Combined effects of caffeine and malnutrition on the development of the trigeminal nuclear center: autoradiographic and biochemical studies.

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.

Combined effects of caffeine and malnutrition during pregnancy on suckling behavior in newborn rats.

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.

Effects of caffeine on the DNA and protein synthesis of the protein-energy malnourished neonatal cardiac muscle cells in culture.

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.

Effect of caffeine and zinc on DNA and protein synthesis of neonatal rat cardiac muscle cell in culture.

The effect of caffeine and/or zinc on DNA and protein synthesis of purified neonatal-rat ventricular cardiac myocytes was studied. Caffeine (0.2-2 mM) inhibited both DNA and protein synthesis of the cells. Addition of EDTA in the growth medium inhibited both DNA and protein synthesis. Without caffeine and in the presence of lower concentrations of caffeine (0.2 mM) in the growth medium, 10 microM of zinc concentration reversed DNA synthesis, which was inhibited by the chelating agent (EDTA). Higher concentrations of caffeine (2 mM) in the growth medium completely abolished sensitivity of cardiac myocytes to zinc. Additional zinc supplementation to the growth medium of cardiac myocytes did not alter the rate of protein synthesis. The present results suggest that the effect of caffeine on cardiac myocytes may be associated with the zinc-dependent enzymes involved in DNA synthesis.

Neuronal development in the trigeminal mesencephalic nucleus of the duck under normal and hypothyroid states: I. A light microscopic morphometric analysis.

Light microscopic morphometric procedures were used in order to examine the effects of propylthiouracil (PTU) on the development of the mesencephalic nucleus of the trigeminal nerve in the duck. A single vascular injection of a 0.2% solution of PTU was administered at a dosage of 2 microliter/gm embryo weight on embryonic day nine (E9). Control embryos received a similar dose of Ringer's solution. The following parameters of cytodifferentiation of cells of the mesencephalic nucleus of V were studied: somal area profiles, nuclear area, and nuclear cytoplasmic ratios. In addition, the frequency of beak clapping was recorded from E16. Significant differences were observed in somal area profiles in the experimental group at E16 and E18 and in nuclear area profiles from E16 through hatching. Beak activity in the experimental embryos was drastically reduced. It is concluded that PTU induces a retardation in the differentiation of cells of the mesencephalic nucleus of V which may lead to behavior deficits as evidenced by reduction of beak activity. These observations provide a basis for the study of interactions between thyroid hormone and specific neuronal systems in the emergence of an adaptive function.

Development of the spinal tract of the trigeminal nerve and its relation to early fetal behavior in rats under normal and hypothyroid conditions.

The effects of hypothyroidism on the development of the spinal tract of V and its relation to early fetal behavior were studied in rats from day 16 through day 20 of gestation. Hypothyroidism was induced by the administration of 0.5% Propylthiouracil mixed with rat diet beginning from day seven through term. The thyroid glands of treated and untreated control fetuses of the same age groups were examined in histologic sections. The position, size, and caudal extent of the ophthalmic and maxillomandibular divisions of the spinal tract of V were analyzed in detail with the aid of graphic reconstructions from serial sections of representative cases of both control and experimental fetuses. The ophthalmic and maxillomandibular divisions of the spinal tract of V at 15 days in the control group of fetuses extended into the second and fourth cervical spinal segments respectively, while by 19 days, both divisions of the spinal tract of V extended as far as the fourth cervical segment. In the treated group of fetuses, the ophthalmic and maxillomandibular divisions were greatly reduced in area as well as in their caudal extent into upper cervical levels compared to control groups at corresponding stages of development. At the behavioral level, the frequency of individual movements of the head, forelimbs and mouth in the treated group of fetuses was significantly reduced compared with control fetuses of the same age. Combination types of movements of head, mouth and forelimbs were severely affected both quantitatively and qualitatively in the treated group from day 18 of gestation age. It could be demonstrated that the caudal extent of the spinal tract of V corresponded very closely to the behavior repertoire seen in the fetuses of that age. We propose (1) that the spinal tract of V, in its relation with the upper cervical spinal cord levels which contain motor neurons of the spinal accessory nucleus and motor neurons which innervate the dorsal neck muscles, could play a decisive role in the integration of head and shoulder movements in early stages of development, and (2) that thyroid hormone may play a crucial role in the normal development of the spinal tract of V which is manifest in its caudal growth into upper cervical levels of the spinal cord.

