Spatiotemporal Development of the Orexinergic (Hypocretinergic) System in the Central Nervous System of Xenopus laevis.

The present immunohistochemical study represents a detailed spatiotemporal analysis of the localization of orexin-immunoreactive (OX-ir) cells and fibers throughout development in the brain of the anuran amphibian Xenopus laevis, a model frequently used in developmental studies. Anurans undergo remarkable physiological changes during the early life stages, and very little is known about the ontogeny and the localization of the centers that control functions such as appetite and feed ingestion in the developing brain. We examined the onset of the orexinergic system, demonstrated to be involved in appetite regulation, using antibodies against mammalian orexin-A and orexin-B peptides. Simultaneous detection of orexins with other territorial markers was used to assess the precise location of the orexinergic cells in the hypothalamus, analyzed within a segmental paradigm. Double staining of orexins and tyrosine hydroxylase served to evaluate possible interactions with the catecholaminergic systems. At early embryonic stages, the first OX-ir cells were detected in the hypothalamus and, soon after, long descending projections were observed through the brainstem to the spinal cord. As brain development proceeded, the double-staining techniques demonstrated that this OX-ir cell group was located in the suprachiasmatic nucleus within the alar hypothalamus. Throughout larval development, the number of OX-ir cells increased notably and a widespread fiber network that innervated the main areas of the forebrain and brainstem was progressively formed, including innervation in the posterior tubercle and mesencephalon, the locus coeruleus, and the nucleus of the solitary tract where catecholaminergic cells are present. In addition, orexinergic cells were detected in the preoptic area and the tuberal hypothalamus only at late prometamorphic stages. The final distribution pattern, largely similar to that of the adult, was achieved through metamorphic climax. The early expression of orexins in Xenopus suggests important roles in brain development in the embryonic period before feeding, and the progression of the temporal and spatial complexity of the orexinergic system might be correlated to the maturation of appetite control regulation, among other functions.

Development of prostaglandin endoperoxide synthase expression in the ovine fetal central nervous system and pituitary.

In this study, we tested the hypothesis that prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145days of gestational age (DGA), 1 and 7days postpartum lambs, and from adult ewes (n=4-5 per group). mRNA and protein were isolated from each region, and expression of prostaglandin synthase-1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNA's were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNA's were upregulated at 120 DGA. Tissue concentrations of PGE(2) correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus.

Fetal onset of general movements.

Perinatal qualitative assessment of general movements (GMs) is a tool to evaluate the integrity of the young nervous system. The aim of this investigation was to study the emergence of GMs. Fetal onset of GMs was studied sonographically in 18 fetuses during the first trimester of uncomplicated in vitro fertilization (IVF) pregnancies in weekly assessments. The earliest motility consisting of small and simple sideways bending (SB) of head and/or rump starts at 7 wk, lasting about 1 s. Between 7 and 8.5 wk, motility differentiates further into movements in which also one or two arms or legs become active; movements are still slow, small, and in one direction, but the duration increases to a few seconds. The transition into GMs at 9-10 wk is characterized by variation in participating body parts and amplitude, speed, and direction during longer periods of time. Between 9 and 13 wk, simple and stereotyped SBs and GMs may coexist. At 9 wk, the incidence of SBs decreases (p = 0.01) and that of GMs increases (p = 0.006). The data suggest that initial simple fetal motility is generated by spinal and brainstem circuitries, and the emergence of complex and variable GMs denotes the onset of supraspinal modulation of this spinal and brainstem activity.

Development of leptin-sensitive circuits.

Energy homeostasis is achieved by the integration of peripheral metabolic signals by neural circuits. The organisation and function of neural circuits regulating energy homeostasis has been the subject of intense investigation and has led to the definition of a core circuitry in the hypothalamus that interacts with key regions in the brain stem, which appear to mediate many of the effects of the adipocyte-derived hormone leptin on feeding and energy balance. Recent data on the ontogeny of these pathways indicate that, in rodents, these feeding circuits primarily form during neonatal life and remain structurally and functionally immature until 3 weeks of life. Our understanding of the mechanisms promoting the formation of these critical circuits has been advanced significantly by recent evidence showing that neonatal leptin acts as a neurotrophic factor promoting the development of projections from the arcuate nucleus of the hypothalamus. Together with an expanding literature on the role of nutritional factors to affect health, these discoveries may contribute to our understanding on perinatally acquired predisposition to later disease, such as obesity and diabetes.

