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Abstract Background The insulin-like growth factor 2 (IGF2) and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR) has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity. DNA methylation in peripheral blood (n = 315) at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs), analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC) at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression. Results The principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units) positively correlated with skin fold thickness (at four CpG units) (P-values between 0.04 to 0.001) and subcutaneous adiposity (P = 0.023) at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC. Conclusions As greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we hypothesize that the HC may be a more sensitive marker of early life programming of the IGF axis and of fetal physiology than birth size. To verify this, investigations of the dynamics of IGF2/H19 methylation and expression from birth to adolescence are required.

Genetic risk for depressive disorders is poorly understood despite consistent suggestions of a high heritable component. Most genetic studies have focused on risk associated with single variants, a strategy which has so far only yielded small (often non-replicable) risks for depressive disorders. In this paper we argue that more substantial risks are likely to emerge from genetic variants acting in synergy within and across larger neurobiological systems (polygenic risk factors). We show how knowledge of major integrated neurobiological systems provides a robust basis for defining and testing theoretically defensible polygenic risk factors. We do this by describing the architecture of the overall stress response. Maladaptation via impaired stress responsiveness is central to the aetiology of depression and anxiety and provides a framework for a systems biology approach to candidate gene selection. We propose principles for identifying genes and gene networks within the neurosystems involved in the stress response and for defining polygenic risk factors based on the neurobiology of stress-related behaviour. We conclude that knowledge of the neurobiology of the stress response system is likely to play a central role in future efforts to improve genetic prediction of depression and related disorders.

Glucose administration is associated with memory enhancement in healthy young individuals under conditions of divided attention at encoding. While the specific neurocognitive mechanisms underlying this 'glucose memory facilitation effect' are currently uncertain, it is thought that individual differences in glucoregulatory efficiency may alter an individual's sensitivity to the glucose memory facilitation effect. In the present study, we sought to investigate whether basal hypothalamic-pituitary-adrenal axis function (itself a modulator of glucoregulatory efficiency), baseline self-reported stress and trait anxiety influence the glucose memory facilitation effect. Adolescent males (age range = 14-17 years) were administered glucose and placebo prior to completing a verbal episodic memory task on two separate testing days in a counter-balanced, within-subjects design. Glucose ingestion improved verbal episodic memory performance when memory recall was tested (i) within an hour of glucose ingestion and encoding, and (ii) one week subsequent to glucose ingestion and encoding. Basal hypothalamic-pituitary-adrenal axis function did not appear to influence the glucose memory facilitation effect; however, glucose ingestion only improved memory in participants reporting relatively higher trait anxiety. These findings suggest that the glucose memory facilitation effect may be mediated by biological mechanisms associated with trait anxiety.

Introduction: Behavioural evidence supports the notion that oral glucose ingestion enhances recognition memory judgements based on recollection, but not familiarity. The present study sought to clarify and extend upon these behavioural findings by investigating the influence of glucose administration on event-related potential (ERP) components that are thought to be differentially mediated by recollection and familiarity processes in healthy adolescents., Methods: In a within-subjects design, participants performed a recognition memory task, during which time electroencephalogram (EEG) was recorded, subsequent to ingestion of either (a) glucose or (b) placebo in a counterbalanced order., Results: Response times during the recognition memory task were observed to be faster for the glucose condition, relative to a placebo control. Further, glucose ingestion was associated with an enhanced left parietal old/new ERP effect (a marker of recollection) and an enhanced mid-frontal old/new ERP effect (known to be mediated by familiarity)., Discussion: These findings (a) support the results of previous research that the 'glucose memory facilitation effect' can be extended to healthy adolescents, but (b) suggest that glucose enhances both the recollection and familiarity components of recognition memory. The observed ERP profile has important implications for the proposal that glucose specifically targets the hippocampus in modulating cognitive performance.

