Prenatal Day Research Articles

EX-1, a surface antigen of mouse neuronal progenitor cells and mature neurons

Using membrane fragments of PCC7-Mz1 embryonal carcinoma cells, an established in vitro model of neural differentiation (Lang et al., J. Cell Biol., 109 (1989) 2481–2493), we have raised monoclonal antibodies (mAb) against developmental stage-specific cell surface antigens. As shown by double-immunofluorescence labeling studies, employing differentiating PCC7-Mz1 cells, primary cultures of mouse cerebellum cells and cryosections of mouse brain and other tissues, rat mAb anti-mouse EX-1 recognizes a membrane protein which is exclusively expressed by cells of the neuronal cell lineage. EX-1-expressing neuronal precursor cells were identified by double labeling with antibodies directed against stem cell markers or BrdU, EX-1-expressing postmitotic neurons by labeling with antibodies directed against phenotypic markers. In the developing mouse brain, the EX-1 antigen is expressed in the neuroepithelium already at prenatal day 8, i.e. clearly before the onset of mature neuron-specific marker expression. Increasing co-expression with the latter is observed from embryonic day E10 throughout neuronal maturation, but not with markers of other cell types tested. From these studies, the EX-1 antigen is the earliest marker for the mouse brain neuronal cell lineage so far discovered.

Developmental expression of G-proteins and adenylyl cyclase in peripheral olfactory systems. Light microscopic and freeze-substitution electron microscopic immunocytochemistry.

Light microscopic immunohistochemistry coupled with freeze-substitution electron microscopic immunocytochemistry was used to localize alpha-subunits of G-proteins and type III adenylyl cyclase in developing rat olfactory epithelia. Some cilia immunoreacted with antibodies to GS alpha and type III adenylyl cyclase as early as prenatal day 15 (E15; E1 = sperm-positive), but immunolabelling with antibodies to Golf alpha was not observed until E16. From then on numbers of receptor cells with immunolabelled cilia increased for all three probes. Immunoreactivity for antibodies to the olfactory signal-transduction proteins tended to parallel cilium development, though Golf alpha lags somewhat behind. Newly formed cilia labelled along their lengths, whereas mature cilia labelled predominantly along their long distal parts. Dendritic knobs and ciliary necklaces showed little or no labelling. While at E22 most multiciliate cells immunolabelled with antibodies to Gs alpha, Golf alpha, and type III adenylyl cyclase, not all of these cells labelled with antibodies to olfactory marker protein. Olfactory axons immunoreacted more intensely than epithelial surface structures with antibodies to Gs alpha at E15; the reverse occurred by about E18. Immunoreactivity with antibodies to alpha-subunits of the G-proteins Go, Gq/G11, and Gi was also found as early as E15. Antibodies to Go alpha labelled receptor cell dendritic knobs and cilia during development only. Antibodies to Gi alpha labelled Bowman's glands, whereas those to Gq alpha/G11 alpha bound to receptor cell cilia and axons (primarily vomeronasal), and supporting cell microvilli. We propose that Gs is the predominant G protein in cilia of immature olfactory receptor cells, while Golf is predominant in cilia of mature cells. Axonal immunoreactivity for some G-protein antibodies suggests G-protein participation in processing of olfactory axon and/or axon terminal-bound signals.

Surface antigens of cell subpopulations in prenatal rat brain are expressed in a characteristic non-random pattern on their ethylnitrosourea-induced malignant counterparts

