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Six groups of pregnant dams were each fed a 20%, 12%, and 6% protein diet with and without caffeine (2 mg/100 g b.wt), starting on day 7 of gestation. Dams of each group were injected with two successive daily doses of 3H-thymidine in an overlapping series beginning on day 7 and ending on day 16 of gestation. At day 15 postbirth, brains of the offspring were removed, fixed and embedded. Neurogenesis in the rat trigeminal motor nucleus was examined by radiogram in serial sections. In general, the peak production of the neurons of the motor nucleus of V in the malnourished group of the control was on embryonic (E) days E10 + 11, whereas that of the caffeine group was E11 + 12. In the normally nourished group, peak production in the control was E9 + 10, whereas in the caffeine group it was E11 + 12. Trigeminal areas from the brains of pups of each group, which were kept frozen from our previous studies on suckling behavior (23), were used to analyze DNA, protein and cholesterol levels in the present study. At day 1 caffeine's effects on DNA, protein and cholesterol concentrations of the trigeminal area between the groups were different, depending upon the nutritional status. At day 15, those parameters in the caffeine group were greater although no caffeine was added to the lactating dams' diet. This study again demonstrated that prenatal caffeine intake in combination with protein-energy malnutrition produces permanent effects on the trigeminal nuclear center indicated by autoradiography and changes in biochemical parameters.

Six groups of pregnant dams were fed a 20%, 12%, and 6% protein diet with and without caffeine (2 mg/100 g b.wt.), starting on day 7 of gestation. At day 18 of gestation, randomly selected dams of each group were used to record prenatal fetal behavior. The remaining dams were continuously fed their respective diets until the birth of their pups. Upon delivery, newborn pups from the dams fed a 20%, 12%, or 6% protein diet with caffeine were placed with foster dams that the dietary regimen during gestation was a 20%, 12%, or 6% protein diet, respectively. Dams fed a noncaffeine diet, along with their newborns, were fed their respective diets until day 15. Suckling behavior tests for newborns were conducted on days 2, 8, and 15. On day 15, both nondeprived and deprived newborn rats were studied. Caffeine in combination with a malnourished diet has different effects on general activity in prenatal stages compared to postnatal stages. Our findings support the view that prenatal exposure to caffeine may produce greater effects because: a) caffeine and its metabolites pass freely into the embryo and attain a concentration slightly lower than in the maternal plasma; and b) caffeine may be poorly metabolized during pregnancy, causing an accumulation in the fetal tissues. Prenatal caffeine at the dosage we have used in combination with malnutrition may produce lasting metabolic alterations in the nervous system related to the emergence of suckling behavior and general motor activity.

1. The growth of cardiac cells derived from newborn rats whose dams were either malnourished or malnourished with caffeine during pregnancy was inhibited in culture over the period of 5 days as compared to that of the normally nourished cells. 2. Cells derived from malnourished rats with caffeine added to their diets showed a greater inhibition than those from the malnourished rats not given caffeine. 3. Both DNA and protein synthesis showed an inhibition due to caffeine in a dose-dependent manner using normally nourished cells. 4. In the presence of exogenous 2 mM caffeine, the degree of percent inhibition of DNA and protein synthesis of cells derived from rats malnourished with caffeine was less than that from the rats with malnutrition alone. 5. The present data indicated that malnutrition combined with caffeine during pregnancy exerted a greater negative effect on the nature of cell growth than malnutrition alone and these cells became less sensitive to exogenous caffeine.