Background: Although there are recognised associations between psychological and immune function, the effects of maternal depressive symptoms on fetal immune development have not been investigated., Methods: This study examined the relationship between maternal depression scores as assessed by the Beck Depression Inventory (BDI) in the second trimester and measure of neonatal immune function measured in cord blood. This study was conducted in a cohort of women (n=83) who had received either fish oil containing 3.7 g/day n-3 polyunsaturated fatty acid (n-3PUFA) or a placebo from 20 weeks gestation as part of a randomised controlled trial., Results: At 20 weeks gestation, prior to the intervention, 22% of women in the study manifested mild to moderate depressive symptoms (BDI > or =10). Neonates of these women had higher lymphoproliferative responses to a range of stimuli (including egg ovalbumin and cat allergen) compared with neonates of women with normal BDI scores (<10). These neonates also showed higher spontaneous cytokine production including (IL-6 and IL-10) and higher stimulated cytokine responses to both bacterial antigens and allergens. These patterns were evident after allowing for maternal age and education, parity, gestation, infant gender, delivery method and neonatal n-3/n-6 PUFA status., Conclusion: This exploratory study supports the notion that maternal mood in pregnancy may have the potential to influence fetal immune development. Further studies are needed to determine the significance of this.

The Cre/LoxP system provides a powerful tool to investigate gene function in vivo. This system requires Cre-recombinase expressing mouse lines that permit control of gene recombination in a tissue-specific and time-dependent manner. To allow spatio-temporal gene deletion in specific central nervous system (CNS) neuronal populations, we generated mice with a tamoxifen-inducible Cre (Cre-ER(T)) transgene under control of the Scl/Tal1 neural promoter/enhancer -0.9E3 (-0.9E3CreER(T) transgenic mice). Using Cre-reporter mice we have shown that tamoxifen-mediated Cre-ER(T) recombination in -0.9E3CreER(T) mice recapitulated the anticipated expression pattern of Scl in the caudal thalamus, midbrain, hindbrain, and spinal cord. Cre-mediated recombination was also effectively induced during embryogenesis and marked the same population of neurons as observed in the adult. Additionally, we identified a tamoxifen-independent constitutively active -0.9E3CreER(T) mouse line that will be useful for gene deletion during early neurogenesis. These -0.9E3CreER(T) mice will provide tools to investigate the role of neuronal genes in the developing and mature CNS. CNS., ((c) 2007 Wiley-Liss, Inc.)

Abstract The basic helix-loop-helix (bHLH) transcription factor Scl displays tissue-restricted expression and is critical for the establishment of the haematopoietic system; loss of Scl results in embryonic death due to absolute anaemia. Scl is also expressed in neurons of the mouse diencephalon, mesencephalon and metencephalon; however, its requirement in those sites remains to be determined. Here we report conditional deletion of Scl in neuronal precursor cells using the Cre/LoxP system. Neuronal-Scl deleted mice died prematurely, were growth retarded and exhibited an altered motor phenotype characterized by hyperactivity and circling. Moreover, ablation of Scl in the nervous system affected brain morphology with abnormal neuronal development in brain regions known to express Scl under normal circumstances; there was an almost complete absence of Scl-null neurons in the hindbrain and partial loss of Scl-null neurons in the thalamus and midbrain from early neurogenesis onwards. Our results demonstrate a crucial role for Scl in the development of Scl-expressing neurons, including gamma-aminobutyric acid (GABA)ergic interneurons. Our study represents one of the first demonstrations of functional overlap of a single bHLH protein that regulates neural and haematopoietic cell development. This finding underlines Scl's critical function in cell fate determination of mesodermal as well as neuroectodermal tissues.

Tumor growth is dependent in part on "neoangiogenesis." Functional involvement of bone marrow (BM)-derived cells in this process has been demonstrated. However, it remains controversial as to whether tumor endothelium itself is BM derived. Here we sought to address this issue with an endothelial-specific, inducible transgenic model. We generated Cretransgenic mice (endothelial-SCL-Cre-ER(T)) using the tamoxifen-inducible Cre-ER(T) recombinase driven by the 5' endothelial enhancer of the stem cell leukemia (SCL) locus. These mice were intercrossed with Cre reporter strains in which beta-galactosidase (LacZ) or enhanced yellow fluorescent protein (EYFP) are expressed upon Cre-mediated recombination. After tamoxifen administration, endothelial LacZ staining was observed in embryonic and adult tissues. Cre-mediated recombination was also observed in newly generated tumor endothelium. In adult BM cells we could only detect trace amounts of recombination by flow cytometry. Subsequently, BM from endothelial-SCL-Cre-ER(T);R26R mice was transplanted into irradiated recipients. When tumors were grown in recipient mice, which received tamoxifen, no tumor LacZ staining was detected. However, when tumors were grown in endothelial-SCL-Cre-ER(T);R26R mice 3 weeks after the cessation of tamoxifen treatment, there was widespread endothelial LacZ staining present. Thus, this genetic model strongly suggests that BM cells do not contribute to tumor endothelium and demonstrates the lineage relation between pre-existing endothelium and newly generated tumor endothelial cells.