Abstract Selective induction of neural tumors in the rat by single-dose exposure of the immature nervous system to ethylnitrosourea (EtNU) is a model for the study of cell lineage-, differentiation stage-, and carcinogen-dependent mechanisms in neuro-oncogenesis. Overall yields and relative frequencies of different types of neural tumors vary with the developmental window chosen for the EtNU-pulse. Precursor cells belonging to different neural lineages and targeted by the carcinogen at distinct developmental stages may thus bear a differential risk of malignant conversion. To specify subpopulations of neural precursors in fetal (prenatal day 18) BDIX-rat brain, four monoclonal antibodies (mAbs) recognizing cell surface differentiation antigens were used: mAb RB13–2 directed against O-acetylated gangliosides and binding to approximately 36% of fetal brain cells (FBC); mAb RB13–6 recognizing a 130 kDa glycoprotein (expressed by approximately 8% of FBC); and mAbs RB21–7 and RB21–15 which bind, respectively, to embryonal neural cell adhesion molecules (N–CAM) and a 24 kDa protein (expressed by approximately 55% and 12% of FBC). Antigen expression profiles were compared with those of 14 primary brain tumors and 16 malignant neural cell lines, all of which had been induced by EtNU on prenatal day 18 in vivo. Monoclonal antibodies RB13–2 and RB21–7 did not bind to any of the tumors or cell lines. In contrast, mAbs RB13–6 and RB21–15 both reacted with 14/14 tumors, and with 16/16 and 10/16 cell lines, respectively. Expression of the latter antigens might thus specify lineage-specific stages of FBC development/differentiation particularly susceptible to EtNU-induced malignant transformation. Two-color fluorescence analyses revealed three subsets of FBC binding mAb RB13–6 (RB13–2 +/ RB13–6 +/ RB21–15 −; RB13–2 −/RB13–6 +/RB21–15 −; and RB13–2 −/ RB13–6 +/RB21–15 +), representing successive stages of differentiation.

Early development of macrophages in intact and organ cultured hearts of rat embryos

Macrophage precursors are present early in embryonic life, being demonstrable in placental and embryonic connective tissues of rats at the neurula stage and as potential macrophages in the brain, liver, and lungs near the onset of organogenesis. We examined the development of macrophages in the heart and the possibility that they initially appear at sites of programmed cell death (apoptosis). Precursors were recognized by the binding of peroxidase-coupled Griffonia simplicifolia isolectin B4 (GSA) on the cell membrane. Their capacity for conversion into macrophages was assayed in organ cultures; confirmation of the progeny as bona fide macrophages was obtained from their responses to particle exposure and macrophage colony-stimulating factor (M-CSF). GSA+cells were first seen on gestational day 12 (4 mm embryos) as 2-3 cycling, nonvacuolated cells located in cardiac tissue outside the blood vessels. This population increased to approximately 12 cells by day 14 (9 mm embryos). Two-thirds were distributed along the bulbus cordis in the jellylike endocardium and a more densely cellular connective tissue closer to the aortic arches where apoptotic sites are expected to develop. Such sites were not found in serial glycol methacrylate sections through our 14-day specimens, although in whole heart explants of this age an area of necrosis developed along the prospective line of bulbar endocardial fusion on the second day of organ culturing, and by then macrophages were fairly abundant. Organ culturing of 13-day embryonic hearts also yielded large, highly vacuolated, GSA+mononuclear phagocytes. After a few days in culture most of the macrophages migrated onto the medium where they formed a tight corona of cells about the explants. They readily ingested iron oxide particles and concentrated supravitally administered neutral red in their vacuoles. Macrophages from 14-day cultures exposed to M-CSF developed significantly larger coronas than macrophages from explants grown in serum-rich control medium (p < 0.001). In the presence of cytokines, moreover, these cardiac macrophages survived as many as 100 (92 "postnatal") days. Macrophage precursors first appear in embryonic rat hearts well before they are needed to clear debris generated by programmed cell death and are capable of rapid conversion into outright phagocytic cells as early as the 13th prenatal day.

Prenatal Photoperiod and the Timing of Puberty in the Female Lamb1

In female sheep, photoperiod regulates the timing of the transition to adulthood. We tested the hypothesis that photoperiod very early in development influences the timing of the pubertal LH rise that initiates sexual maturation. The first experiment was designed to determine the influence of day length information perceived before birth by varying prenatal photoperiod experience. Two groups that experienced either increasing or constant long days prenatally, and then a gradually decreasing photoperiod postnatally, reached puberty at the same age (Prenatal Increase, 20.4 +/- 0.5 wk vs. Prenatal Long Days [LD], 19.4 +/- 0.8 wk). Puberty in these groups was much earlier than in two control groups exposed to the same photoperiods, but beginning at birth, for 13 wk (Postnatal Increase, 29.6 +/- 1.0 wk; Postnatal LD, 26.2 +/- 1.3 wk). In the second experiment, the role of prenatal photoperiod in timing sexual maturity was also examined through the use of treatments with greater contrast. Lambs were exposed prenatally to either decreasing or increasing day lengths. Beginning at birth, both groups were exposed to a decreasing photoperiod. Although only half of the lambs in each group exhibited the pubertal LH rise, those that attained puberty did so at the same age (Prenatal Decrease, 14.8 +/- 1.0 wk vs. Prenatal Increase, 14.8 +/- 0.3 wk). We therefore conclude that day length cues experienced postnatally predominantly time sexual maturation in the female lamb.

