were not lower than the mean values of the control population. These animals may represent those individuals in which normal (control) populations would have cerebral DNA and protein levels well above the average (by 1 to 2 SD), but here, because of prenatal irradiation, were about average. If so, this lack of effect of irradiation cannot be caused by protection against radiation. However, at present, a differential repair mechanism of protection in those animals (or their mothers) that showed little or no effect of irradiation cannot be excluded.

Quantitative studies of mitoses: hippocampus of neonatal rat.

In continuation of our quantitative studies of mitoses in 15-day fetal rat brain, we have now studied mitoses in hippocampus of neonatal rat. In contrast to cortex in which neuroblast mitoses terminate prenatally, hippocampus in rodents has been known to exhibit mitoses well past birth. Our present study suggests that at birth (rat) 61% of these mitoses are situated in the medioventral tip of the germinal (ventricular and sub-ventricular) zone of lateral ventricle. However, the remaining 39% of mitoses may not be neural but represent mitoses of brain capillaries, similar to "deep mitoses" which we reported in 15-day fetuses. Like these, some of the neonatal mitoses are situated not in the germinal layer of the ventricle, but 44 +/- 21 microns deeper, near Ammon's horn or dentate gyrus; also, as in these previous studies, the spindles of all deep mitoses are not parallel to ventricular or hippocampal structures but perpendicular, presumably to promote their radial expansion known for capillaries at that age. Possibilities are considered that capillary cells are produced in excess and eventually undergo natural "capillary death", to follow natural "neuronal death".

Selective sensitive period in chick embryo: toxic effect of exogenous neurotransmitters.

The phenomenon of sudden mortality in the chick embryo, induced by the exogenous neurotransmitters norepinephrine, epinephrine, dopamine, serotonin, carbachol, and by the beta-blockers propranolol and atenolol, is described. When introduced singly into eggs (albumen) in doses of 0.1 mg per egg, these substances induced highly significant, sharply increased mortality (60% to 100%) when the introduction was on embryonic (incubation) days 8 to 12 (hereafter called E8 and E12), but none if introduction was earlier. These ages follow the appearance of functional receptors (or receptor-effectors) for these substances. However, the involvement of receptors here has not been proved. Solvent alone (Ringer's solution for chick) had no effect, indicating that the procedures used were in themselves not lethal. Possibly, the above neurotransmitters became lethal to the embryo because they were introduced in excess of the amounts which were produced and needed at that time by the embryo for optimal development.

Toxic effects of phencyclidine on developing chick embryo brain.

We studied the effects of Phencyclidine (PCP, Angel Dust) on the developing chick embryo brain. In Group-1, the eggs were injected with PCP on the 7th day of incubation and the embryo brains were studied on the 10th day. In Group-2, eggs were injected twice; first on the 7th day and then on the 10th day of incubation. Group-2 brains were then studied on the 16th day of incubation. PCP significantly depressed the development of embryo brains. Cerebral hemisphere weight, total protein and total DNA were significantly lower on day 10 of incubation in Group-1. Similar results were observed in Group-2. Concomitantly, the concentration of brain serotonin at day 10 was also significantly reduced when PCP was injected into the eggs on the 7th day of incubation. Since serotonin has been reported to influence development of the chick embryo brain, the present finding of the effect of PCP on brain development might be a secondary phenomenon. The possible implications of the effects of PCP on human brain development are also discussed.

Quantitative studies of mitoses in fetal rat brain: orientations of the spindles.

In continuation of our quantitative studies on 15-day fetal rat brain development, evidence was obtained of ventricular mitoses whose spindles are perpendicular to the surface of the ventricle. In most locations the proportion (ratio) between cells with perpendicular mitoses and predominant cells with mitoses parallel to ventricle is only 0.064 (+/- 0.057): 1; however, in two specific locations of lamina terminalis this proportion is much higher: 2.006 (+/- 1.24): 1 and 0.634 (+/- 0.42): 1. Total mitotic counts in these two locations were also higher: 1.8 and 1.5 X higher than the average elsewhere in cerebral hemispheres. It is suggested that these perpendicular orientations are not accidental but serve some specific purpose: either they give rise to a different cell population, or they represent a stage preparatory for cell migration.

Effect of intrauterine positions on fetal brain development in the rat.

The effect of the position on the horn on fetal brain development was studied in 2276 rats (248 litters), by assessing the intrauterine location of those fetuses which had the highest values ('maximal fetuses') of the following parameters: body weight, brain weight, brain DNA (cell number), brain protein and the ratios brain weight/body weight and brain protein/brain DNA. The fetuses were at term (21 days) or at 16-18 days postcoitum. The total numbers of animals in the right and in the left uterine horn were essentially the same: 4.64 and 4.54, respectively. For each parameter, the locations of maximal fetuses were, on the average, symmetrical with respect to the center of each horn. The distributions of maximal fetuses along each horn followed specific patterns, which were different for each horn but showed similar locations of peaks and depressions for various parameters. However, the differences between the peaks and depressions were not statistically significant. It is concluded that both horns are equipotent, and all the positions on the horn are also essentially equipotent for production of animals maximal with respect to parameters studied. This also suggests that occasional cases of outstanding high fetal brain development occur from causes other than a favorable position on the uterine horn.

Quantitative studies of mitoses in cerebral hemispheres of fetal rats.

In continuation of our study of 15-day fetal rat brain development, evidence was obtained of a single layer of mitoses, situated away from the ventricle at a mean depth of 75% of the thickness of the cell layer of cerebral hemispheres ('deep mitoses'). Their number is 17.9% of ventricular mitoses and their spindles are perpendicular to spindles of ventricular mitoses. Their distribution is not correlated with distribution of ventricular mitoses. The fate and destination of these deep mitotic cells is at present unknown; the possibility that they are mitoses of the capillaries has been discussed.