The basic helix-loop-helix (bHLH) transcription factor stem cell leukaemia (SCL) is a 'master regulator' of haematopoiesis, where SCL is pivotal in cell fate determination and differentiation. SCL has also been detected in CNS, where other members of the bHLH-family have been shown to be indispensable for neuronal development; however, no detailed expression pattern of SCL has so far been described. We have generated a map of SCL expression in the embryonic and adult mouse brain based on histochemical analysis of LacZ reporter gene expression in sequential sections of brain tissue derived from SCL-LacZ knockin mice. The expression of LacZ was confirmed to reflect SCL expression by in situ hybridisation. LacZ expression was found in a range of different diencephalic, mesencephalic and metencephalic brain nuclei in adult CNS. Co-localisation of LacZ with the neuronal marker NeuN indicated expression in post-mitotic neurons in adulthood. LacZ expression by neurons was confirmed in tissue culture analysis. The nature of the pretectal, midbrain and hindbrain regions expressing LacZ suggest that SCL in adult CNS is potentially involved in processing of visual, auditory and pain related information. During embryogenesis, LacZ expression was similarly confined to thalamus, midbrain and hindbrain. LacZ staining was also evident in parts of the intermediate and marginal zone of the aqueduct and ventricular zone of the fourth ventricle at E12.5 and E14. These cells may represent progenitor stages of differentiating neural cells. Given the presence of SCL in both the developing brain and in post-mitotic neurons, it seems likely that the function of SCL in neuronal differentiation may differ from its function in maintaining the differentiated state of the mature neuron.

Background: We have previously developed a quantitative calibrated PCR assay to measure cytokeratin 19 (CK19) expression in haematopoietic tissue in order to detect systemic micrometastases., Patients and Methods: Serial measurements of CK19 expression in bone marrow of patients with primary breast cancer were performed at operation, at 3 weeks and 6 months postoperatively., Results: Reference values for CK19 expression were established by analysing bone marrow samples from 48 healthy female volunteers or patients without epithelial cancer. Samples from breast cancer patients with CK19 values above the upper reference limit were considered positive. Bone marrow samples taken at operation were positive in 29 out of 141 patients (20.6%) and remained positive in 12, turned negative in 4 and were unavailable in 13 at 6 months postoperatively., Conclusion: Serial measurements increase the reliability of detecting micrometastases perioperatively. Further studies are in progress to evaluate the relationship between elevated CK19 values and clinical outcome.

Objective: We developed a quantitative reverse-transcription polymerase chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue., Design and Methods: Our method incorporates a calibrated PCR with an internal competitor and an external standard., Results: The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples., Conclusion: To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.

An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluorescent labelling, to separate and detect the PCR products. Calibration curves were constructed from a serial dilution of CK19 cDNA coamplified with a fixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carried out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoint cluster region (BCR) gene expression. The assay is sensitive, detecting < 10 CK19 transcripts, and reproducible with a coefficient of variation of approximately 10%. CK19 expression showed overlapping values when measured in samples from peripheral blood and bone marrow in operable breast cancer patients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts.