Ontogeny of gubernacular contraction and effect of calcitonin gene-related peptide in the mouse

The mouse gubernaculum undergoes inguinoscrotal migration in the first postnatal week and also shows rhythmic contractions in organ culture in response to calcitonin generelated peptide (CGRP). This study aimed to investigate the ontogeny of gubernacular contractile activity and effect of CGRP in organ culture in relation to the normal time of migration. Two hundred eighty gubernacula from male mice of 17 and 19 days' gestation, and of postnatal days 0, 2, 4, 7, 10, and 14 were studied. Half were cultured with CGRP 714 nmol/L, and the other half were cultured without CGRP as controls. All were examined daily (for 7 days) for contractions. In the control group, the cumulative percentages of contractile gubernacula increased from 5% to 100% with age; in the CGRP group, these values ranged from 65% to 100%. There were significant differences between the two groups from prenatal day 17 to postnatal day 4 ( P values ranged from less than .001 to less than 0.05, respectively). With increasing age in the CGRP group, the highest contractile rates were observed at the fewest number of days in culture. From prenatal day 17 to postnatal day 4, the gubernaculum had increasing endogenous contractility, but there was low endogenous contractility without exogenous CGRP. Contractility of gubernacula was enhanced strongly by exogenous CGRP, and the culture days of the peak contractile rates lined up in the reverse order. These results suggest that the days of optimal gubernacular contraction with CGRP in vitro are in keeping with the days of natural migration in vivo. This is consistent with the hypothesis that CGRP (released from the genitofemoral nerve) is important for gubernacular migration.

Morphological assessment of grafted rat and mouse cortical neurons: A light and electron microscopic study

The morphology of cortical neurons grafted into (or near) the rat striatum was studied by means of intracellular Lucifer yellow injections in fixed slices. Rat donor syngeneic cortical tissue (from postnatal day 1 old rats; AO strain) as well as mouse donor xenogeneic cortical tissue (prenatal day 19; C3H/HE strain) were grafted as solid pieces into 8-12 week-old rats (AO strain). Recipients of mouse xenografts were immunosuppressed with a monoclonal antibody against the interleukin-2 receptor. After perfusion and sectioning of the graft-containing areas, individual slices were incubated in the DNA stain 4.6-diamidino-2-phenylindole (DAPI) to visualize the cell nuclei. Grafts could be easily identified by a surrounding rim of astrocytes which outline the border between grafted and host tissue. Grafted cortical neurons were intracellularly filled with Lucifer yellow, DAB-photoconverted, and further processed for light and electron microscopy. In general, no cortical lamination could be observed in the grafted rat and mouse cortical tissue, but neurons were loosely packed throughout the graft. Two major cell types could be identified in all grafts investigated so far. The majority resembled those described as spiny neurons (85%), which could be further classified into pyramid-like, spiny stellate-like or fusiform spiny neurons, with somata ranging between 15 and 25 microns in diameter. The remaining 15% resembled non-spiny neurons with either a multipolar basket-like or fusiform morphology. Dendrites of spiny and non-spiny neurons, which could extend to distances up to 400 microns, were never seen to cross the astrocytic border, but some main axon and axonal collaterals of spiny neurons were found to leave the graft. On the basis of light microscopic observations no difference was found between mouse and rat grafted cortical neurons. The results of this study show that grafted cortical neurons retain some of the characteristic features of neurons in the intact adult cerebral cortex, although there appears to be a greater preponderance of spiny neurons in grafted tissue. This may reflect an immaturity of the grafted tissue or a response to the striatal environment.

