Neuroactive steroids in the brain and age and sex specificity of behavior and anxiety: An experimental study

Changes in the brain’s neuroactive steroid levels, behavior in the open field, and the anxious-phobic status of male and female rats in the course of development have been studied. An increase in the motor and exploratory activity and emotionality in rats of both sexes in the pubertal period and a decrease in their values in mature and old animals have been detected. Anxiety has no sexual dimorphism in adult animals; it is significantly higher in males than in females in the prepubertal and pubertal periods of development and is higher in old females than in males of the same age. An increase in the level of corticosterone in some brain structures in maturing and old rats has been found; the testosterone concentration increases in one-month-old and adult animals but decreases in old individuals, while the estradiol concentration in all studied brain structures of male and female rats was low in all periods of postnatal life. Correlation analysis has shown modulation by steroid hormones of the changes in behavioral responses during development.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

The fatty acid composition of the tissues of streptozotocin-diabetic rats

The authors studied acute changes in the fatty acid composition of the tissues of streptozotocin-diabetic rats. They found that streptozotocin diabetes led to changes in the total lipids fatty acid spectrum in serum and in tissues (liver, adipose tissue, renal cortex diaphragm). After only 7 days' diabetes there was an increase in the percentual proportion of saturated fatty acids and a decrease in the amount of polyene fatty acids in the serum and in all the above tissue of diabetic animals. Palmitic acid (16:0) participated in the increase in the proportion of saturated fatty acids in all the given tissues, while stearic acid (18:0) played a role in the increase in the renal cortex and the serum. Among the monoene acids, there was a drop in the proportion of palmitoleic acid (16:1) in the adipose tissue and serum and in the amount of oleic acid (18:1) in the renal cortex, liver and muscle. Linoleic acid (18:2) played a role in the decrease in the proportion of polyene acids in all the given tissues and the serum, while arachidonic acid (20:4) was involved in the drop in the renal cortex, liver and muscle. The results show that diabetes leads to changes in the fatty acid composition of the renal cortex and muscle, as well as of the liver and adipose tissue. At present it is not yet clear whether there is an absolute decrease in the proportion of essential fatty acids, or whether diabetes is characterized by an increase in the amount of lipids in both serum and tissues.     

Effects of ammonium sulphate application on the rhizosphere,fine-root and needle chemistry in aPicea abies (L.) Karst. stand

Rhizosphere, fine-root and needle chemistry were investigated in a 28 year old Norway spruce stand in SW Sweden. The uptake and allocation pattern of plant nutrients and aluminium in control plots (C) and plots repeatedly treated with ammonium sulphate (NS) were compared. Treatments started in 1988. Current year needles, one-year-old needles and cylindrical core samples of the LFH-layer and the mineral soil layers were sampled in 1988, 1989 and 1990. Compared to the control plots, pH decreased significantly in the rhizosphere soil in the NS plots in 1989 and 1990 while the SO₄-S concentration increased significantly. Aluminium concentration in the rhizosphere soil was generally higher in the NS plots in all soil layers, except at 0–10 cm depths, both in 1989 and 1990. Calcium, Mg and K concentrations also increased after treatment with ammonium sulphate. Ammonium ions may have replaced these elements in the soil organic matter. The NS treatment significantly reduced Mg concentrations in fine roots in all layers in 1990. A similar trend was found in the needles. Ca concentrations in fine roots were significantly lower in the NS plots in the LFH layer in 1990 and the same pattern was found in the current needles. The N and S concentrations of both fine roots and needles were significantly higher in the NS plots. It was suggested that NS treatment resulted in displacement of Mg, Ca and K from exchange sites in the LFH layer leading to leaching of these cations to the mineral soil. Further application of ammonium sulphate may damage the fine roots and consequently adversely affect the water and nutrient uptake of root systems.     