Melatonin receptors in human fetal brain: 2-[(125)I]iodomelatonin binding and MT1 gene expression.

The purpose of this study was to identify sites of action of melatonin in the human fetal brain by in vitro autoradiography and in situ hybridization. Specific, guanosine triphosphate (GTP) sensitive, binding of 2-[(125)I]iodomelatonin was localized to the leptomeninges, cerebellum, thalamus, hypothalamus, and brainstem. In the hypothalalmus, specific binding was present in the suprachiasmatic nuclei (SCN) as well as the arcuate, ventromedial and mammillary nuclei. In the brainstem specific binding was present in the cranial nerve nuclei including the oculomotor nuclei, the trochlear nuclei, the motor and sensory trigeminal nuclei, the facial nuclei, and the cochlear nuclei. The localization of MT1 receptor subtype gene expression as determined by in situ hybridization matched the localization of 2-[(125)I]iodomelatonin binding. No MT2 receptor subtype gene expression was detected using this technique. Thus, melatonin may act on the human fetus via the MT1 receptor subtype at a number of discrete brain sites. A major site of action of melatonin in both fetal and adult mammals is the pars tuberalis of the pituitary gland. However, no 2-[(125)I]iodomelatonin binding or melatonin receptor gene expression was detected in the pituitary gland in the present study, indicating that the pituitary, particularly the pars tuberalis, is not a site of action of melatonin in the human fetus.

T2-Weighted fast MR imaging with true FISP versus HASTE: comparative efficacy in the evaluation of normal fetal brain maturation.

OBJECTIVE: This study compares the relative efficacy of two fast T2-weighted MR imaging techniques-fast imaging with steady-state free precession (true FISP) and half-Fourier acquisition single-shot turbo spin-echo (HASTE)-in the evaluation of the normal fetal brain maturation during the second and third trimesters of gestation. SUBJECTS AND METHODS: The brain maturation of 10 normal nonsedated fetuses (5 during the second trimester and 6 during the third trimester of gestation [1 fetus underwent 2 examinations]) was examined by both techniques using a Vision+ 1.5-T MR system. We specifically looked for developing events, including white matter myelination, neuronal migration, and cortical sulcation. Image quality was graded according to the presence or absence of undesirable blurring. RESULTS: The specific absorption rate was lower for true FISP than for HASTE by a factor of 3 at equivalent imaging conditions. HASTE and true FISP provide comparable image quality in the second trimester when myelination of the cerebrum has not begun. Neuronal migration could be recognized as hypodense bands on both sequences during the second trimester. Myelination beginning at the third trimester was better delineated with true FISP than with HASTE because of point spread function-related blurring effects inherent in HASTE that hampered visualization of short-T2 structures. Cortical sulcation was well delineated by both sequences. CONCLUSION: With relatively superior image quality and significantly lower radiofrequency absorption than HASTE, true FISP is a safer and more effective alternative in the prenatal evaluation of normal fetal brain.

Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex.

In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.

Characterisation of GABA(A) receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone.

Progesterone metabolites acting via GABA(A) receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA(A) receptors in fetal, newborn and adult sheep brains using [(35)S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity (K(D)) and density (B(MAX)) in the brainstem were not different in fetal, newborn (1-2 days old) and adult brains. In the hypothalamus K(D) and B(MAX) increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K(D) and B(MAX) increased progressively between 85 days and term ( approximately 147 days gestation), and were then not different from postnatal and adult values. The K(i) values for the GABA(A) receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [(35)S]TBPS binding in the presence of 5 microM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA(A) receptors are present in the fetal brain from at least 85 days gestation; (ii), 3alpha-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA(A) receptor binding parameters. Steroid modulation of the GABA(A) receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.

Ontogeny of immunoreactive prostaglandin endoperoxide synthase isoforms in ovine fetal pituitary, hypothalamus and brainstem.