High-performance liquid chromatographic analysis of methylation changes of CCGG sequence in brain and liver DNA of mice during pre- and postnatal development

The change of the methylation of CpG in the CCGG sequence of brain and liver DNAs of mice during late fetal and suckling periods was determined by high-performance liquid chromatography using a reversed-phase column and 0.1 M phosphate buffer (pH 6.0) as the mobile phase. The tissue DNA was digested with the restriction enzyme, MspI, and was labeled at the 5′-end with [γ- 32P]ATP. The cpm% of deoxycytidine 5′-monophosphate (5mdCMP) in total CpG dinucleotides was calculated from the equation 5mdCMP/total CCGG (cpm%) = (5mdCMP) MspI,cpm /{(5mdCMP) MspI,cpm + (dCMP) MspI,cpm } × 100. The brain DNA exhibited a significant decrease in CpG methylation at prenatal day 18 but little change after birth. This marked decline of 5mdCMP in the CCGG sequence may be associated with the increase of enzymes before birth. The liver DNA showed considerable change during the late prenatal period. The observed changes of CpG methylation in liver DNA are indicative of the corresponding alterations of enzymes, multinucleate cells and hepatocytes. The results obtained indicate that both brain and liver cells have the development-associated changes in the conformation and transition of DNA around the time of birth.

In vitro differentiation of E‐N‐CAM expressing rat neural precursor cells isolated by FACS during prenatal development

Most fetal rat brain cells expressing the embryonal, highly sialylated form of the cell adhesion molecule N-CAM (E-N-CAM) are precursor cells, as judged from the absence of marker molecules specific for mature neural cell types. However, the detection of E-N-CAM+ cells in frozen sections does not provide information on the lineage-specific differentiation of these cells during development. To investigate their differentiation behaviour in vitro, E-N-CAM+ cells were isolated at different times of brain development by fluorescence-activated cell sorting (FACS), using a monoclonal antibody (Mab RB21-7) which specifically recognizes polysialic acid (PSA) residues on E-N-CAM. Double-immunofluorescence analyses showed that the majority of E-N-CAM+ cells isolated on prenatal days 15 to 18 differentiated into neurons while a small subset of Mab RB21-7 binding cells proved to be astrocytic precursors and/or bipotential. The proportion of E-N-CAM+ astrocytic precursors increased during later development (prenatal day 22) concomitantly with the onset of gliogenesis. While conversion of E-N-CAM to mature forms of N-CAM was never observed in neurons during cultivation, E-N-CAM+ cells of the astrocyte lineage switched to N-CAM soon after the onset of GFAP expression. A lineage-specific transition of E-N-CAM to mature N-CAM expression is, therefore, suggested for these astrocytic progenitor cells during rat brain development.

The differentiation of entero-endocrine cells of pre- and postnatal rats: light and electron microscopy and immunocytochemistry.

The differentiation of cholecystokinin/gastrin (CCK/GA)-, glucagon (GLU)- and somatostatin (SOM)-positive cells in the pre- and postnatal rat duodenum was investigated with a special emphasis on the relationship to that of the fibromuscular intestinal wall by light and electron microscopy and immunocytochemistry. Between prenatal days 16 and 21, contacts of mesenchymal cells with the epithelium are frequent, and the differentiation of the entero-endocrine cells which occasionally display a secretory function becomes advanced in close proximity to an extensive capillary network in the underlying connective tissue. CCK/GA-positive cells can be detected by immunocytochemistry in the intestinal epithelium at prenatal day 18. Other types of entero-endocrine cells are not detected until prenatal day 19. Quantitative analysis demonstrates that the volume density of CCK/GA-positive cells per 100 microns2 cytoplasmic area increases sharply at postnatal day 5 relative to earlier stages. This may be mainly due to suckling of the neonatal rat.