Natural incidence of interruptions in the internal elastic lamina of caudal and renal arteries of the rat

Interruptions in the internal elastic lamina (IEL) have been quantified by light microscopy in the caudal arteries of different strains of rats and in caudal and renal arteries of (i) male and female virgin rats up until 2 years of age, (ii) repeatedly bred rats and (ii) hypertensive rats. Results showed that gaps in the IEL exist in caudal arteries of adult males in all strains studied, but to a lesser extent in the hairless mutant. In the virgin Wistar rat these interruptions in the IEL form with age in both caudal and renal arteries, and are more numerous (i) in the male than in the female in both arteries, and (ii) in the caudal than in the renal artery in both sexes. Repeated breeding in the Wistar rat abolishes the sex difference in incidence of IEL gaps in both renal and caudal arteries. The Sprague-Dawley breeder rat is more susceptible than the Wistar breeder to their formation in the renal artery. Hypertension in the male consistently increases the formation of IEL gaps in the renal but not in the caudal artery. The importance of local factors, e.g. hemodynamics, in the formation of these defects in the IEL, and their possible relationship with the development of arteriosclerosis is discussed.     

Levels of linoleic acid and saturated and monounsaturated fatty acids in selected lipid fractions of the serum and platelets in patients with coronary arterial disease

The levels of serum fatty acids in the serum and in the serum fractions of cholesterol esters (ECH) and triglycerides (TG) and the levels of these acids in these fractions of platelets were compared in healthy controls and in patients with clinically manifested coronary arterial disease. Decreased level of linoleic acid was found in the serum and in the ECH fraction of the serum in the patients, with a rise in the level of palmitic acid in the ECH fraction of the serum of these patients. The level of linoleic acid in the ECH and TG platelet fractions in these patients was not different from that in the healthy controls, while in the platelet TG fraction of the patients the level of palmitoleic acid was raised, and the level of oleic acid was increased in the platelet ECH fraction.     

Ovulation and the movement of the conceptus in the first 35 days of pregnancy in thoroughbred mares.

Data from 937 Thoroughbred mares, when analysed as a single population, showed no significant difference between the activity of the right and left ovaries and the frequency of location of 35-day pregnancies in the right and left uterine horns. Nor were any differences shown when the data were analysed in years or sire groups. However, significantly more ovulations occurred in the right ovary in September and December and in the left ovary in October and November. Whereas the left and right ovulations were equal in lactating and dry mares, there was a highly significant increase in the number of pregnancies in the right uterine horn in dry mares and in the left uterine horn in lactating mares.     

Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.

Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.     

Sex and estrous cycle-dependent differences in glial fibrillary acidic protein immunoreactivity in the adult rat hippocampus

Sex differences in the morphology and function of the hippocampus have been reported in several species, but it is unknown whether a sexual dimorphism exists in glial fibrillary acidic protein (GFAP) expression in the rat hippocampus. We analyzed GFAP immunoreactivity in the hippocampus of intact adult male rats as well as in females during diestrus and proestrus phases of the estrous cycle. We found that in CA1, CA3, and dentate gyrus, GFAP immunoreactivity was higher in proestrus females as compared with males and diestrus females. In CA1, a similar GFAP immunoreactivity was found in males and in diestrus females, but in dentate gyrus, males presented the lowest GFAP content. Interestingly, differences in astrocyte morphology were also found. Rounded cells with numerous and short processes were mainly observed in the hippocampus during proestrus whereas cells with stellate shape with few and long processes were present in the hippocampus of males and diestrus females. The marked sex and estrous cycle-dependent differences in GFAP immunoreactivity density and in astrocyte number and morphology found in the rat hippocampus, suggest the involvement of sex steroid hormones in the sexually dimorphic functions of the hippocampus, and in the change in its activity during the estrous cycle.     

In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius Ham. (rosy barb).