Cell formation in the motor nucleus and mesencephalic nucleus of the trigeminal nerve of rats made hypothyroid by propylthiouracil.

Neurogenesis in the motor and mesencephalic nuclei of the trigeminal nerve was examined using autoradiographic techniques. Two groups of pregnant rats (control and experimental) were injected with two successive daily doses of 3H thymidine in an overlapping series starting from day nine of gestation in order to label in their progeny, the dividing precursor of neurons of the motor nucleus and mesencephalic nucleus of the trigeminal nerve. Control group of rats was raised on a standard diet, while the experimental group was made hypothyroid by propylthiouracil (PTU). At postnatal ages ranging between 20-30 days in the pups of both the control group and experimental group, the percentage of cells labelled and the proportion of cells added during each embryonic day were determined quantitatively throughout the rostro-caudal extent for both motor and mesencephalic nuclei. The neurons of the mesencephalic nucleus undergo their final cell divisions between gestational days 9 and 10 (E9 and E10). More than 80% of the population is generated by E10. The neurons of the motor nucleus undergo their final cell divisions between E9 and E11, and nearly 88% of the cells is generated by E11. In the thyroid deficient rats, in both nuclear centers, only 61% of the cells is generated by E12, and labelled cells are observed even as late as E18 and E19. In the hypothyroid state, there is a significant lengthening of the proliferative period. On the basis of absolute datings and duration of neuron production, it is postulated that in normal development, thyroid hormone determines the duration of the proliferative period, and push cells into the differentiative phase by taking them out of the proliferative phase.

Effects of induced thyroid deficiency on the development of suckling behavior in rats.

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.

Abnormal differentiation of selected nuclear centers in the brain of a duck embryo associated with partial duplication of the primitive streak.

In a control set of duck embryos, an abnormal duck embryo of 16 days incubation was found which had two beaks as the only outward sign of duplication. The beaks were of equal size, each with upper and lower bills. Bill-clapping movements were absent. The embryo had two normal eyes placed one on either side of the head, and the rest of the body was normal in external appearance. Sections through the head revealed further duplication of the fore-, mid-, and hindbrain divisions. The medial half of each of the embryonic brain divisions, however, was greatly reduced. Two additional optic primordia were also noticed in sections, each of which was reduced to a mass of tissue representing a lens and a much-folded pigment epithelium. The orbital tissues associated with the rudimentary eyes were greatly disorganized. Abnormal differentiation associated with duplication of the brain divisions was determined by cell counts in selected nuclear centers. Cell numbers in each case appeared to be remarkably proportional to the size of the innervation field. Our data, based on cell counts in the nuclear centers chosen for this study in the abnormal embryo and normal control embryos of the same age, are consistent with the hypothesis that cell survival in related parts of the nervous system may be regulated by their peripheral field of innervation.

Inhibition of axoplasmic transport in the developing visual system of the rat-III. Electron microscopy and Golgi studies of retino-fugal synapses and post-synaptic neurons in the dorsal lateral geniculate nucleus.

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.

Ultrastructural and histochemical observations in the developing iris musculature in the chick.