Role of mesenchymal cells in the neovascularization of the rabbit phallus

The neovascularization of the rabbit phallus at ages between prenatal days 15 and 21 was investigated by light- and electron microscopy, computer-aided light microscopic reconstruction, and immunocytochemistry. The phalli are embedded by an abundance of mesenchymal cells, which are in contact with the neighboring ones or with the endothelial lining of growing capillaries. They often form solid cell cords that eventually make contact with the growing capillaries. The computer-aided reconstruction of the serial light micrographs reveals that these cell cords are involved in connecting the adjacent capillaries. The incorporation of such mesenchymal cell projections into the endothelial lining, occasionally conjugated with simple attachment devices, is frequently observed by transmission and scanning electron microscopy. The contact areas between the mesenchymal and endothelial cells show immunoreactions of fibronectin. These results indicate the successive transformation of mesenchymal cells to endothelial cells of the growing capillaries. As endothelial cells of the growing capillaries show mitotic proliferation, such vasoformative mesenchymal cells seem to be involved in the acceleration of the neocapillarization of the rabbit phallus. Fibronectin actively produced in the mesenchymal cells may participate in their migration and the mechanical linkage with the endothelial cells.

Preproenkephalin mRNA expression in the developing and adult rat brain

[Met 5]-Enkephalin is derived from the protein precursor, proenkephalin A, which in turn is encoded by the preproenkephalin (PPE) gene. [Met 5]-Enkephalin is not only a putative neuromodulatory substance, but also serves as a growth factor (= opioid growth factor, OGF). OGF exerts an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative and differentiative events. This study examined the relationship of PPE mRNA expression to late prenatal and postnatal rat brain development. Northern blot analysis of the whole brain and cerebellum showed that message is present in the fetal nervous system on prenatal day 15 (the earliest timepoint examined), is expressed at relatively similar levels within each tissue during the first 2 postnatal weeks, and reaches adult levels by the beginning of the 3rd postnatal week. In situ hybridization methodology revealed that PPE mRNA was prominent in areas associated with cell generation. Message was found in sites of primary (i.e., ventricular region) and secondary (e.g., external germinal layer of the cerebellum) cellular replication, as well as in discrete foci of cell proliferation (e.g., medullary layer of the cerebellum). PPE mRNA was also present for varying periods of time in postmitotic cells. During development, a number of patterns (decrease, increase, and no perceptible change) of PPE mRNA could be detected in relationship to the fetal/neonatal period. Given the strong evidence (e.g., regulation of cell proliferation and differentiation, temporal and spatial patterns of peptide and zeta opioid receptor) that enkephalin immunoreactivity is associated with proliferating and differentiating neurons and glia, these results suggest that the source of [Met 5]-enkephalin is both autocrine and paracrine in nature.

Pre‐ and postnatal development of rabbit foliate papillae with special reference to foliate gutter formation and taste bud and serous gland differentiation

The epithelial downgrowth arises in the presumptive foliate papilla region of the tongue approximately at prenatal day 22, and the distal portion of the primary epithelial cell cords transforms to the bifurcated serous gland and excretory duct between prenatal day 30 and postnatal day 2. The excretory ducts extend upward through the primary epithelial cell cords at rather random intervals and are open to the tongue surface between postnatal days 1 and 2. In this process, successive connections between the adjacent ascending excretory ducts occur mainly due to desquamation of the keratinized lining cells of the ducts, resulting in the formation of the foliate gutter. Taste bud primordia appear in the primary epithelial cell cords by nerve penetration through the basal lamina from prenatal day 30 and nearly fully formed taste buds facing the ascending excretory ducts have been already observed before the foliate gutter formation is completed. In the differentiation process of chemoreceptor (type III) cells from less-differentiated basal (type IV) cells, subsurface cisterns of endoplasmic reticulum are occasionally present where nerve endings make contact. Since subsurface cisterns have been considered to be involved in reciprocal or efferent synaptic transmission, it is reasonable to consider that these morphological specializations may take a role in neurotrophic signals for differentiation of the chemoreceptor cells.

Expression Patterns of Rat Somatostatin Receptor Genes in Pre‐ and Postnatal Brain and Pituitary

The relative abundances of mRNAs encoding four different somatostatin receptors were examined using PCR techniques during postnatal development of the rat brain and hypophysis. In most tissues, somatostatin receptor 1 and 4 mRNAs are more abundant than those encoding somatostatin receptor 2 and 3. Transcript levels of somatostatin receptor subtype 4 are relatively high in the cortex, hippocampus, and striatum, those of subtype 1 in the cortex and brainstem, and those of subtype 3 in the cerebellum. In situ hybridization revealed the presence of significant amounts of somatostatin receptor 1 mRNA, as early as prenatal day 14, in the trigeminal ganglion and in the neuroepithelial layers surrounding the lateral, third, and fourth ventricles. In the developing cortex a morphological change in the sites of somatostatin receptor 1 gene expression occurs; mRNA is present superficially in the cortex at prenatal stages, appears in all layers shortly after birth, and in adult rats is restricted to the deep cortical layers. In the cerebellum, somatostatin receptor 1 mRNA levels are highest around birth, declining thereafter. In contrast, cerebellar somatostatin receptor 3 transcripts are absent at birth, become detectable around postnatal day 7, and reach a maximal level during maturation.

