NGFI-B mRNA Research Articles

PS-B04-8: YM750, AN ACAT1 INHIBITOR, REDUCES ALDOSTERONE SECRETION BY DECREASING ADRENAL CYP11B2 EXPRESSION

Primary aldosteronism (PA) is a refractory hypertension that accounts for approximately 5%-10% of hypertensive patients. aldosterone-producing adenomas and idiopathic hyperaldosteronism are known causes of PA. In aldosterone-producing adenomas, depolarization due to somatic mutations of ion channels in adrenocortical cells is considered the cause of aldosterone oversecretion. Recently, there have been reports on the expression of various cholesterol-metabolizing enzymes in aldosterone-producing adenomas, suggesting that intracellular cholesterol metabolism is very important for aldosterone secretion. We present here the possibility that an inhibitor of Acetyl-CoA acetyltransferase (ACAT1), an enzyme that converts cholesterol to cholesteryl esters, may inhibit aldosterone secretion by depolarization in adrenal cells. Potassium chloride and YM750, an ACAT1 inhibitor, were added to human adrenocortical carcinoma, H295R cells. Then, we measured aldosterone synthase gene, CYP11B2 mRNA expression level. As a result, the expression level of CYP11B2 decreased. Both NURR1 and NGFIB mRNA expression levels also decreased. Additionally, aldosterone production was also measured and found to be significantly decreased in the YM750 added group. Next, angiotensin II was added and the inhibitory effect of YM750 was examined. The results showed that CYP11B2 expression was not decreased. This indicates that YM750 decreased the induction of CYP11B2 expression in a potassium chloride-induced stimulus-specific manner. The experimental results suggest that YM750 specifically inhibits CYP11B2 gene expression by cell depolarization and suppresses aldosterone secretion. However, there are few reports of cholesterol metabolism affecting CYP11B2 gene expression. In this study, we found that intracellular cholesterol metabolism may affect CYP11B2 gene expression. We plan to investigate the relationship between ACAT inhibition and CYP11B2 gene expression in detail mechanisms in the future.

Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

Differences in NGFI-B, Nurr1, and NOR-1 expression and nucleocytoplasmic translocation in glutamate-treated neurons

NGFI-B (NR4A1, Nur77 or TR3) together with Nurr1 (NR4A2) and NOR-1 (NR4A3) constitute the NR4A subgroup of orphan nuclear receptors. They play critical roles in proliferation, differentiation, survival and apoptosis in different cell types, including neurons, immature T-cells, and different cancer cells. As ligand-independent and constitutively active receptors, the diverse biological activities of NGFI-B, Nurr1 and NOR-1 depend on their levels of expression, post-translational modifications and subcellular localization. Nuclear localization of the NR4A proteins leads to transcriptional activity, whereas NGFI-B and recently also NOR-1 have been shown to induce apoptosis by a more direct mechanism when localized at mitochondria. In the present study we investigated mRNA expression and subcellular translocation of the NR4A proteins during glutamate excitotoxicity in rat cerebellar granule neurons. NGFI-B and Nurr1 mRNA, but not NOR-1 mRNA, were induced by treatments associated with calcium influx, although their regulation seemed to be different. NR4Agfp fusion proteins showed a predominant nuclear localization in untreated cells. After glutamate treatment NGFI-Bgfp translocated to cytosol and mitochondria within a few hours, whereas Nurr1gfp translocation was delayed, and NOR-1gfp mainly stayed in the nucleus. Subcellular targeting of NGFI-B seems to be tightly regulated, as a single mutation of threonine 142 altered NGFI-Bgfp localization. Differences in expression and subcellular translocation of NGFI-B, Nurr1, and NOR-1 may reflect different functions in neurons in glutamate excitotoxicity.

Differential glucocorticoid effects on stress-induced gene expression in the paraventricular nucleus of the hypothalamus and ACTH secretion in the rat

Although previous studies have examined the extent to which adrenocorticotropic hormone (ACTH) secretion depends on endogenous glucocorticoid levels, few have examined the parallel glucocorticoid dependency of gene expression within the corticotropin releasing hormone (CRH) neuron containing subregion of the hypothalamic paraventricular nucleus (PVN). This study examined resting and stress-induced expression of three immediate early genes (c-fos, zif268, and NGFI-B mRNAs) and two phenotypic restricted immediate early genes that code for ACTH secretagogues (CRH and arginine vasopressin [AVP] hnRNAs) in the PVN of adrenalectomized (ADX) rats given either 0.9% saline to drink for 5 days or saline with corticosterone (CORT; 25 μg/ml). CORT-containing saline was replaced with saline 18 h before testing to ensure clearance of CORT at the time of testing. Dependent measures were examined 0, 15, 30, 60, or 120 min after 30 min restraint. Compared to sham surgery, ADX produced a large upregulation of basal ACTH secretion but only a trend for an increase in basal PVN CRH and parvocellular (mp) PVN AVP hnRNA expression, and a marked augmentation of restraint-induced ACTH secretion and the expression of all five genes examined. CORT containing saline partially normalized basal and restraint-induced ACTH secretion and restraint-induced AVP hnRNA, c-fos mRNA, and zif268 mRNA in the PVN in ADX rats. In contrast, expression patterns of restraint-induced PVN CRH hnRNA and NGFI-B mRNA were not different between ADX rats with or without CORT replacement. Given that there was no circulating CORT present at the time of restraint challenge in either group of ADX rats, the differential impact of CORT replacement on restraint-induced PVN gene expression must reflect differential dependency of the expression of these genes in the PVN on the prior presence of CORT.