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/l cadmium chloride) and in vitro (10(-6) M cadmium chloride). 2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (AlP) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AlP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills. 3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and GPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills. 4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family

Cells expressing mRNA for hippocampus-derived neurotrophic factor (HDNF/NT-3) or brain-derived neurotrophic factor (BDNF) were identified by in situ hybridization. In the rat brain, HDNF mRNA was predominantly found in pyramidal neurons in CA1 and CA2 of the hippocampus. Lower levels of HDNF mRNA were found in granular neurons of the dentate gyrus and in neurons of the taenia tecta and induseum griseum. BDNF mRNA-expressing cells were more widely distributed in the rat brain, with high levels in neurons of CA2, CA3, and the hilar region of the dentate gyrus, in the external and internal pyramidal layers of the cerebral cortex, in the claustrum, and in one brainstem structure. Lower levels were seen in CA1 and in the granular layer of the hippocampus, in the taenia tecta, and in the mammillary complex. In peripheral tissues, HDNF mRNA was found in glomerular cells in the kidney, secretory cells in the male rat submandibular gland, and epithelial cells in secondary and tertiary follicles in the ovary. Cells expressing BDNF mRNA were found in the dorsal root ganglia, where neurons of various sizes were labeled.     

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.     

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Total nitrogen and free amino acids in Morus alba stems from autumn through spring

During leaf senescence and abscission, total nitrogen in leaves of mulberry ( Morus alba L. ev. Shin-ichinose) declined substantially whereas total nitrogen in buds, bark and stem wood increased markedly, suggesting translocation of nitrogen from senescent leaves in the autumn. After leaf abscission the winter buds and stems remained almost unchanged with respect to fresh and dry weight and total nitrogen until bud break in spring. In burst buds these parameters then increased drastically during the new growth while they decreased markedly in stems. Free arginine in the stem bark accumulated in parallel with the accumulation of total nitrogen in buds and stems in the autumn. Accumulation of proline in the wood, bark and buds also started in October but continued even after leaf-fall, increasing until mid-January (wood), mid-February (bark) and the new growth (buds). Prior to and in the early stage of bud break, proline in bark and wood decreased significantly and arginine in stem bark decreased slightly. Simultaneously, proline and arginine in the dormancy-releasing buds and asparagine, aspartic acid and glutamic acid in the buds and stems increased appreciably, suggesting that this increase in free amino acids was mainly derived from free amino acids (proline and arginine) stored in stems. The resulting marked decrease in total nitrogen and the drastic increase in asparagine in the stems and sprouting buds/new shoots were primarily due to a breakdown of protein stored in stems.     

Adrenomedullin gene expression and peptide levels in the heart and blood vessels of streptozotocin-diabetic rats.

The aim of the present study was to assess the changes in gene expression and peptide adrenomedullin (AM) levels in cardiovascular and other tissues in the streptozotocin-diabetic rats. For this purpose, diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/Kg body weight). Half of the diabetic rats were subcutaneously injected with insulin in the afternoon (4 units/day) one week after STZ injection until the day before killing. Control rats received only saline injection. AM mRNA was determined in cardiovascular and other tissues of streptozotocin-diabetic rats using solution-hybridization-RNase protection assay. Circulating AM and peptide AM in cardiovascular and other tissues were estimated using a specific radioimmunoassay. There were increases in preproAM mRNA levels in the left and right ventricles and in the thoracic aorta in both the 2-week and 4-week diabetic rats, but not in the two atria, the mesenteric artery and the lung. In the 2-week diabetic rats, there were decreases in AM contents in the two atria and the lung but an increase in the thoracic aorta. In the 4-week diabetic rats, there were bigger decreases in the atria and also a decrease in the left ventricle. The plasma AM levels were not changed but there was an increase in the excretion of AM in the urine. Our results suggest a possible increase in AM release in the heart and the thoracic aorta in the 2-week and 4-week diabetic rats.     