Parturition is initiated in sheep by an increase in the activity of the fetal hypothalamus-pituitary-adrenal (HPA) axis. Prostaglandins, known to augment the activity of this endocrine axis, have long been proposed as involved in the initiation of paturition. We have previously demonstrated that endogenously produced prostanoids augment the activity of the HPA axis, and we have proposed that the increased production of prostanoids within the fetal brain or pituitary at the end of gestation might be involved in the initiation of parturition. An important regulatory step in the biosynthesis of prostanoids is the activity of prostaglandin endoperoxide synthase (PGHS). The present study was designed to test the hypothesis that the abundance of one or both isoforms of PGHS (PGHS-1 and PGHS-2) increase in brain and/or pituitary at the end of gestation. We used immunoblot analysis to measure the abundance of immunoreactive PGHS-1 and PGHS-2 in pituitary, hypothalamus and brainstem collected from fetuses of known gestational ages. We found that the abundance of PGHS-1 was weakly but significantly increased at the end of gestation in the pituitary and brainstem. The abundance of PGHS-2, on the other hand, increased exponentially in the pituitary and hypothalamus with highest concentrations found in term fetuses. We conclude that these enzymes are developmentally regulated in pituitary and in brain regions important for HPA axis control. We speculate that the increased enzyme's abundance results in increased prostanoid biosynthesis near term, and is a link in the chain of events which initiates parturition.

Ontogeny of estrogen sulfatase activity in ovine fetal hypothalamus, hippocampus, and brain stem.

Ovine parturition is initiated by increases in fetal hypothalamus-pituitary-adrenal (HPA) axis activity, which in turn increase placental estrogen biosynthesis and ultimately increase uterine contractility. In addition to the action in the uterus, estrogens augment fetal ACTH secretion. In late gestation, estrone sulfate is more abundant in fetal plasma than is unconjugated estrone. We studied hypothalamus, hippocampus, and brain stem tissue from fetal, neonatal, and adult sheep to test the hypothesis that the ovine brain contains estrogen sulfatase activity. We found that the activity in the hippocampus was significantly increased in late-gestation fetuses compared with both younger and older animals. No significant change in either hypothalamus or brain stem was revealed; however, the activity in all brain areas was high. Immunohistochemistry revealed the presence of estrogen sulfatase in the paraventricular nucleus of the hypothalamus, the nucleus of the solitary tract, and the rostral ventrolateral medulla. We conclude that ovine fetal hypothalamus, hippocampus, and brain stem contain estrogen sulfatase activity and that the activity in the hippocampus is developmentally regulated.

Dynamic expression of SEK1 suggests multiple roles of the gene during embryogenesis and in adult brain of mice.

Stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, plays a key role in a variety of cellular processes. It is well established that SAPK/JNK activation is controlled by SEK1/MKK4, an up-stream MAP kinase kinase. To gain insight into the role of SEK1 during embryonic development and in adult life, we examined the temporal and spatial patterns of sek1 expression in mice by using in situ hybridization and immunohistochemical study. Dynamic changes of sek1 expression were observed during embryogenesis. Strong sek1 expression was detected in most of the central nervous system and in liver and thymus during early stages of development. While the sek1 expression in nervous system increases over time, expression in fetal liver and thymus gradually decreases as embryogenesis proceeds. High level of the sek1 expression in the central nervous system was persisted throughout postnatal development and remained at a stable level in adult brain. These observations provide an anatomical basis for the vital role(s) of SEK1 in development, for example, in hepatogenesis and/or neurogenesis. Although SEK1 was widely expressed in adult brain, more strong expression of the sek1 was observed at layers 2 and 6 in cerebral cortex, in Purkinje cells of cerebellum, and also in hypothalamic nuclei. The strongest expression of the sek1 was found in the CA3 region of hippocampus, the region being highly vulnerable to exitotoxicity-induced apoptosis in kainate-treated animal models. Interestingly, SEK1 was localized not only in cytoplasm but in dendrites and/or in nucleus of neurons depending on the regions of adult mouse brain. Taken together, these results suggest multiple roles of the SEK1 during embryogenesis and in adult brain.

Immunoreactive thromboxane synthase is measurable in ovine fetal hypothalamus as early as 86 days' gestation.