This study was designed to examine adrenocortical function in old (30 months) and young (6 months) male Brown Norway rats. The following observations were made. First, stress induced a higher pituitary adrenocorticotropic hormone (ACTH) response in the aged male Brown Norway rats than in young rats, while peak circulating corticosterone (CORT) levels were not different. Moreover, this type of "repeated" stress involving subcutaneous injection and blood sampling at various time points by pinching the tail vein, evoked a prolonged ACTH and CORT response in the aged animal. Second, exogenous ACTH1-24 administered to dexamethasone-pretreated Brown Norway rats, used as an in vivo challenge test for adrenocortical function, resulted in a delayed CORT response in the aged rats. The termination of the CORT response to ACTH, however, was not different between young and old rats. Third, ACTH1-24 stimulation of adrenocortical cells in vitro showed a tendency to a reduced CORT output, when these cells were obtained from old animals. Fourth, adrenalectomy (ADX) differentially affected pituitary ACTH release at both ages. The initial post-ADX ACTH surge was more pronounced in the aged animals. Beyond 4 days post-ADX the old Brown Norway rats did not show the pronounced afternoon peak in circulating ACTH as was observed in the young animals. This study demonstrates that during the aging process a deficiency in adrenocortical function develops in the male Brown Norway rat. This deficiency involves a less efficient stress-induced activation of adrenocortical output of CORT having enhanced pituitary ACTH release as one of the consequences.(ABSTRACT TRUNCATED AT 250 WORDS)

A retrospective study on the thyroid status of 832 infants, born in Panaga Hospital in Brunei, was conducted. Despite a high degree of ethnic variability, screening for congenital hypothyroidism (CHT) included a fixed T4 and a TSH reference interval, based on a population of Malay infants. We tested their reliability for this heterologous group of infants. New T4 and TSH intervals were determined for each ethnic group and compared, revealing false positive and false negative judgements made during the period of study. Caucasian infants showed significantly higher T4 and TSH serum levels than all other ethnic groups. Regarding T4, most false positive judgements were found among the Malay infants. False negative judgements were detected among the Caucasian female infants. A new reference scheme was recommended, consisting of reference values that are applicable in neonatal thyroid screening of all infants in Panaga Hospital, regardless of their ethnic origin.

1. In the brain, glucocorticoids bind to both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). These receptors show clearly distinct developmental patterns in the infant rat. 2. Low levels of GRs are present around the time of birth throughout the brain. Concentrations rise slowly, and do not achieve adult levels until the third week of life, approximately. GR affinity for corticosterone is higher perinatally than at later ages. Receptor microdistribution changes dramatically during ontogeny. In particular, certain regions, such as the suprachiasmatic nucleus of the hypothalamus, express high levels of receptor only during the first week of life. GRs may show impaired capacity to undergo transformation and/or nuclear translocation during the second postnatal week. Environmental manipulations during early ontogeny (e.g., early handling) may have permanent effects on GR capacity. 3. MRs are present at very low concentrations in the first days of life. Binding capacity rises rapidly thereafter and resembles that found in the adult by the end of 1 week. Neither binding affinity in vitro nor overall distribution changes with age. As in the adult, low doses of corticosterone, in vivo, bind mainly to the MRs. Levels of corticosterone are low and relatively unperturbable in the intact infant rat. It is likely, therefore, that most of the physiological actions of this hormone during this period are mediated by the MR.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary of old (30 months) and young (3 months) male Brown Norway rats. Adrenocorticotropin hormone (ACTH) and corticosterone (B) were measured following exposure to novelty and to a conditioned emotional stimulus in blood samples sequentially obtained from chronically cannulated animals. Mineralocorticoid (MR) and glucocorticoid (GR) receptors were quantified by radioligand binding assay and in situ hybridization. The receptor binding constants were determined in tissue of rats that were adrenalectomized 24 hours previously, whereas gene expression was measured in the brain of intact animals. Aged Brown Norway rats showed a small but significant elevation in basal circulating ACTH level. The conditioned emotional stimulus, rather than the exposure to novelty, triggered a more than two-times higher ACTH response in the aged compared to the young rat. The termination of the stress-induced ACTH response seemed to proceed more efficiently in the aged rat. Basal and stress-induced total plasma B level did not differ in the young and old rats. The latter showed a 65% lower binding capacity of corticosteroid-binding globulin (CBG). Interestingly, in the aged rat the stress-induced rise in free circulating plasma B level was not elevated, but only prolonged. The hippocampus of aged rats displayed a decrease of maximally 44% in the apparent Bmax of MR, but no change in GR number. The Bmax of GR showed a 40% reduction in the hypothalamus and a 50% reduction in the anterior pituitary. GR affinity was considerably increased in the anterior pituitary, but was unchanged in the hippocampus and hypothalamus. Old age affected MR and GR gene expression differentially. GR mRNA was significantly reduced in cell field CA3 (-42%), CA4 (-41%) and the dentate gyrus (-26%) of the dorsal hippocampus, but did not change either in hippocampal cell field CA1 or in the hypothalamic paraventricular nucleus (PVN) of the old rat. There was no significant difference in MR mRNA between young and aged rats in the different cell fields of the hippocampus. The aged rat, therefore, is characterized by site- and receptor-specific changes in binding constants as well as by changes in receptor transcription and translation. The data demonstrate that in the old Brown Norway rats, a conditioned emotional stimulus results in enhanced pituitary ACTH release.(ABSTRACT TRUNCATED AT 400 WORDS)