The effects of propylthiouracil (PTU) on the ontogeny of suckling behavior in rats were examined. The drug was given at two dosage levels of 0.3% and 0.5% respectively mixed with rat diet throughout gestation and suckling. The thyroid glands of treated fetuses and pups and of untreated control animals of the same age groups were monitored by histologic examination. At the behavioral level, the frequency of individual movements of head, forelimbs and mouth was significantly reduced in treated fetuses. The combination movements of head, mouth and forelimbs showed severe deficits both quantitatively and qualitatively for all ages in the experimental group from day 18 of gestation. Hypothyroid pups of dams raised on 0.5% PTU were unable to attach to the nipple of the mother and died within a few days. Pups of dams raised on 0.3% PTU showed longer latencies for nipple attachment, and their gross motor movements of rooting and suckling were greatly impaired. These results have been discussed in relation to the development of suckling behavior to indicate that, during ontogeny, some decisive step in the integration of individual movements takes place in utero from day 18 of gestation. This coincides with the establishment of pituitary thyroid relationship, which is continued through postnatal stages.

Quantitative light and electron microscopy, together with Golgi methodology, were used to study alterations in retino-fugal terminals and postsynaptic neurons within the dorsal lateral geniculate nucleus of the rat at various intervals following inhibition of axoplasmic transport in the optic nerve induced by intraocular injections of colchicine from 1-20 days postnatal. Colchicine concentrations used in this study ranged from 10-5 M-10-2 M. These were selected on the basis of our measurements of axon transport suppression described in the preceding article. 61. The volume of the nucleus was determined by section planimetry and reconstruction. Growth of the contralateral dorsal lateral geniculate nucleus was significantly retarded by intraocular colchicine at 1 and 5 days of age, only achieving a volume of between 61-75% of the normal or the control (ipsilateral) nucleus depending upon dosage. Application of colchicine at 10 days of age resulted in minimal stunting of nuclear growth, (79-93% of normal). Mean numbers of neurons in the contralateral and ipsilateral nucleus remained stable throughout all postnatal ages examined suggesting that nuclear volume loss was caused principally by a reduction in the amount of neuropil. Golgi impregnation displayed dendritic stunting in relay neurons characterized by narrowing of the portion of the shaft between 40-80 micrometer from the soma and a reduced incidence of spinous protrusions, particularly those shown by other studies to engage the retino-fungal terminal. 27,69 A concomitant Sholl76 analysis of dendritic branching in relay neurons demonstrated no significant differences in the number of intersections between normal and experimental nuclei. No alterations were observed in intrinsic neurons. Electron microscopy of postsynaptic neurons following concentrations ranging from 10-4 M at birth revealed altered patterns of granular endoplasmic reticulum in many cells characterized by reduced numbers of cisternae and scattered instances of cisternal dilation, together with enhanced infolding of the nuclear membrane at 20 days postnatal. Those animals which were given 5 X 10-3 M-10-2 M colchicine demonstrated an increased incidence of cisternal dilation, loss of ribosomes, disruption of the nuclear membrane and occasionally, complete degeneration. A similar array of alterations took place following intraocular injection at 5 days of age; however, animals receiving colchicine at 10 days postnatal displayed minimal alterations in relay neurons. Synaptic glomeruli, which contain the retinofugal terminal, displayed dose and age-dependent reduction in the size of the presynaptic element of the complex following intraocular colchicine, together with fewer post-synaptic spinous protrusions. Synaptic vesicles remained normal in appearance and distribution and our quantitative analysis demonstrated no loss of such terminals in accordance with colchicine concentrations which were previously found not to be lethal to retinal ganglion cells and optic axons.

Opossum fetuses undergo most of their neurological development within the mother's pouch. This extrauterine growth offers unique opportunities not shared by placenta mammals for early behavior studies. Described here are an apparatus and technique for observing the behavior of pouch young. The apparatus restrains the mother and retracts the pouch flaps within an environmentally controlled setting similar to that inside the mother's pouch, allowing the opossum young to be observed in an almost natural state. The investigator has ready access to the young pouch through side windows that have slotted curtains to prevent sudden changes in the chamber environment. Because this apparatus provides a simple noninvasive approach, fetal behavior can be observed long-term, encompassing the entire sequence of behavior development within a chosen time-interval.