Thromboxane A2 (TxA2) augments hypothalamus-pituitary-adrenal axis activity in both fetal and adult animals. We have proposed that TxA2 acts as a neuromodulator within the brain to stimulate the release of corticotropin releasing hormone (CRH) or arginine vasopressin (AVP) into the hypophyseal-portal blood. We performed the present experiments to identify immunoreactive thromboxane synthase (TxS) within fetal brain regions and to quantify developmental changes in the TxS immunoreactivity measurable within those regions. We found that immunoreactive TxS was present in fetal hypothalamus, pituitary, brainstem, and lung. In fetal hypothalamus, we found immunoreactive TxS in three identifiable molecular weights, approximately 65, 42, and 35 kD. In fetal pituitary and lung, we found the 65 and 35 kD forms, and in the brainstem we found only the 35 kD form. In fetal pituitary, there was a clear ontogenetic change in TxS immunoreactivity. The 42 kD TxS immunoreactivity was not present in the youngest fetal sheep studied (86-90 days' gestation), but was expressed in the other age groups (125-128, 135-139, 141-term, and postnatal ages). The other molecular weight forms appeared to increase in the older fetuses, but the changes were not significant. In the hypothalamus, all three forms of TxS were measurable at all ages, and there was no significant change in relative abundance. We conclude that immunoreactive TxS is present in the fetal brain throughout the last half of fetal gestation, but that the significance of multiple molecular weight forms is not clear.

A long-lasting change in the excitability of fetal brain neurons following activation of dam's hypothalamus in rats.

The influence of the dam's brain on the electrical activity of brain neurons was examined in the rat fetus while the fetus was still connected to the dam by the umbilical cord. Under urethan anesthesia, the spontaneous or glutamate-induced discharge of single brain stem neurons in fetuses was decreased by electrical stimulation of the dam's hypothalamus (Hyp). The changes occurred 21-122 s after start of the stimulation and persisted throughout the recording until the cells were lost after 2.4-9.5 min. The effects of electrical stimulation of dam's midbrain reticular formation (MRF) were much weaker and of shorter duration compared with Hyp stimulation. The dam's blood pressure was increased immediately after the beginning of MRF or Hyp stimulation. The change was always greater with MRF than with Hyp stimulation. Activation of the dam's MRF and Hyp had no marked influence on the blood flow in the fetal brain. These results indicate the presence of a functional interaction between the brain of the dam and that of its fetus mediated through the placenta.

Characterization of 11 beta-hydroxysteroid dehydrogenase activity in fetal and adult ovine tissues.

11 Beta-hydroxysteroid dehydrogenase (11 beta-HSD) converts 11-hydroxycorticosteroids to 11-oxocorticosteroids, thereby influencing the availability of bioactive cortisol or corticosterone in target tissues. The activity of this enzyme was investigated in sheep by: (1) measuring relative 11 beta-HSD activities in kidney, liver and placenta, and in various areas of the brain (hypothalamus, hippocampus, and brainstem); (2) characterizing the optimum pH of activities in the tested tissues; (3) investigating the possible effect of gonadal steroids on 11 beta-HSD activity in adult hypothalamus and kidney; and (4) investigating possible developmental changes in activities in the tested tissues. The optimum pH in liver and placenta was pH 9-10, whereas the optimum pH in kidney was pH 7-8. In tissues from adult ewes, 11 beta-HSD activity was highest in liver (84.6 +/- 3.8%) and kidney (49.8 +/- 11.6%), lower but measurable in pituitary (38.8 +/- 3.7%), and near the limit of detection in hypothalamus and hippocampus (2.7 +/- 0.9% and 3.2 +/- 0.8% respectively). Liver, kidney and pituitary from late-gestation fetal sheep contained activities which were similar to those in the adult (76.9 +/- 4.5%, 66.0 +/- 6.7% and 26.3 +/- 3.0% respectively). Activity in the pituitary was not related to fetal gestational age. Placenta also contained measureable 11 beta-HSD activity (21.4 +/- 4.7%). However, no activity was detected in hypothalamus (-1.7 +/- 0.2%), hippocampus (-0.2 +/- 0.6%) or brainstem (-1.0 +/- 0.6%) in late-gestation fetal or neonatal sheep. Enzyme activities in kidney and hypothalamus did not change significantly when the circulating concentrations of ovarian steroids were altered over a 1-3-week period. It is concluded that the ovine kidney, liver and placenta, but not hypothalamus or cerebral cortex, contain 11 beta-HSD activity. In addition, there is no change in 11 beta-HSD activity between late-gestation fetal life and adult life, and the relative activities are not altered by the ovarian steroid milieu.

