Early morphological and neurochemical changes of the bed nucleus of stria terminalis (BNST) in gestational protein-restricted male offspring.

BACKGROUND: The bed nucleus of the stria terminalis (BNST) is a structure with a peculiar neurochemical composition involved in modulating anxietylike behavior and fear. AIM: The present study investigated the effects on the BNST neurochemical composition and neuronal structure in critical moments of the postnatal period in gestational protein-restricted male rats' offspring. METHODS: Dams were maintained during the pregnancy on isocaloric rodent laboratory chow with standard protein content [NP, 17%] or low protein content [LP, 6%]. BNST from male NP and age-matched LP offspring was studied using the isotropic fractionator method, Neuronal 3D reconstruction, dendritic-tree analysis, blotting analysis, and high-performance liquid chromatography. RESULTS: Serum corticosterone levels were higher in male LP offspring than NP rats in 14-day-old offspring, without any difference in 7-day-old progeny. The BNST total cell number and anterodorsal BNST division volume in LP progeny were significantly reduced on the 14th postnatal day compared with NP offspring. The BNST HPLC analysis from 7 days-old LP revealed increased norepinephrine levels compared to NP progeny. The BNST blot analysis from 7-day-old LP revealed reduced levels of GR and BDNF associated with enhanced CRF1 expression compared to NP offspring. 14-day-old LP offspring showed reduced expression of MR and 5HT1A associated with decreased DOPAC and DOPA turnover levels relative to NP rats. In Conclusion, the BNST cellular and neurochemical changes may represent adaptation during development in response to elevated fetal exposure to maternal corticosteroid levels. In this way, gestational malnutrition alters the BNST content and structure and contributes to already-known behavioral changes.

Chromosome segregation and cytokinesis in bacteria.

Substantial progress has recently been made in the understanding of chromosome partitioning and cytokinesis in bacteria. The biochemical properties of some key protein components involved in these processes are beginning to emerge. New evidence supports the recently developed notion that, in prokaryotic cells, basic cell biological processes rely on the activity of previously unidentified cytoskeletal-like elements.

The distinction between retractor and protractor muscles of the freshwater snail's male organ has no physiological basis.

Many animals are equipped with organs that can be everted, a notable example being male copulatory organs. The ability to protrude or evert an organ generally requires protractor and retractor muscles. Male copulatory behaviour of the pond snail Lymnaea stagnalis (L.) involves eversion (protraction) and retraction of the relatively large penis-carrying organ. For this preputium, protractor and retractor muscle bands have been defined, which implies eversion and retraction through the activity of these muscle bands. However, no physiological data are available that confirm that the terms protractor and retractor are appropriate. To test whether eversion and retraction are possible without protractor and/or retractor muscle bands, lesion experiments were performed. The results show that with either one or several muscle bands lesioned, snails were still capable of everting their preputium and using it for copulation. However, the majority of animals that had six or more muscle bands lesioned were unable to retract its preputium. Hence, retractor muscle bands serve their designated function whereas protractor muscle bands do not. We therefore suggest that a different terminology is used in which all muscle bands are retractors and, based on their location, are either called distal or proximal retractors. The findings furthermore indicate that the preputium muscle bands are normally contracted, possibly in a catch state, retaining the organ inside without high-energy expenditure.

Effects of atrazine on female Wistar rats: morphological alterations in ovarian follicles and immunocytochemical labeling of 90 kDa heat shock protein.

Fertility in female mammals may be affected by a variety of endocrine disrupters present in the environment. Herbicide atrazine is an example of endocrine disrupter employed in agriculture, which disrupts estrous cyclicity in rats. Aiming to characterize morphologically the effect of low and sublethal doses of atrazine on the ovaries of Wistar rats, in an effort to determine the possible intrafollicular target site through which this herbicide acts adult females were submitted to both subacute and subchronic treatments. Additionally, immunocytochemical labeling of 90 kDa heat shock protein (HSP90) was performed in order to evaluate the role played by this protein in the ovary, under stressed conditions induced by herbicide exposure. The results indicated that atrazine induced impaired folliculogenesis, increased follicular atresia and HSP90 depletion in female rats submitted to subacute treatment, while the subchronic treatment with low dose of atrazine could compromise the reproductive capacity reflected by the presence of multioocytic follicle and stress-inducible HSP90.

Anxiety-like behavior and structural changes of the bed nucleus of the stria terminalis (BNST) in gestational protein-restricted male offspring.