Histochemical, immunofluorescence, and ultrastructural differences in fetal cartilage among three genetically distinct chondrodystrophic mice

The severe lethal chondrodystrophies in man result in a common clinical syndrome including shortening of the face, mandible, and limbs. Studies of three lethal chondrodystrophic mutants in mice, viz., chondrodysplasia (cho), cartilage matrix deficiency (cmd), and disproportionate micromelia (Dmm), which share this syndrome, were performed with the aim of identifying histochemical, immunofluorescence, or ultrastructural differences which might exist among these hereditary cartilage disorders. We examined limb cartilage epiphyses from day 18 normal and mutant fetuses and observed repeatable, mostly qualitative differences. All observations were made relative to the normal control. Histochemical staining of matrix proteoglycan was moderately decreased in cho and Dmm cartilage and markedly decreased in cmd when compared to the normal control. Staining of matrix collagen was irregular in distribution in cho, increased in cmd, and decreased in Dmm. Immunofluorescence of proteoglycan was increased in the matrix of cho and Dmm and decreased in cmd. Immunofluorescence of type II collagen was heterogeneous and moderately decreased in the matrix of cho, increased in cmd, and markedly decreased in Dmm. Immunofluorescence of link protein in cho was localized in the cellular-pericellular region as in the normal and appeared increased in the matrix of cmd and Dmm. Immunofluorescence of chondronectin was localized in the cellular-pericellular region and appeared normal in all three mutants. Major differences in cellular and matrix ultrastructure were observed among the mutants, including a decreased frequency of small-diameter collagen fibrils in cho and Dmm, increased density of collagen fibrils in cmd, and dilated RER in Dmm. These observations demonstrate that distinct structural and possibly molecular differences exist among the chondrodystrophies. In the case of cmd, the differences correlated with a previously reported molecular defect, viz., absence of core protein of cartilage specific proteoglycan in the cartilage of this mutant. It is anticipated that the methods used in the present study can be applied to humans in case classification and in identifying potential mouse-human correlates.     

Comparative distribution of substance P (SP) and cholecystokinin (CCK) binding sites and immunoreactivity in the brain of the sea bass (Dicentrarchus labrax).

Specific binding sites for cholecystokinin (CCK) and substance P (SP) were detected in the brain of a marine teleost fish, the sea bass, after in vitro incubation of tissue sections with the tritiated peptides and light microscopic autoradiography. Specific binding sites for [3H]-CCK were detected in the dorsal and ventral telencephalon, in the preoptic, tuberal and posterior hypothalamus, in the optic tectum, in the valvulla cerebelli, in the vagal lobe and further in a dorsal location in the medulla oblongata. Areas rich in [3H]-SP binding were located in the ventral telencephalon, in the entire hypothalamic and thalamic region, in the midbrain tegmentum, in the optic tectum, in the valvulla cerebelli and in the medulla oblongata. The distribution of these binding sites seemed to match fairly well with the location of the corresponding immunoreactive elements, although some minor mismatches could be observed. These autoradiographic findings provide the first anatomical evidence for the presence of CCK-like and SP-like binding sites in the brain of a teleost fish.     

Effect of a cholesterol-biosynthesis inhibitor on the fatty acid composition of phospholipids in the serum and tissues of rats

1. Changes produced by a cholesterol-biosynthesis inhibitor, trans-1,4-bis-(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944), in the total fatty acids in the liver and brain, and in phospholipids in the serum, liver, heart, brain and lungs from male rats, have been studied. 2. Treatment with AY-9944 produced the following changes in the fatty acid composition: (a) a marked decrease in the percentage of linoleic acid and an increase in oleic acid in the total fatty acids in the liver; (b) in the serum, an overall decrease in the percentage of linoleic acid in neutral lipids and phospholipids; (c) an increased content of linoleic acid in the beta-acyl chain of phosphatidylcholines in the liver and in sphingomyelins in the brain and lungs; (d) an increased content of palmitic acid and oleic acid in the beta-acyl chain of phosphatidylcholine in the liver, heart and lungs; (e) an increased content of phosphatidylcholines and sphingomyelins, together with an increased percentage of saturated fatty acids in these phosphatides in the lungs. 3. Changes in the phosphatides and the production of foam cells in the lungs suggest that AY-9944 may be of use in the study of the alveolar membrane.