Corticotropin releasing factor mRNA and peptide levels are differentially regulated in the developing ovine brain.

The regulation of CRF mRNA and protein in the developing ovine brain has been studied to assess the hypothesis that CRF is differentially regulated in the hypothalamus (Hypo), hippocampal-amygdala complex (H & A), frontal cerebral cortex (FCC) and brainstem (BS). We used a quantitative RNase protection assay and radioimmunoassay to determine mRNA and peptide concentrations, respectively, from the last third of gestation until term (i.e., from 95 to 142 days gestation (dg); term approximately 145 days). The major findings from this study are: (1) Hypothalamic CRF mRNA was increased by 2-fold in 140-142 dg fetuses compared to 128-138 and 95-123 dg fetuses; P = 0.016. (2) In the hypothalamus of 140-142 dg fetuses, there was a 2.5-fold increase in CRF mRNA derived from polyadenylation at poly(A) sites 2, 3 or 4; P = 0.005. (3) In 128-138 dg fetuses, CRF mRNA in the frontal cortex was 2-fold higher than in the other brain regions during this time period; P = 0.008. (4) CRF peptide concentrations in the Hypo were 2.5-fold higher in 140-142 dg fetuses compared to 95-106 and 128-138 dg fetuses; P = 0.007. (5) CRF peptide concentrations in the frontal cortex were 5.5-fold higher in 140-142 dg fetuses compared to fetuses at 95-106 dg; P = 0.004. (6) CRF peptide concentrations in the H & A were 5-fold higher in 140-142 dg fetuses compared to 95-106 dg fetuses; P = 0.029. The results from the present study demonstrate for the first time that CRF mRNA and peptide are differentially regulated in a region-specific manner during development.

Culture of hypoglossal cells, dissociated from foetal and new-born rats.

Primary cultures of dissociated cells from brainstem cranial nuclei have not been described in the literature. The present paper shows that dissociated rat posterior brainstem cells, as well as cells from the hypoglossal nucleus, taken from both foetal and postnatal animals, can be maintained in long-term culture. This can be achieved by using a DMEM/F-12 medium with defined supplements, with or without foetal calf serum. Under such conditions, the growth of neuritic processes as well as the formation of neural networks can be observed. The different cell types present in the cultures can be identified by immunohistochemistry with antibodies raised against neuron specific enolase, glial fibrillary acidic protein, galactocerebroside and choline-acetyl transferase. As previously demonstrated for other brain-dissociated neurones maintained in cultures, the use of molecules, involved in cell adhesive mechanisms, can modify the morphological properties of the growing cells. This was particularly observed when poly-L-lysine and laminin were used as substrata.

Brainstem auditory evoked potentials in the fetal sheep, in utero.

We studied brainstem auditory evoked potentials (BAEPs) in 8 fetal sheep in utero, ranging in gestational age from 105 to 142 days gestation (normal term 147 days). We could not elicit BAEPs prior to 117 days of gestation. After this age rapid maturation was seen, with three discernible peaks observed prior to 120 days and five peaks after 120 days. A significant (P less than 0.05) gestational age related fall in peak latencies and interpeak latencies was observed. The rate of stimulus presentation that could be tolerated without significant changes in wavepeak latency or amplitude also increased with advancing gestational age. In older fetuses (greater than 125 days), where a differentiated electrocorticogram (ECOG) was observed, differences were seen in latency and amplitude of several of the BAEP wavepeaks dependent upon the state. In high voltage ECOG states the amplitudes of wave IV and V were significantly (P less than 0.05) greater than in the low voltage ECOG state and the latencies of wave I, II and V were significantly (P less than 0.05) longer in low as compared to high voltage ECOG state. The BAEP, being obtainable over very short periods of time, appears to provide a useful indice of neural maturation for the sheep fetus in utero.

Fetal brain development in response to iodine deficiency in a primate model (Callithrix jacchus jacchus).