In situ hybridization was used to study the neuroanatomical distribution of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression during development in the rat. This study was based on incubation of adjacent sections of brains from 2-, 8-, 12-, 16-day-old and adult (3 months) rats with 35S-labelled cRNA probes. These probes are transcribed from 513 and 500 basepair cDNA fragments with little homology from rat brain MR and rat liver GR respectively. Different patterns of expression were found in the brain of MR and GR during ontogeny. At postnatal day (pnd) 2, a high density of labelled MR mRNA was found in all pyramidal (CA1-4) and granular (dentate gyrus) cell fields of the hippocampal structure, the anterior hippocampus and indusium griseum, and cortex layer II. Modest to high labelling of MR mRNA was observed in the subfornical organ and the anterior hypothalamus. A variety of other telencephalic regions anterior and posterior of bregma exhibited modest to weak intensity of labelled MR mRNA. The diencephalon virtually lacked labelled MR mRNA. At older postnatal ages including the adult age, this regional distribution of radiolabelled MR mRNA did not change. At pnd 2, abundant radiolabelled GR mRNA was found widespread over the tel- and diencephalon, with the highest density observed in cell field CA1 and CA2 of the hippocampus and the parvocellular division of the hypothalamic paraventricular nucleus. Modestly labelled GR mRNA was observed in various hypothalamic and thalamic nuclei, basal ganglia, the lateral septum and the amygdala. At older postnatal ages and in adulthood, the intensity of labelled GR mRNA became progressively stronger in the hippocampus. Moreover, we observed a trend towards a more condensed and narrow band of cell bodies in the hippocampus for both MR and GR mRNA during ontogeny. A semi-quantitative comparison of the intensity of both labelled mRNA's performed at each age revealed a significantly lower expression of GR than MR mRNA in the CA3 cell field at pnd 2. At pnd 8 and 12, the amount of GR mRNA was significantly lower in the dentate gyrus and the CA3, whereas in adulthood, less GR mRNA was measured in all pyramidal and granular cell fields. The present study demonstrates that MR and GR genes are expressed in early postnatal development in a pattern resembling that in adulthood. As is the case in the adult brain, there is more MR than GR mRNA in the hippocampus during ontogeny, especially in the CA3 cell field and the DG.

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary in old (30 months) and young (3 months) male Brown Norway rats. The data demonstrate that circulating ACTH rather than the corticosteroid plasma level was elevated under basal conditions and following stress. Furthermore, a reduction of mineralocorticoid receptor (MR) number in the hippocampus and of glucocorticoid receptor (GR) number in the hypothalamus and the pituitary correspond to increased neuroendocrine responsiveness and negative feedback following stress. The changes in receptor binding do not parallel the changes in the amount of MR and GR mRNA measured with in situ hybridization. This suggests that the processing rather than the receptor gene expression is affected in senescence.