Irises from chick embryos and from 2- and 3-week-old chicks were studied using ultrastructural and histochemical methods in order to clarify the relationship between cell loss in the ciliary ganglion and the establishment of permanent peripheral connections between the ciliary neurons and the iris muscle. The iris muscle undergoes morphological and biochemical differentiation between 11 and 13 days of incubation. This period coincides with the critical period in the development of the ciliary ganglion when massive cell degeneration occurs. During this period, the iris develops typical sarcomeric structure, with AChE activity in the nuclear envelope, Golgi, and the "T' system. At 15 days of incubation AChE activity is found localized in discrete areas on the muscle fiber, forming specific neuromuscular junctions. Between 13 and 15 days of incubation, there is a shift in the localization of AChE activity in the iris muscle, from the sarcoplasmic structures to the junctional membranes. Few synaptic terminals are observed in the iris musculature prior to 11 days of incubation. There is a marked increase in the number of synaptic terminals between 11 and 13 days of incubation which also coincides with the period of cell loss in the ciliary ganglion. The establishment of neuromuscular junctions at 15 days of incubation corresponds with the period when the number of neurons in the ciliary ganglion has attained the adult level. The time table of the events described above, leads us to conclude that during development only those neurons in the ciliary ganglion which make peripheral contacts survive, and only such contacts differentiate into mature neuromuscular junctions on the iris muscle. This will imply that neurons which are doomed to die, although they may send out fibers to the periphery, do not make peripheral contacts before death.

An apparatus for observing behavior of pouch-young opossums.

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.

An analysis of the integrity of the brachial motor unit in the dystrophic chick embryo.

In New Hampshire chickens, the primary clinical symptom of dystrophy is limitation of wing motility. Examination of the brachial-level motor unit in chick embryos homozygous for dystrophy reveals abnormalities in both muscular and neural components. Wing motility in these embryos is abnormal as early as six days, and there is a corresponding lack of differentiation of the pectoralis major muscle. The findings suggest that delayed development of brachial-level neuronal pathways is responsible for the decreased wing motility and early degeneration of the pectoral muscle.

Neural crest and placodal contributions in the development of the glossopharyngeal-vagal complex in the chick.

By using the method of quail-to-chick transplantation of neural crest in one series (VNG) and placodal ectoderm in a second series (VPG) we were able to determine the relative contribution of cranial neural crest and placodal ectoderm to the formation of the Glossopharyngeal-vagal complex. In chimeric embryos, quail cells originating from cranial neural crest grafts of postotic levels end up in the root ganglia, while quail cells originating from placodal ectoderm of postotic levels end up in the trunk ganglia. The results clearly indicate that the caudal levels of the medulla and rostral cervical segments represent the site, and the neural crest the source, for the neurons of the root ganglia. The neurons form a homogenous population of the small-cell type. This clearly rules out any contribution to the root ganglia from placodal ectoderm. On the basis of our experiments, it is also concluded the the neurons of the trunk ganglia are purely placodal in origin and are composed of a populatin of cells of the large-cell type. Our experiments also provide convincing evidence for a neural crest origin for Schwann cell and ganglionic Satellite cells.

On the origin of the ciliary ganglion in birds studied by the method of interspecific transplantation of embryonic brain regions between quail and chick.

The development of the ciliary ganglion with reference to the site and cells of origin have been investigated by the method of quail-to-chick transplantation of embryonic fore-, mid-and hindbrain regions, and by the method of transplantation of cranial neural crest from specific brain levels. In chimaerical embryos, quail cells originating from the graft end up in the ciliary ganglion, only when the graft is from the midbrain level of quail embryo donors. In fore- and midbrain grafts the ciliary ganglia of chimaerical embryos are composed of chick cells only. The results indicate that the mesencephalon is the principal site for the precursor cells of the ciliary ganglia and clearly rules out any contribution to the ganglia from either the forebrain or hindbrain levels. In interspecific transplantation of cranial neural crest, quail cells originating from the graft are observed consistently in the ciliary ganglion of the operated side when the grafted neural crest material is derived from the mesencephalon of quail embryo donors. On the basis of the evidence provided, it is concluded that mesencephalon is the principal site and the cranial neural crest of this level the source from which the precursor neurons of the ciliary ganglia are derived.

Neuronal adjustments in developing nuclear centers of the chick embryo following transplantation of an additional optic primordium.