The common cotton-eared marmoset (Callithrix jacchus jacchus) has been used for the first time as a primate model to study the effects of dietary iodine deficiency on fetal brain development. Paired male and female marmosets were fed a low-iodine diet of maize, peas, meat meal, Torula yeast, maize oil and added vitamins, minerals and amino acids for 6 months before mating. Offspring from first and second pregnancies were compared with offspring from control marmosets fed the same diet but supplemented with iodine. Severe iodine deficiency in the fetus at birth was evident by reduced plasma thyroxine levels, increased plasma thyroid stimulating hormone levels, increased thyroid weight and reduced thyroid iodine content. Thyroid histology revealed hyperplasia, hypertrophy and absence of colloid material in the follicles. Iodine deficiency caused a reduction in the weight of the fetal brain and in particular the cerebellum. Brain cell number was reduced in the cerebellum and brainstem but cell size was reduced in the cerebral hemispheres. Histology of the brain revealed morphological changes in the cerebellum and cerebral hemispheres. In the-cerebellum there was: an increase in the thickness of the external germinal layer indicative of impaired cell acquisition; a decrease in total area; a decrease in molecular layer area; and an increase in Purkinje cell (Pc) linear density due to a reduction in the length of the Pc line. The decrease in molecular layer area and increase in Pc linear density imply diminished ascending and lateral extension of Pc dendrites. Changes in the cerebral hemispheres consisted of an increase in the density of neuronal cell bodies in the granular band and a decrease in synaptic counts in the layer between the pia mater and supragranular band of the visual cortex. Offspring from second pregnancies compared to those from first pregnancies were more severely affected and associated with lower plasma levels of maternal and fetal thyroxine. These findings indicate the importance of maternal and fetal thyroid function in relation to fetal brain development in the primate.

The onset and development of descending pathways to the spinal cord in the chick embryo.

The ontogenetic development of afferent (supraspinal and propriospinal) as well as efferent (ascending) fiber connections of the spinal cord was examined following the injection of horseradish peroxidase (HRP) or wheat germ agglutinin HRP (WGA-HRP) into the cervical and lumbar spinal cords (or brains) of embryos ranging in age from 4 to 14 days of incubation. A few cells were first reliably retrogradely labelled in the pontine reticular formation on embryonic day (E) 4 and E5 following the injection of WGA-HRP into the cervical and lumbar spinal cord, respectively. Propriospinal projections to the lumbar spinal cord, originating from brachial spinal cord, were found by E5, and from the cervical spinal cord by E5.5. Ascending fibers arising from neurons in the lumbar spinal cord could be followed to rostral mesencephalic levels in E5 embryos. Thus, the earliest supraspinal, propriospinal, and ascending fiber connections appear to be formed almost simultaneously. Retrogradely labelled cells were found in the raphe, reticular, vestibular, interstitial, and hypothalamic nuclei in E5.5 embryos following lumbar injections of WGA-HRP. Except for neurons in cerebellar nuclei, all the cell groups of origin that project to the cervical spinal cord of posthatching chicks were also retrogradely labelled by E8. There was a delay in the time of appearance of the projections from various regions of the brain stem to the lumbar versus the cervical spinal cord, ranging from 0.5 to 7 days, but typically of about 3 days duration. A large number of cells located in the ventral hypothalamic region, just dorsal to the optic chiasma, were found to be labelled following cervical HRP injection between E6 and E10. These cells may represent transient projections that are present only during embryonic stages since no labelled cells were found in this region in the newly-hatched chick.

Relationship between phenolamines and catecholamines during rat brain embryonic development in vivo and in vitro.

p-Octopamine and phenylethanolamine are present in the embryonic rat brain earlier than catecholamines. These phenolamines are localized mainly in the hypothalamus, where the level of p-octopamine is very high. The parallel developmental study of the activities of dopamine beta-hydroxylase, 3,4-dihydroxyphenylalanine decarboxylase, tyrosine hydroxylase, and monoamine oxidase shows that phenolamines are present in significant amounts in the hypothalamus until tyrosine hydroxylase and monoamine oxidase become catalytically active. The culture of embryonic hypothalamus at different ages shows that no tyrosine hydroxylase and monoamine oxidase activities can be detected if the tissue is cultured before 15 days. This clearly indicates that all the enzymes related to catecholamine biosynthesis are not triggered at the same time during the development of the rat brain. These results are discussed on the basis of the physiological importance of phenolamines in mammals and of the use of the developing rat brain as a model for the study of the onset of the catecholaminergic system and the decline of the octopamine.