Corticosteroids interact with receptors in the central nervous system. These receptors display heterogeneity and can be distinguished as corticosterone- and aldosterone-binding mineralocorticoid receptors and dexamethasone-binding glucocorticoid receptors. Ligand specificity of mineralocorticoid receptors for either corticosterone or aldosterone seems to be determined by co-localized transcortin and the enzyme, 11 beta-hydroxysteroid dehydrogenase. Aldosterone-selective mineralocorticoid receptors appear to be present in the circumventricular organs and the AV3V region of the hypothalamus and mediate behavior that is driven by salt appetite. Highest concentrations of mineralocorticoid receptors are found in neurons of the hippocampus. These limbic mineralocorticoid receptor sites mediate tonic influences of corticosterone on brain processes. Glucocorticoid receptors bind corticosterone with a tenfold lower affinity than do mineralocorticoid receptors, and are widely distributed in neuronal and glial cells of the brain. Glucocorticoid receptors are involved in the termination of the stress response (negative feedback). Studies involving measurement of glucocorticoid receptor mRNA and binding sites have revealed that glucocorticoid receptors are subject to autoregulation. After ADX, glucocorticoid receptor concentration increases, but is reduced after chronic stress, chronic administration of glucocorticoids, and at senescence. A diminished glucocorticoid receptor concentration may compromise the negative feedback action exerted by glucocorticoids after stress. After ADX, mineralocorticoid receptor binding is acutely up-regulated and reaches its maximum between 7 and 24 hours post-ADX. Mineralocorticoid receptor mRNA level shows a transient increase following ADX. Long-term ADX has no effect on the mineralocorticoid receptor concentration, but, interestingly, chronic dexamethasone treatment results in an up-regulation of mineralocorticoid receptors. Mineralocorticoid receptor level is decreased at senescence, but this age-related decrement can be reversed by chronic treatment with the ACTH4-9 analog, ORG 2766. Functionally, mineralocorticoid receptors and glucocorticoid receptors are involved in different aspects of the organization of the stress response, and in conjunction they control the stress responsiveness of the animal.

Quantitative in vitro autoradiography, cytosol receptor assay in punched brain tissue, and immunocytochemistry have revealed that the glucocorticoid receptor is present in the rat supraoptic nucleus (SON). Based on its binding characteristics the receptor appears to be the type II glucocorticoid receptor. With the use of a monoclonal antibody against purified liver glucocorticoid receptor, immunostaining was found in magnocellular neurosecretory neurons in the SON, but not in magnocellular neurons in the paraventricular nucleus. Immunoreactive cells seem to be concentrated in ventral parts of the SON where vasopressin cells were previously shown to be located. One to 2 weeks after bilateral adrenalectomy, there was a substantial decrease in glucocorticoid receptor immunostaining in magnocellular as well as other types of neurons in various brain regions. Administration of synthetic glucocorticoids (RU 28362 or dexamethasone) induced a robust increase in the intensity of immunostaining in cell nuclei of neurosecretory cells. The presence of glucocorticoid receptors in the SON suggests that glucocorticoids may affect vasopressin synthesis or/and secretion through a direct action on magnocellular neurons.