Physiological and pathological changes in levels of the two small stress proteins, HSP27 and alpha B crystallin, in rat hindlimb muscles.

The two small stress proteins, HSP27 and alpha B crystallin, are expressed widely in normal rat tissues and abundantly in skeletal muscle. In order to clarify the physiological significance of these stress proteins, the changes in their levels were determined immunochemically, in the slow-twitch soleus muscle and fast-twitch extensor digitorum longus muscle or rectus femoris muscle of growing rats, and in those of adult rats during denervation and tenotomy. HSP27 was quantitated by specific immunoassay, similar to that for alpha B crystallin, with antibodies raised in rabbits against purified rat HSP27. In adult rats, HSP27 was present at high levels in tissues composed of striated muscle, and it was present at much higher levels in the soleus muscle than in the rectus femoris or extensor digitorum longus muscle, as is alpha B crystallin. However, in rats of perinatal age (from prenatal day 2 to postnatal day 3), levels of HSP27 in the rectus femoris muscle were enhanced like those in the soleus muscle, reaching the maximum levels at postnatal day 3. Thereafter HSP27 in the fast-twitch muscle showed a steep decrease. The increase in alpha B crystallin in the hindlimb muscles was also observed in the perinatal period. However, alpha B crystallin concentrations in the soleus muscle of perinatal rats were as low as those in rectus femoris muscle. The transection of the sciatic nerve resulted in decreases in the levels of HSP27 and alpha B crystallin in the soleus muscle of adult rats, together with increases in the levels of the two proteins in the extensor digitorum longus muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Strain differences in the control incidences of morphological abnormalities in the rat whole embryo culture

Since the animal supplier to this laboratory stopped carrying the rat strain used for the previous 7 yr, a number of other strains from this supplier were tested to determine their suitability for use in the whole embryo culture system. The in vitro development from prenatal days 9.5 to 11.5 of rat conceptuses of the strain Kfm:WSA (A) was compared with that of strains Ibm:WSA (B), HanIbm:WIST (C) and Ibm:RORO (D) cultured under identical methodological conditions. The parameters for conceptal growth (yolk-sac diameter, embryonic crown-rump and head lengths) after 48 hr of culture of strains A, B and D were similar. These measurements were smaller in the conceptuses of strain C than in those of strain A, but embryonic differentiation (morphological score and somite numbers) was not delayed in comparison with strain A. The overall frequencies of conceptuses with dysmorphogenic events in strains A and B were 3.1 and 4.4%, respectively. In contrast, in strains C and D 30.0 and 22.6% of the conceptuses assessed had morphologically abnormal features. Both values were statistically significantly higher than the incidence in strain A. The types and frequencies of anomalies observed were different in the different strains tested. The results from conceptuses of strain B (derived from strain A) indicate that this strain is suitable for the culture model established in this laboratory, whereas strains C and D are not useful without methodological changes or adaptations. This study indicates that strain differences should be kept in mind when setting up the whole embryo culture system.

Cytochemical and immunocytochemical demonstration of acetylcholinesterase of the prenatal rat lower limb.

Acetylcholinesterase (AChE) activities in the prenatal rat lower limb were investigated by both cytochemistry and immunocytochemistry. Results indicate that the epidermal cells show immunoreactions of AChE at a limited stage at prenatal day 15, and mesenchymal cells which are occasionally in contact with the basal lamina or with the adjacent myotubes begin to show AChE activities at prenatal day 17. Such AChE-positive mesenchymal cells, involved in the formation of the muscular tissues, have almost disappeared in the subepidermis by prenatal day 19. This suggests that AChE independent of the neuromuscular system may be involved in the mesenchymal cell differentiation especially in the inductive process during myogenesis.