Animal evidence has suggested that maternal emotional and nutritional stress during pregnancy is associated with behavioral outcomes in offspring. The nature of the stresses applied may differ, but it is often assumed that the mother's hippocampus-hypothalamic-pituitary-adrenal (HHPA) axis response releases higher levels of glucocorticoid hormones. The bed nucleus of the stria terminalis (BNST) is in a pivotal position to regulate the HHPA axis and the stress response, and it has been implicated in anxiety behavior. In the current study, to search whether BNST structural changes and neurochemical alterations are associated with anxiety-related behavior in adult gestational protein-restricted offspring relative to an age-matched normal protein diet (NP) rats, we conduct behavioral tests and, BNST dendritic tree analysis by Sholl analysis, associated to immunoblotting-protein quantification [11ß-HSD2, GR, MR, AT1R, 5HT1A and 5HT2A, corticotrophin-releasing factor (CRH) and CRH1]. Dams were maintained either on isocaloric standard rodent chow [with NP content, 17% casein or low protein content (LP), 6% casein] chow throughout their entire pregnancy. Here, in rats subjected to gestational protein restriction, we found: (a) a significant reduction in dendritic length and impoverished dendritic arborization in BNST neurons; (b) an elevated plasmatic corticosterone levels; and (c) associated with enhanced anxiety-like behavior when compared with age-matched NP offspring. Moreover, altered protein (11ß-HSD2, GR, MR and type 1 CRH receptors) expressions may underlie the increase in anxiety-like behavior in LP offspring. This work represents the first demonstration that BNST developmental plasticity by maternal protein restriction, resulting in fine structural changes and neurochemical alterations that are associated with modified behavioral states.

Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

Impact of long-term high-fat diet intake gestational protein-restricted offspring on kidney morphology and function.

Emerging evidence highlights the far-reaching consequences of high-fat diet (HFD) and obesity on kidney morphological and functional disorders. In the present study, we aim to evaluate the effects of early HFD intake on renal function and morphology in maternal protein-restricted offspring (LP). LP and normal protein-intake offspring (NP) were fed HFD (LPH and NPH, respectively) or standard rodent (LPN and NPN) diet from the 8th to 13th week of age. Blood pressure, kidney function, immunohistochemistry and scanning electron microscopy were analyzed. Increased total cholesterol and low-density lipoprotein serum levels were observed in LPH offspring. The adiposity index was reduced in the (LPN) group and, conversely, increased in the NPH and LPH groups. Blood pressure was higher beyond the 10th week of age in the LPH group compared with the other groups. Decreased urinary sodium excretion was observed in LP offspring, whereas the HFD-treated groups presented a decreased urine pH in a time-dependent fashion. The LPN, NPH and LPH groups showed increased expression of type 1 angiotensin II (AngII) receptor (AT1R), TGF-ß1, collagen and fibronectin in the kidneys. Moreover, the adult fetal-programmed offspring showed pronounced effacement of the podocyte foot process associated with the rupture of cell membranes and striking urinary protein excretion, exacerbated by HFD treatment. To the best of our knowledge, this is the first study demonstrating that young fetal-programmed offspring submitted to long-term HFD intake have increased susceptibility to renal structural and functional disorders associated with an accentuated stage of fibrosis and tubular dysfunction.

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl)phthalate.

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.

Interleukin-15 expression in atherosclerotic plaques: an alternative pathway for T-cell activation in atherosclerosis?

T-cell activation in atherosclerotic plaques is thought to be initiated by plaque-derived antigens, such as oxidized LDL (oxLDL). An alternative pathway of T-cell activation independent of antigen stimulation, mediated by the cytokine interleukin (IL)-15, was recently described. We investigated IL-15 expression in atherosclerotic plaques in relation to plaque morphology, inflammatory cells, T-cell activation, and oxidation-specific epitopes by use of immunohistochemistry. In situ hybridization was used to evaluate IL-15 mRNA expression. We also studied the proliferative response of plaque-derived T-cell lines to IL-15 in vitro using [(3)H]thymidine incorporation. Fresh-frozen specimens were classified as fibrous (n=9), fibrolipid (n=8), and lipid-rich (n=14) plaques; normal vessels (n=4) served as reference. Expression of IL-15 mRNA and protein was found almost solely in fibrolipid and lipid-rich plaques, associated with oxLDL-positive macrophages. Sequential immunostains revealed colocalization between IL-15- and CD40L-positive T cells. Moreover, plaque-derived T-cell lines were highly responsive to IL-15. Hence, IL-15 could provide a pathway for antigen-independent T-cell activation.

Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Pericentral expression pattern of glucokinase mRNA in the rat liver lobulus.

The spatial distribution of glucokinase mRNA (GK mRNA) in rat liver was studied by in situ hybridization under normal and inducing conditions. GK mRNA was first detectable in the liver parenchyma of neonatal rats of 1.5 days. The density of grains decreases in a central-portal direction. This pattern remains essentially unchanged up to 15 days, after which the adult type of distribution gradually starts to develop, i.e. low density of grains indicating low levels of GK mRNA, in which no gradient of expression could be visualized. Within 2 h after an oral glucose load to starved animals, the GK mRNA expression pattern changed from hardly detectable to a clear gradient with the highest grain density around the terminal central venules. Within 6 h relatively high levels of grains, almost homogeneously distributed across the liver lobule, were observed. Glucocorticosteroid treatment also induced GK mRNA in the pericentral area. It is concluded that the observed induction pattern qualifies GK mRNA as a pericentral mRNA suggesting that the pericentral expression pattern of the protein is primarily regulated at the pretranslational level.

Hepatic glutaminase mRNA is confined to part of the urea cycle domain in the adult rodent liver lobule.