In vivo and in vitro autoradiography with radiolabeled corticosteroid analogs as well as immunocytochemistry with monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to determine the presence and the topography of two corticosteroid receptor systems (type I and type II) in hamster and rat brains. In the rat, the in vivo autoradiograms clearly revealed the retention by the type I receptor of tracer amount of [3H]corticosterone, primarily in the CA1 and CA2 cell field, dentate gyrus and lateral septum. In the hamster, tracer doses of [3H]cortisol were retained not only in the CA1, CA2, dentate gyrus and lateral septum, but also at high level in the CA3 and CA4 areas. In both species, immunocytochemistry showed the widespread distribution of the type II receptor sites in areas such as the hippocampus, lateral septum, hypothalamus (particularly in the paraventricular nucleus), thalamus and cortex (these results were also reflected in the in vitro autoradiography). Strong cell nuclear glucocorticoid immunoreactivity (type II-IR) was observed in the CA1 and CA2 (as well as CA3 and CA4 in the hamster) pyramidal neurons. In the hippocampus of intact animals, type II-IR was seen in the neuronal cell nuclei. Adrenalectomy caused a depletion of the type II-IR signal from the cell nucleus, which returned 1 h following subcutaneous administration of RU 28362 to adrenalectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The localization of the glucocorticoid receptor (GR) (type 2) in the rat brain was studied with immunocytochemistry using a monoclonal antibody against the rat liver GR. Strong GR immunoreactivity (GR-ir) was observed in neurons of limbic and brainstem structures known to be associated with the stress-activated circuitry, which suggest that these sites are responsive to glucocorticoid feedback. The intracellular localization of GR-ir was examined in CA1 and CA2 pyramidal neurons of the hippocampus. In intact rats GR-ir is predominantly present in the cell nucleus. Adrenalectomy (ADX) caused a slow depletion of the GR-ir signal from the cell nucleus until near detection limits at two weeks postsurgery. At that time, 1 h after administration to longterm ADX rats the synthetic glucocorticoid (type 2) agonist RU 28362 as well as a moderate and high dose of corticosterone (CORT) markedly enhanced the cell nuclear GR-ir. The type 2 antagonist RU 38486 also caused an increase of GR immunostaining in cell nuclei upon acute administration to ADX rats. The mineralocorticoid aldosterone (ALDO) and a low dose of CORT, which bind almost exclusively to type 1 corticosteroid receptors, were ineffective. In conclusion, our data suggest that in the hippocampal CA1-2 neurons type 1 and type 2 corticosteroid receptors may coexist. The steroid-induced changes in cell nuclear immunoreactive GR staining intensity suggest possible cytoplasmic-cell nuclear translocation of GR and/or exposure of immunogenic GR domains.

The distribution of vasotocin (VT)-immunoreactive neuronal perikarya and fibers in the canary (Serinus canaria) was investigated with immunohistological techniques. The results suggest that most VT-stained cell bodies are located in three diencephalic regions. First, a large number of densely packed neurons are found in the paraventricular nucleus (PVN) and the anterior preoptic nucleus. Neurons here vary widely in size and shape. Small-size rounded neurons and large-size multipolar neurons appear to concentrate in separate subdivisions. Second, a series of loosely organized cell groups of medium- to large-size cells occurs in the lateral parts of the hypothalamus. These aggregates of neurons apparently correspond to subdivisions of the supraoptic nucleus (SON). Third, diffusely distributed, lightly stained cells are found dorsal to the paraventricular nucleus in the dorsal diencephalon. A number of cells of this group seem to be located in the basal septal area and bed nucleus of the stria terminalis. Immunoreactive fibers and varicosities concentrate in brain regions that are associated with neuroendocrine, autonomic, and limbic functions. Axons from the PVN and SON form compact bundles of the hypothalamohypophysial tract in the lateral hypothalamus and then funnel into the internal zone of the medium eminence (ME). Furthermore, a heavy innervation seems to be present in the palisadal, external zone of the ME. A substantial number of fibers appear to leave the PVN toward extrahypothalamic areas. Most extrahypothalamic VT fibers innervate telencephalic and brainstem regions that are thought to be involved in mediation of limbic and autonomic functions. These areas include the lateral and medial septum, the lateral habenula, the substantia grisea centralis, the area ventralis (Tsai), the locus coeruleus, raphe nuclei, the nucleus tractus solitarii, and lateral medulla. In addition, fibers with immunoreactivity for VT innervate structures such as the optic tectum and the nucleus ovoidalis that have been implicated in sensory processing of visual and auditory information. Finally, VT fibers and varicosities occur in centers including the nucleus robustus archistriatalis and nucleus intercollicularis that have been implicated in vocal control.

The cellular localization of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) gene expression in the rat hippocampus was studied by in situ hybridization using 35S-labeled RNA-probes, complementary to either 513 bases of the rat brain mineralocorticoid receptor (MR)-mRNA or 500 bases of the rat liver glucocorticoid receptor (GR)-mRNA. Neurons in CA1, CA2, and the dentate gyrus expressed both receptor genes at high levels. The MR-mRNA was demonstrated in all pyramidal cell fields (CA1-4) of the hippocampal formation and the granular neurons of the dentate gyrus. In contrast, GR-mRNA was mainly restricted to CA1 and CA2 pyramidal cell fields and the dentate gyrus. This pattern of hybridization was found to agree with the cellular distribution of the two types of corticosteroid receptors detected previously in the hippocampus by autoradiography of the radio-labeled receptors and by immunocytochemistry of the receptor protein. These observations suggest that the corticosteroid receptors described previously as type 1 and type 2 are encoded by MR- and GR-mRNA, respectively. Although both the MR and GR genes are co-expressed in some hippocampal neurons, the unique patterns of distribution of the two receptor mRNAs in the hippocampal formation suggest that the genes for these receptors are differentially regulated. Moreover, the microanatomy of MR and GR expression provides insight into molecular mechanisms underlying the characteristic action of various steroids on behaviors involved in stress and circadian regulation.