Ontogeny of zeta (ζ), the opioid growth factor receptor, in the rat brain

Opioid growth factor (OGF), [Met 5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (ζ) opioid receptor to perform its function. Using [ 3H]-[Met 5]enkephalin, the ontogeny of the ζ receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (E15). In the whole brain, binding capacity ( B max) was two-fold greater at E15 than at E18 adn E20. The quantity of ζ receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8–10. The B max receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be observed. The binding affinities of the whole brain and cerebellum postnatal day 6 increased body weight, the B max of the ζ receptor in the whole brain and cerebellum (but not the K d), and increased the number of layers of germinal cells in the cerebellum. These results define the temporal expression of the ζ receptor in the rat brain, as well as some regulatory properties, and support the concept that the ζ opioid receptor is primarily related to the proliferation of cells in the nervous system.

Developmental changes in nerve growth factor level in rat serum.

In serum, nerve growth factor (NGF) forms a complex with alpha 2-macroglobulin (alpha 2M), which formation inhibits the immunoreactivity between NGF and its antibodies. For measuring the serum level of NGF, it is thus necessary to liberate NGF from the NGF-alpha 2M complex and prevent reformation of such complex. The pretreatment of rat serum with 1 M guanidine hydrochloride for a few hours and operation of the enzyme immunoassay (EIA) in the presence of guanidine hydrochloride provided a reliable means for determination of the NGF level in serum. By this procedure we followed the serum NGF level in rats developmentally. It increased from prenatal day 2 to postnatal day 5 and decreased slightly at postnatal week 3, thereafter remaining constant throughout adulthood. In pregnant rats, the NGF level in serum increased threefold to fivefold before birth and then decreased rapidly. These data suggest that serum NGF level may reflect the demand for this molecule during establishment of the peripheral nervous system.

Macrophage development: IV. Effects of blood factors on macrophages from prenatal rat lung cultures.

Effects of colony-stimulating factors M-CSF, GM-CSF, G-CSF, and IL-3 were assessed on cells of macrophage lineage present in organ cultured 14-day prenatal rat lungs. Treatment groups were compared between one another and against control lungs grown on standard medium containing 40% fetal bovine serum without added factors, where a monoculture of macrophages rapidly develops from precursors present at explantation, leading to appearance of a large mature population on the pleural surface outside the lungs. Studies were carried out in living cultures and by light and electron microscopy using peroxidase-coupled isolectin B4 of Griffonia simplicifolia to identify macrophages and their precursors. In the first experiment, 14-day prenatal lung explants (14 + 0 days) containing macrophage precursors but not matured cells were exposed to individual CSFs for 7 days in an attempt to determine whether precursors are committed irrevocably to the macrophage line or can be altered by exposure to factors promoting significant granulocyte development. In succeeding experiments, 4- and 7-day-old cultures (14 + 4, 14 + 7 days) containing matured macrophages were targeted to see whether macrophage survival can be extended beyond expectations in controls and whether mitotic activity is stimulated. Recombinant CSFs were used at dosage levels known to promote colony formation in vitro (200-1,000 CFU/ml). Cultures exposed from prenatal day 14 to M-, GM-, G-CSF, or IL-3 yielded a monoculture of macrophages without exception. Populations developed in the presence of M- or GM-CSF were much larger than in controls or cultures grown with the other blood factors. GM-CSF-exposed cultures produced by far the largest macrophages, among them many multinucleate giant cells. Macrophages developed in the presence of G-CSF were also significantly larger than controls. Growth of the mature macrophage population was greatly stimulated by exposure to M-CSF or GM-CSF but not by IL-3 or G-CSF. Mitotic figures were noted in the coronas of emerged cells surrounding stimulated cultures, compared to none in the controls. Ultrastructurally, macrophages stimulated by M-CSF retained a mature appearance like macrophages in control, IL-3, and G-CSF treatment groups, whereas many in the GM-CSF group became less differentiated. As to long-term survival, a single 14-day explant was grown for 8 days on standard medium (the equivalent date for birth), then placed in a soft agar medium containing M-CSF.(ABSTRACT TRUNCATED AT 400 WORDS)