Following transplantation of an additional optic primordium into the orbital mesenchyme of chick embryos of approximately 2 days of incubation age, the changes in cell number in the ciliary ganglion, accessory oculomotor and trochlear nuclei were studied at various stages of development. Cell counts were made at 1-day intervals from days 9 through 15 for ciliary ganglion, and from days 13 through 15 for the accessory oculomotor and trochlear nuclei. Cell counts for the ciliary ganglion on days 9 and 11 were similar on the operated and control sides which suggests that grafting of an additional optic primordium, and thus enlarging the periphery, is not involved in the control of proliferation. Comparison of the number of cells for the ciliary ganglia and the accessory oculomotor nuclei at days 13 and 15 showed an increase on the affected side ranging from 8 to 27%, and 9 to 33% respectively. We interpret this increase on the experimental side as a reduction in the number of degenerating cells that occur in normal development, as a result of an enlargement of the peripheral field of innervation. Three cases showed an increase in the number of cells in the trochlear nucleus ranging from 9 to 29%. This increase was attributed to an increase in the size of the superior oblique muscle of the operated side as determined by volumetric measurements. On the basis of the evidence we conclude that an enlarged periphery acts by regulating the level of naturally occurring cell death by reducing the amount of cell loss, leading to a corresponding increase in final cell number.

Determination of the embryonic origin of the mesencephalic nucleus of the trigeminal nerve in birds.

The precise site of origin and the cells of origin of the mesencephalic nucleus of the trigeminal nerve in birds have been studied by the method of growing embryonic fore-, mid- and hind brain grafts inside a millipore filter chamber filled with amniotic fluid, upon a host chorioallantoic membrane, and by the method of transplantation of cranial neural crest of the mesencephalon between embryo-donors and duck embryo-hosts. The growth and differentiation of grafts grown in the chambers resemble very closely those of normal embryos at corresponding stages of development. Data obtained from a total of 42 grafts representative of the three species of birds used in this study, show that the cells of the mesencephalic nucleus are present only in grafts of embryonic midbrain and totally absent in forebrain and hindbrain grafts. The presence of cells of the mesencephalic nucleus in midbrain grafts suggests that these neurons arise chiefly from the developing mesencephalon. Histological examination of such grafts has shown that these neurons are observed in the leptomeningeal tissue overlying the midbrain region and extend into the various laminae of the optic tecta. A progressive increase in the size of the cells from an immature state confined to the mesodermal tissue to fully mature neurons in the stratum griseum periventriculare of the optic tectum is observed. This is also indicative of a downward migration of these cells. In a few embryonic midbrain grafts mature cells clearly identifiable as belonging to the mesencephalic nucleus are observed in the leptomeningeal tissue and outside the external limiting membrane of the neural tube. These appear to be neurons which had failed to migrate. The resemblance of these latter neurons to those in the stratum griseum periventriculare of the optic tectum lend further support to the hypothesis that the neurons of the mesencephalic nucleus are derived from neural crest of the developing mesencephalon in birds. In interspecific transplantation experiments of cranial neural crest of the mesencephalon, the quail cells occur in the form of clusters and appear to migrate towards the ventricular surface independent of the migration of the cells of the tectum which takes place simultaneously. Three stages in the development of the grafted neural crest material of the quail are observed based on the perikaryal diameter of the cells, and the accumulation of Nissl material in the cytoplasm. The smallest cells are located more superficially near the pial surface, and the larger mature neurons are observed in the stratum griseum periventriculare of the optic tectum. All the layers of the optic tectum including the neuroepithelium on the experimental side are comprised entirely of duck cells...

An experimental inquiry into the central source of preganglionic fibers to the chick ciliary ganglion.

Degenerative changes in the entire third nerve nucleus were studied following selective removal of the ciliary ganglion, usually of the right side, in day-old chicks. The operated animals were allowed to survive three, six, nine, and twelve days after ciliary ganglionectomy. Retrogarde degenerative changes proceed rapidly between three and nine days post-operatively, and are strictly limited to the ipsilateral accessory oculomotor nucleus. The changes are at first observed in the medial division of the accessory oculomotor nucleus, followed by the lateral division of the accessory oculomotor nucleus. By nine days following the operation, both the divisions of the accessory oculomotor nucleus are virtually depleted of almost all neurons. The other components of the oculomotor complex were not affected at any stage by ciliary ganglionectomy. These observations provide conclusive evidence that the accessory oculomotor nucleus is indeed the avian homologue of the Edinger-Westphal nucleus.