The ontogeny of the Type 2 glucocorticoid receptor (GR) in the rat brain was examined using a monoclonal antibody raised against the rat liver GR. Marked changes both in the intensity and in the localization of GR immunoreactivity (GR-ir) were found to occur as a function of age and brain area examined. First, GR-ir was high perinatally and decreased to a low intensity of immunostaining around postnatal day 12 (pnd 12). Thereafter, GR-ir increased to a moderate intensity, which resembled adult levels by pnd 20 in most brain areas. Second, in some regions, such as the hippocampal CA3-4 pyramidal cell fields and the suprachiasmatic nucleus of the hypothalamus, GR-ir was only clearly present during the first postnatal week. Third, in the hippocampus, GR-ir localization showed a distinctive developmental trend towards greater compactness within the CA1-2 pyramidal cell fields and a greater restriction of immunoreactive staining to these cell fields with exclusion of the adjoining areas. Fourth, adrenalectomy reduced overall GR-immunopositive staining, which could be reversed by administration of the selective glucocorticoid agonist, RU 28362. Our results suggest that during ontogeny the glucocorticoid receptor system displays considerable plasticity. Such plasticity may provide a basis for understanding the role of glucocorticoids during brain development.

In vitro cytosol binding, receptor autoradiography with radiolabelled corticosteroid analogs, and immunocytochemistry with monoclonal antibodies have revealed the presence of two receptor systems for corticosteroids in rat and hamster brains. The type I receptor is found mainly in the hippocampal region, and in the hamster it binds cortisol (F) and corticosterone (B) with similar affinity while in the rat (a species which unlike the hamster secretes solely B) the type I receptor shows high affinity to B and not to F. The type II receptor is more widely distributed in the brain and it binds to F (hamster) or B (rat) with affinity 4-6-fold lower than to the type I. in vivo, the hamster type I and II retain F much more than B while those in the rat show the opposite. In conclusion, the present study clearly indicates species-specificity in type I and type II receptor systems in these animals. Furthermore, the type I receptor displays in vivo stringent preference for retention of the animal's predominantly circulating corticosteroid (F in hamster, in B in rat).

The effect of frontal deafferentation of the medial basal hypothalamus on pro-opiomelanocortin (POMC) gene expression was studied in the intermediate lobe (IL) and anterior lobe (AL) of the pituitary gland in the absence and presence of corticosterone (CORT). The lesion in the basal hypothalamus removed neural inputs to the IL and induced the glucocorticoid receptor (GR) in this tissue. The GR was visualized in the denervated IL by immunocytochemistry. Induction of the GR had a slow onset and was detectable at 3 weeks after lesion, but not at one week after the lesion. In order to study the effect of IL denervation on pro-opiomelanocortin (POMC) gene expression, the level of messenger RNA specifically encoding POMC was measured 1 and 3 weeks after lesion, in the IL and AL. In adrenalectomized (ADX) animals, the changes in POMC mRNA levels were not significant 1 week after lesion in the IL. Three weeks after denervation there was a 3-fold decrease in POMC mRNA in the IL in ADX rats which was blocked by chronic CORT replacement via subcutaneously implanted pellets. In the AL, CORT reduced the level of POMC gene expression in both the lesioned and control animals. It is concluded that (1) removal of neural input induces GR in the denervated IL cells; (2) with the appearance of the GR, POMC gene expression in the IL becomes sensitive to circulating glucocorticoids; (3) under these conditions, CORT may stimulate POMC gene expression in the IL as opposed to its inhibitory effect in the AL.