Differential effects of antifolate on the development of brain parts in chick embryos.

A quantitative study was made of the differential effects of the antifolate amethopterin (methotrexate) on the development of cerebral hemispheres, optic lobes and cerebellum in chick embryos. Most surviving embryos show a significant under-development at day 18 of incubation. Different brain parts respond differently (disproportionately) to the treatment: cerebellum--most drastically, optic lobes--least drastically (net weights). Within the cerebellum, Purkinje and other large cells respond less drastically than small cells. The embryo thus had highly significantly changed ratios of weights of various brain parts and of numbers of various cerebellar cells. Several mechanisms to explain these differential effects have been discussed.

Litter size, maternal parameters, and brain and body parameters of neonatal rats.

The present work is concerned with the effect of natural litter size in the rat on the brain and body parameters of individual newborns, with particular reference to those newborns that were 'outstanding' (i.e., had parameters higher than mean +/- 2 SD). Correlations between litter size on one side, and length of gestation, number of stillborns, maternal weight at conception and maternal food consumption were also studied. To our knowledge, no previous report presented a similar integrated study on such a large number of rats (288 litters, 2,725 newborns) and wide range of litter size (2-17).

Effects of prenatal and chronic undernutrition on aging and survival in rats.

The effect on aging and survival of mild (2/3 of ad lib) chronic prenatal and postnatal undernutrition over 10 generations, or prenatal undernutrition only has been studied in rats. Both regimes of undernutrition used decreased body weights of animals as compared to the controls. Both undernourished groups had lower cholesterol levels and tumor incidence than the controls. The effects on survival depended not only on the time period of undernutrition but also on the age of the animals examined: chronic undernutrition resulted in lower survival of young adult animals (4-8 months) but higher survival than the controls in old age. Prenatal undernutrition only had no effect on young adult animals, gave higher survival than the controls at ages 8-18 months but considerably lower survival than controls in the old age: the underdevelopment of vital organs whose cells proliferate only before birth might have been the cause of such decreased longevity.

Quantitative study of fetal brain development in individual rats.

The development of cerebral hemispheres was quantitatively studied in twenty 15-day-old rat fetuses from two liters. Thicknesses, areas and mitotic counts (per mm length of ependyma) were not different of left and right hemispheres (parasagittal paraffin sections). On the other hand, among individual fetuses of the same litter, and areas of cerebral hemispheres differed as much as 2 standard deviations, and these values were not correlated with fetal weights (age). It appears that individual fetuses of the same litter and age may differ in the extent of their brain development.

Quantitative studies of variabilities and of nutrient partition between cerebral hemispheres, optic lobes, cerebella and bodies of individual chick embryos.

The extent of variability in weight, DNA content and protein content of cerebral hemispheres, optic lobes and cerebella of individual brains has been studied in newly hatched chicks in which these parts can be easily delineated and quantitatively dissected out. The variabilities were within narrow ranges; nevertheless, for some individuals the above parameters were significantly higher. Correlations between such parameter values for egg and body weights, and different brain parts of the same animals were also studied. Body weights were significantly correlated with egg weights. Cerebellar weights were significantly correlated with both egg weights and body weights, but cerebral hemispheres weights and optic lobes weights were not. Thus, larger body necessitates larger cerebellum, and this variability appears to be environmental (amount of egg nutrients) rather than genetic. On the other hand, in the chick larger body does not necessitate larger cerebral hemispheres and larger optic lobes.

Distribution of nutrients between fetal brain body during rat development.

During prenatal development, the distribution of nutrients between brain and body is influenced by many factors. Fetal and neonatal ratios may serve as indices of the final outcome; brain weight/body weight (Rw), brain DNA/body DNA (RDNA) and brain protein/body protein (Rprot; similar to Rw). These ratios decrease with fetal age but this dependence on age disappears towards term. Within one age, these ratios show considerable variability (up to 14%), from litter to litter, and within litters. The variability is not due to variation in water contents ( less than 1%). An extensive study of 2,089 normal newborns revealed that 7.5% had Rw values significantly higher than the mean for the population. Of these, 35% had normal body weights but significantly higher brain weights. Thus, they do not represent previously studied cases of general (brain and body) overdevelopment; rather, they represent cases of favorable brain versus body growth, i.e. distribution of nutrients between brain and body that is more favorable for the brain. Such a favorable distribution may be caused by maternal factors if it affects entire litters (3% of all affected) or by fetal factors, if it affects only individuals within a litter (49%).

Maternal nutrient storage and efficiency in production of fetal brain tissue in rats.

Maternal 'efficiencies' in production of fetal tissue, especially brain tissue, were studied in controls and in chronically (9 generations) undernourished rats. These 'efficiencies' were formulated as the ratio of a neonatal parameter (body) weight, brain weight, DNA, or protein) to the food consumed during pregnancy. Mean or total values (for the entire litter) of the above parameters were used in computation of the above ratios. It was found that all these ratios were highly significantly lower for control animals than for undernourished. The gain in maternal body weight (postpartum versus day 0 of pregnancy), i.e., nutrient storage, was found to be significantly lower or even negative in the undernourished group, but increased through generations. We interpret these results as follows. Undernourished animals mobile their nutrient reserves, avoid deamination of essential amino acids, and improve their intestinal absorption of nutrients; thus, they are more efficient than normal animals, even though the latter may produce more fetal tissue. These improvements suggest inducible enzymes. Individual mothers in each group vary considerably in their efficiency; the most efficient undernourished mothers may produce offspring that escape undernutrition, or, in the control group, offspring with outstanding values of brain and body parameters.