This in situ hybridization study describes the developmental appearance of the lobular distribution of the mRNA encoding hepatic glutaminase in normal rat liver. Glutaminase has been proposed to provide the urea cycle with ammonia [Häussinger and Gerok (1983) Eur. J. Biochem. 133, 269-275]. Hence, the (developmental) pattern of expression of the mRNA would be expected to be closely linked to that of the urea cycle enzymes. From embryonic day 20 onward, hepatic glutaminase mRNA can be detected along the entire porto-central axis, with predominant expression in the portal area. In the adult phenotype, which is acquired at the end of the first postnatal week, glutaminase mRNA is no longer present along the entire porto-central distance but has become confined to a relatively small periportal domain in which the expression decreases in a porto-central direction. Thus, in contrast to the large periportal domain, in which the urea cycle enzymes are expressed, the glutaminase mRNA-expressing domain is much smaller and not contiguous with the glutamine synthase mRNA-expressing pericentral domain, leaving a midlobular area that is devoid of glutaminase mRNA. A similar pattern of distribution was found in adult mouse liver. The significance of these observations is that, within the liver lobules, there is an area in which glutaminase is not expressed and, hence, glutamine can not be the substrate for urea synthesis.

Diet- and hormone-induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus.

A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary-treated rats was carried out, using radioactively-labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line with previous findings, GS mRNA is exclusively expressed in a small pericentral compartment, CPS mRNA exclusively in a contiguous large periportal compartment and PEPCK mRNA across the entire porto-central distance. The density of labelling in CPS and PEPCK mRNA-positive hepatocytes decreases in a porto-central direction. Starvation resulted in a reversal of the gradient of CPS mRNA within its periportal compartment; glucose refeeding counteracted this effect. Livers of glucocorticosteroid-treated, starved or diabetic rats also revealed a reversal of the normal gradient of CPS mRNA, but now across the entire porto-central distance. The patterns of expression of GS and PEPCK mRNA remained essentially unchanged, notwithstanding substantial changes in the levels of expression. It is concluded that blood-borne factors constitute the major determinants for the expression patterns of CPS mRNA within the context of the architecture of the liver lobulus.

Messenger RNA sorting in enterocytes. Co-localization with encoded proteins.

This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane-bound glycoprotein, lactase-phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance of these observations is twofold; (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells; and (ii) they imply the presence of two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysomes (CPS and PEPCK mRNA).

The dynamics of the expression of C/EBP mRNA in the adult rat liver lobulus qualifies it as a pericentral mRNA.

A hybridocytochemical approach has been applied to establish whether the gene for the C/EBP mRNA might be involved in the topographical regulation of gene expression in adult rat liver. To that end the spatial distribution of the mRNA of C/EBP has been compared to that of the mRNAs of glutamine synthetase (GS), phosphoenolpyruvate carboxykinase (PEPCK) and glucokinase (GK) in normal adult livers, in livers from dexamethasone-treated animals and in livers from starved animals refed with glucose for 4 h. In normal rat liver, in situ hybridization with a probe for C/EBP mRNA revealed a low density of apparently homogeneously distributed grains, indicating low levels of C/EBP mRNA. In contrast, the livers of the experimentally-treated animals revealed a zonal distribution of the mRNA of C/EBP with the highest density of grains around the central venules. The dynamics of the pattern of expression of C/EBP mRNA are virtually identical to that of the GK mRNA. These data qualify C/EBP mRNA as a pericentral mRNA and suggest a role for the C/EBP protein in the topographical regulation of the expression of the GK mRNA.

Transmitter identification in neurons involved in male copulation behavior in Lymnaea stagnalis.

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Target-dependent differentiation and development of molluscan neurons and neuroendocrine cells: use of parasitisation as a tool.

Specimens of the freshwater snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata show a strongly inhibited development of their reproductive tract. We hypothesised that the effects of the underdevelopment of targets are reflected at the level of the neuronal development of (i) the motor neurons innervating the male copulation organ and (ii) neuroendocrine cells regulating the gonad. We determined the state of neuronal development by measuring cell number, cell size and neuropeptide gene expression. Our results show that the neuronal development of both copulation controlling anterior lobe motor neurons of the right cerebral ganglion and neuroendocrine caudodorsal cells, which produce neuropeptides regulating ovulation, egg laying and accompanying behaviour, are affected in parasitised animals in which their respective target organs were not developed. The cell bodies were smaller and fewer cells were found to express neuropeptide genes compared to those in non-parasitised animals. These effects were not observed in the appropriate controls. Backfills and lesions of the penis nerve have shown that the inhibited development of central motor neurons in parasitised snails is target dependent; neighbouring neurons that have no connection with the male copulation organ are not affected. Our data suggest that this effect is established by target-derived neurotrophic factors that need this connection for being transported to the innervating motor neurons. We propose that the effect on the neuroendocrine caudodorsal cells is mediated by a humoral factor, since they have no known connection with their target. We have shown that the size and gene expression of motor neurons controlling copulation behaviour in the pond snail Lymnaea stagnalis are related to the size of their target, the copulation organ, and depend on the connection with this target.

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level.

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.

Towards quantitative in situ hybridization.

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissue.