Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

A novel G protein-coupled receptor mediating both vasopressin- and oxytocin-like functions of Lys-conopressin in Lymnaea stagnalis.

We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.

Nuclear receptor coregulators differentially modulate induction and glucocorticoid receptor-mediated repression of the corticotropin-releasing hormone gene.

Nuclear receptor coregulators are proteins that modulate the transcriptional activity of steroid receptors and may explain cell-specific effects of glucocorticoid receptor action. Based on the uneven distribution of a number of coregulators in CRH-expressing cells in the hypothalamus of the rat brain, we tested the hypothesis that these proteins are involved as mediators in the glucocorticoid-induced repression of the CRH promoter. Therefore, we assessed the role of coregulator proteins on both induction and repression of CRH in the AtT-20 cell line, a model system for CRH repression by glucocorticoids. The steroid receptor coactivator 1a (SRC1a), SRC-1e, nuclear corepressor (N-CoR), and silencing mediator of the retinoid and thyroid hormone receptor (SMRT) were studied in this system. We show that the concentration of glucocorticoid receptor and the type of ligand, i.e. corticosterone or dexamethasone, determines the repression. Furthermore, overexpression of SRC1a, but not SRC1e, increased both efficacy and potency of the glucocorticoid receptor-mediated repression of the forskolin-induced CRH promoter. Unexpectedly, cotransfection of the corepressors N-CoR and SMRT did not affect the corticosterone-dependent repression but resulted in a marked decrease of the forskolin stimulation of the CRH gene. Altogether, our data demonstrate that 1) the concentration of the receptor, 2) the type of ligand, and 3) the coregulator recruited all determine the expression and the repression of the CRH gene. We conclude that modulation of coregulator activity may play a role in the control of the hypothalamus-pituitary-adrenal axis.

Brain corticosteroid receptor balance in health and disease.

In this review, we have described the function of MR and GR in hippocampal neurons. The balance in actions mediated by the two corticosteroid receptor types in these neurons appears critical for neuronal excitability, stress responsiveness, and behavioral adaptation. Dysregulation of this MR/GR balance brings neurons in a vulnerable state with consequences for regulation of the stress response and enhanced vulnerability to disease in genetically predisposed individuals. The following specific inferences can be made on the basis of the currently available facts. 1. Corticosterone binds with high affinity to MRs predominantly localized in limbic brain (hippocampus) and with a 10-fold lower affinity to GRs that are widely distributed in brain. MRs are close to saturated with low basal concentrations of corticosterone, while high corticosterone concentrations during stress occupy both MRs and GRs. 2. The neuronal effects of corticosterone, mediated by MRs and GRs, are long-lasting, site-specific, and conditional. The action depends on cellular context, which is in part determined by other signals that can activate their own transcription factors interacting with MR and GR. These interactions provide an impressive diversity and complexity to corticosteroid modulation of gene expression. 3. Conditions of predominant MR activation, i.e., at the circadian trough at rest, are associated with the maintenance of excitability so that steady excitatory inputs to the hippocampal CA1 area result in considerable excitatory hippocampal output. By contrast, additional GR activation, e.g., after acute stress, generally depresses the CA1 hippocampal output. A similar effect is seen after adrenalectomy, indicating a U-shaped dose-response dependency of these cellular responses after the exposure to corticosterone. 4. Corticosterone through GR blocks the stress-induced HPA activation in hypothalamic CRH neurons and modulates the activity of the excitatory and inhibitory neural inputs to these neurons. Limbic (e.g., hippocampal) MRs mediate the effect of corticosterone on the maintenance of basal HPA activity and are of relevance for the sensitivity or threshold of the central stress response system. How this control occurs is not known, but it probably involves a steady excitatory hippocampal output, which regulates a GABA-ergic inhibitory tone on PVN neurons. Colocalized hippocampal GRs mediate a counteracting (i.e., disinhibitory) influence. Through GRs in ascending aminergic pathways, corticosterone potentiates the effect of stressors and arousal on HPA activation. The functional interaction between these corticosteroid-responsive inputs at the level of the PVN is probably the key to understanding HPA dysregulation associated with stress-related brain disorders. 5. Fine-tuning of HPA regulation occurs through MR- and GR-mediated effects on the processing of information in higher brain structures. Under healthy conditions, hippocampal MRs are involved in processes underlying integration of sensory information, interpretation of environmental information, and execution of appropriate behavioral reactions. Activation of hippocampal GRs facilitates storage of information and promotes elimination of inadequate behavioral responses. These behavioral effects mediated by MR and GR are linked, but how they influence endocrine regulation is not well understood. 6. Dexamethasone preferentially targets the pituitary in the blockade of stress-induced HPA activation. The brain penetration of this synthetic glucocorticoid is hampered by the mdr1a P-glycoprotein in the blood-brain barrier. Administration of moderate amounts of dexamethasone partially depletes the brain of corticosterone, and this has destabilizing consequences for excitability and information processing. 7. The set points of HPA regulation and MR/GR balance are genetically programmed, but can be reset by early life experiences involving mother-infant interaction. 8. (ABSTRACT TRUNCATED)

A G-protein beta subunit that is expressed in the central nervous system of the mollusc Lymnaea stagnalis identified through cDNA cloning.

We have cloned cDNA encoding a G-protein beta subunit from the central nervous system (CNS) of the mollusc Lymnaea stagnalis. The deduced protein is very homologous to other metazoan beta subunits. Thus, the Lymnaea CNS can be used as a model system to study beta gamma subunits in their native setting since its large neurons can be manipulated and studied relatively easily in vivo.

Neuroanatomical distribution and colocalisation of nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) in rat brain.

The two structurally related nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) proteins have been found to differentially affect the transcriptional activity of numerous nuclear receptors, such as thyroid hormone, retinoic acid and steroid receptors. Because of the numerous effects mediated by nuclear receptors in brain, it is of interest to extend these in vitro data and to explore the cellular distribution of both corepressors in brain tissue. We therefore examined, using in situ hybridisation, whether the relative abundance of these two functionally distinct corepressors differed in rat brain and pituitary. We find that although both N-CoR and SMRT transcripts are ubiquitously expressed in brain, striking differences in their respective levels of expression could be observed in discrete areas of brain stem, thalamus, hypothalamus and hippocampus. Using dual-label immunofluorescence, we examined in selected glucocorticoid sensitive areas involved in the regulation of the hypothalamus-pituitary-adrenal axis activity, the respective protein abundance of N-CoR and SMRT. Protein abundance was largely concurrent with the mRNA expression levels, with SMRT relatively more abundant in hypothalamus and brain stem areas. Colocalisation of N-CoR and SMRT was demonstrated by confocal microscopy in most areas studied. Taken together, these findings are consistent with the idea that the uneven neuroanatomical distribution of N-CoR and SMRT protein may contribute to the site-specific effects exerted by hormones, such as glucocorticoids, in the brain.

Identification of differentially regulated genes in mildly hyperlipidemic ApoE3-Leiden mice by use of serial analysis of gene expression.

Although genes determining lipoprotein homeostasis and atherosclerosis are the subject of intensive investigation, only a subset of these genes is known at present. Hence, we do not have sufficient knowledge to explain the genetic basis of hyperlipidemia in the majority of subjects. Our aim was to identify novel genes and pathways underlying lipoprotein homeostasis by using serial analysis of gene expression. The liver expression profile of mild hyperlipidemic apolipoprotein E3-Leiden (E3L) transgenic mice was compared with that of the wild-type C57BL/6JIco (B6) mice. Over 18 000 liver transcripts of B6 as well as E3L mice were analyzed, representing >9400 unique genes. One hundred seventy-five genes showed altered expression between the strains (P<0.05). Although several of these genes belonged to known metabolic pathways, such as lipoprotein metabolism, detoxification processes, glycolysis, and the acute-phase response, most were novel. Differential gene expression of 8 of 10 genes tested could be confirmed by Northern blot analysis. This inventory of differentially expressed genes will provide a unique basis for detailed studies to gain more insight into their role in lipoprotein homeostasis and atherosclerosis.

The Yin and Yang of nuclear receptors: symposium on nuclear receptors in brain, Oegstgeest, The Netherlands, 13-14 April 2000.

Novel aspects of nuclear receptors and their function in brain were discussed at a recent Symposium in Oegstgeest, The Netherlands. Presentations covered the diversity of these receptors, their target genes, proteins involved in transcriptional regulation, functional consequences of nuclear receptor activation and their relevance for human pathology. By elucidating the signalling pathway of nuclear receptors in brain, potential targets for therapeutic treatment of brain disorders can be identified.

Glucocorticoid feedback resistance.

Glucocorticoid feedback resistance can be inherited or locally acquired. The implications of these two forms of resistance for disease are strikingly different. The inherited form is characterized by enhanced adrenocortical function and hypercorticism to compensate for a generalized deficit in the glucocorticoid receptor gene, but these individuals lack symptoms of Cushing's syndrome. By contrast, resistance acquired at the level of the hypothalamic corticotropin-releasing hormone (CRH) neurons is linked to hypercorticism, which is not compensatory but overexposes the rest of the body and the brain to glucocorticoids. This cell-specific glucocorticoid resistance can be acquired by genetically predisposed individuals failing to cope with (early) life events and causes enhanced vulnerability to disease-specific actions of glucocorticoids. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997; 8:26-33).

Corticosteroids in the brain. Cellular and molecular actions.

The rat adrenal hormone corticosterone reaches the brain and binds to intracellular receptors. These receptors comprise high-affinity mineralocorticoid and lower-affinity glucocorticoid receptors that, upon activation, affect the transcription rate of specific genes. The two receptor types are discretely localized in the brain, with particularly high expression levels in the hippocampus. Here we review recent studies showing that electrical properties and structural aspects of hippocampal principal neurons are specifically regulated by mineralocorticoid- or glucocorticoid-receptor activation. The molecular mechanisms by which these cellular effects could be accomplished are discussed.

Molecular cloning of the rDNA of Saccharomyces rosei and comparison of its transcription initiation region with that of Saccharomyces carlsbergensis.

We have cloned one complete repeating unit of rDNA from Saccharomyces rosei and determined its physical and genetic organization. Heteroduplex analysis of the rDNA units from S. rosei and S. carlsbergensis shows that the nontranscribed spacers are largely nonhomologous in sequence, whereas the transcribed regions are essentially homologous. We also determined the transcription initiation site for the 37S precursor RNA on S. rosei rDNA. Sequence comparison of the region surrounding the site of transcription initiation for the 37S RNA with the corresponding region of S. carlsbergensis revealed extensive homology from position -9 downstream into the external transcribed spacer. Very little homology was observed between position -9 and -55, but some homologous tracts are present upstream from position -55.

Characterization of a putative molluscan insulin-related peptide receptor.

In the pond snail Lymnaea stagnalis (Ls), growth and associated processes are likely to be controlled by a family of molluscan insulin-related peptides (MIP). Here we report on the cloning of a cDNA encoding a putative receptor for these MIP. This cDNA was isolated from Ls via PCR with degenerate oligodeoxynucleotides corresponding to conserved parts of the tyrosine kinase domain of the human insulin receptor and its Drosophila homologue. Many of the typical insulin-receptor features, including a cysteine-rich domain, a single transmembrane domain and a tyrosine-kinase domain are conserved in the predicted, 1607-amino acid (aa) protein. Comparison of the aa sequence of the molluscan receptor to other insulin-receptor sequences revealed strong variations in the percentage of sequence identity for the different domains, ranging from 70% sequence identity in the tyrosine-kinase domain to virtually no sequence identity in the C-terminal sequence. Striking differences are the absence of a clear tetrabasic cleavage site, and the extremely long C-terminus of 308 aa that contains seven Tyr residues. Southern blot analyses at varying stringencies, extensive screening of cDNA- and genomic libraries, and PCR experiments indicate the presence of a single putative MIP receptor. This suggests that the four different MIP may exert their functional role in Ls by binding to the same receptor.

Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.

Expression of the egg-laying hormone genes in peripheral neurons and exocrine cells in the reproductive tract of the mollusc Lymnaea stagnalis.

The neuroendocrine caudodorsal cells play an important role in the control of reproduction in Lymnaea stagnalis. These neurons produce at least nine neuropeptides which are encoded by caudodorsal cell hormone-I and -II genes. The role of some of these peptides in the control of reproduction has been established. The present study demonstrates that the transcription and translation of the caudodorsal cell hormone genes also proceeds abundantly in the reproductive tract of this hermaphroditic animal. In the female part of the reproductive tract neurons were found to express gene I. These neurons are most likely involved in the control of transport of the eggs and egg-masses and in the regulation of secretory activity from the female accessory sex glands. In the male part of the reproductive tract exocrine secretory cells express gene I or gene II. The gene products are secreted into the male duct and transferred to the female copulant during copulation. Furthermore, putative sensory neurons in the skin were found to express gene I. The results indicate that in L. stagnalis the complex process of reproduction is regulated--at least in part--by a set of neuropeptides which are encoded by a small multigene family, viz. the caudodorsal cell gene family.

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

Molecular dissection of corticosteroid action in the rat hippocampus. Application of the differential display techniques.

Both adrenal steroids and glutamate are crucial for hippocampal cell viability. In order to identify adrenal steroid- and glutamate-responsive genes controlling hippocampal cell viability, we have used the PCR-based differential display method. We have described the characteristics of this technique and how it can be automated. Using differential display, we have identified a number of rat hippocampal genes of which the expression is affected by a combination of the glutamate analog kainic acid and adrenalectomy. Administration of kainic acid or removal of the adrenals alone gave a limited number of differentially displayed genes. Therefore, our results indicate that the main mode of corticosteroid receptor-controlled gene expression in the hippocampus is interaction with other transcription factors (e.g., CREB, AP-1) and not by binding to hormone-responsive elements of corticosterone-specific genes. Characterization by multiplex PCR experiments of a differentially displayed fragment of which the expression is increased by the combination of kainic acid and adrenalectomy confirmed our differential display results. Further characterization by DNA sequence analysis of the corresponding full-length cDNA clone revealed a gene product with 91.4% sequence identity with the mouse transcription factor KROX-20, suggesting that we have cloned the rat homolog. This finding suggests a role of KROX-20 in corticosteroid- and kainic acid-controlled hippocampal plasticity.

Circadian variation in BDNF mRNA expression in the rat hippocampus.

BDNF mRNA levels in the hippocampus were studied during the circadian cycle by in situ hybridization. These levels display a circadian pattern, which may be due to regulation by corticosterone. This may have consequences for hippocampal functioning at different time points of the circadian cycle.

Multiple transcripts generated by the DCAMKL gene are expressed in the rat hippocampus.

We have recently cloned a novel Doublecortin CaMK-like kinase (rDCAMKL) cDNA, and a related cDNA called CaMK-related peptide (CARP) from the rat hippocampus. These genes are structurally highly similar to the human DCAMKL-1 gene and doublecortin, a gene associated with X-linked lissencephaly and subcortical band heterotopia. Here we report on the genomic organization of the murine DCAMKL gene and its products. Our results show that DCAMKL and CARP are alternative splice products of the same gene. The DCAMKL gene also generates three alternatively-spliced rDCAMKL transcripts of which we have cloned the corresponding cDNAs and which potentially generate different DCAMKL proteins. In situ hybridization experiments show that the different rDCAMKL transcripts are all expressed in the adult rat hippocampus. We conclude that alternative splicing of the DCAMKL gene may generate different but similar proteins in the adult rat hippocampus thereby regulating different but overlapping aspects of DCAMKL controlled neuronal plasticity.

Differential effects of dopamine depletion on the binding and mRNA levels of dopamine receptors in the shell and core of the rat nucleus accumbens.

In the present study, using quantitative receptor autoradiography and in situ hybridization histochemistry the effects of unilateral 6-hydroxydopamine lesions on the binding density levels of dopamine D1 and D2 receptors and the levels of mRNA encoding D1 and D2 receptors were investigated in the core and shell territories of the nucleus accumbens (Acb) and in the caudate-putamen (CP). The lesions induced contrasting effects on the D1 binding and D1 mRNA in the Acb and CP, i.e. an increase in binding and a decrease in the mRNA levels. For the D2 receptor an increase in both the binding density and mRNA levels was observed. The lesion-induced effects displayed regional differences. For D1 mRNA and D1 and D2 binding, the lesion effect was more pronounced in the core than in the shell of the Acb. For the D2 mRNA levels an increase was observed in the CP but not in the two territories of the Acb. Furthermore, the decrease in D1 mRNA was greater in the rostral than in the caudal parts of the core and shell of the Acb. These results indicate that the core and shell of the Acb and the CP respond differentially to dopamine depletion.

The molecular basis of the neuro-endocrine control of egg-laying behaviour in Lymnaea.

The biosynthesis, axonal transport and release of multiple peptides by the egg-laying controlling caudodorsal cells (CDC) of Lymnaea stagnalis were studied. High performance gel permeation chromatography was used to resolve newly synthesized peptides after pulse-chase experiments with radioactive amino acids. The precursor is a 35 kd polypeptide which is produced in the CDC somata. It gives rise to intermediate products (20 kd, 10 kd and 7 kd) and a number of end products which include a approximately 4.5 kd (the ovulation hormone, CDCH), a 1.5 kd peptide (the autotransmitter) and other peptides (6 kd, 3.5 kd and 2 kd). The end products are transported in neurosecretory granules to the CDC axon terminals in the cerebral commissure where they are released into the medium during electrical discharges of the CDC system. A cDNA clone encoding part of the CDCH precursor was isolated from a Lymnaea CNS cDNA library by differential hybridisation and use of synthetic oligonucleotide probes. Examination of the deduced amino acid sequence indicates that the precursor contains additional peptides, besides CDCH.