Reduction of dopamine-related transcription factors Nurr1 and NGFI-B in the prefrontal cortex in schizophrenia and bipolar disorders

Abnormal cortical and subcortical dopaminergic activities are among the most consistent neuropathological findings in schizophrenia. The molecular mechanisms remain unspecified. NGFI-B and Nurr1 are two closely related transcription factors involved in dopaminergic cell differentiation, maturation, and apoptosis. NGFI-B knockout mice show attenuated behavioral response to dopamine receptor agonists, whereas Nurr1 knockout disrupts midbrain dopaminergic neuron development. To further understand the role of Nurr1 and NGFI-B in schizophrenia and bipolar disorders, we measured Nurr1 and NGFI-B mRNA in the prefrontal cortex Brodmann's areas 9 (BA 9) and BA 46 by in situ hybridization, and the protein levels in BA 9 by Western blotting, of patients with schizophrenia, major depression, and bipolar disorders, and non-psychiatric control subjects ( n = 15 per group). NGFI-B mRNA ( P < 0.05) and protein ( P < 0.01) were significantly lower in patients with schizophrenia (BA 9), and NGFI-B mRNA was lower in bipolar disorder (BA 9 and BA 46) than in the controls. In the deep cortical layers of BA 46, Nurr1 mRNA was significantly ( P < 0.05) lower in patients with bipolar disorder and schizophrenia than in the controls. Nurr1 protein in BA 9 was significantly lower in major depression ( P < 0.05) and lower at a trend level in schizophrenia ( P = 0.056) than in the controls. These data show a deficient prefrontal NGFI-B and Nurr1 expression in schizophrenia and bipolar disorder. Further study may elucidate if and how these deficiencies could be associated with abnormal dopaminergic functions seen in both illnesses.

Diurnal effects of enriched environment on immediate early gene expression in the rat brain

Rodents housed in an enriched environment (EE) show increased neuronal plasticity with enhanced long-term potentiation and memory performance. We report an EE-induced increase in NGFI-A and Krox-20 mRNA expression exclusively during the dark period of the day. In addition, EE-housed rats showed considerable diurnal variation in NGFI-A, Krox-20, and NGFI-B mRNA expression which was absent in single-housed rats. Thus, EE-induced molecular changes are more evident during the dark phase when the rats have higher motor and exploratory activity. This is important to take into account in future studies of molecular mediators of experience-dependent neuronal plasticity.

NGFI‐B (Nurr77/Nr4a1) orphan nuclear receptor in rat pinealocytes: circadian expression involves an adrenergic‐cyclic AMP mechanism

NGFI-B (Nur77/Nr4a1) is a member of a nuclear steroid receptor subgroup that includes the related factors Nurr1 (Nr4a2) and NOR-1 (Nr4a3). These proteins do not have recognized ligands and in fact function independently as orphan receptors with transcriptional regulatory activity. In the present study, expression of the NGFI-B gene in the rat pineal gland was found to exhibit a robust circadian rhythm, with elevated levels of NGFI-B mRNA occurring at night. The rhythm of NGFI-B mRNA is translated into a circadian rhythm of NGFI-B protein, which accumulates in the nucleus of pinealocytes. In addition, there is a parallel marked nocturnal increase in pineal DNA binding activity to a NGFI-B response element (NBRE, AAAGGTCA). Pharmacological studies indicate that NGFI-B mRNA and protein levels are elevated via activation of adrenergic receptors. NGFI-B protein levels are also elevated by dibutyryl cyclic AMP, as in other systems. In the pineal gland, regulation of NGFI-B expression also involves the AP-1 protein Fra-2, based on studies with a transgenic Fra-2 knockdown rat, in which pineal NGFI-B expression increases. This set of observations extends the number of pineal genes that are known to be regulated by Fra-2, and also provides the first indication that a member of the NGFI-B group of nuclear receptors is involved in controlling gene expression in the pineal gland.

Nur-related factor 1 and nerve growth factor-induced clone B in human adrenal cortex and its disorders.

Nerve growth factor-induced clone B (NGFI-B; NR4A1) and Nur-related factor 1 (Nurr1; NR4A2) are members of NGFI-B family of orphan receptors. We recently demonstrated induction of CYP11B2 (aldosterone synthase) by Nurr1 and NGFI-B, suggesting possible important roles of these transcriptional factors in the regulation of adrenocortical steroidogenesis. Therefore, we immunolocalized Nurr1 and NGFI-B in various human adrenal specimens to study their biological significance. In nonpathological adrenal glands (n = 25), Nurr1 and NGFI-B immunoreactivities were detected at high levels in the fetal definitive zone or postnatal zona glomerulosa. NGFI-B immunoreactivity was increased according to development in the zona fasciculata, reaching a level similar to that in the zona glomerulosa in adult adrenal cortex. In adrenocortical neoplasms (n = 44), Nurr1 immunoreactivity was higher in aldosteronoma than in Cushing's adenoma or adrenocortical carcinoma. NGFI-B immunoreactivity was also higher in aldosteronoma than in adrenocortical carcinoma, but was not significantly different among the types of adenoma. Both Nurr1 and NGFI-B mRNA expressions were correlated with their immunoreactivities in adrenocortical neoplasms (n = 23), and mRNA expression of Nurr1 was significantly (P < 0.0001) associated with that of CYP11B2. These results suggest that the expression of Nurr1 and NGFI-B plays an important role in human adrenal cortex and its neoplasms, including possible regulation of steroidogenesis.

Effects of postischemic environment on transcription factor and serotonin receptor expression after permanent focal cortical ischemia in rats

Housing rats in an enriched environment improves functional outcome after ischemic stroke, this may reflect neuronal plasticity in brain regions outside the lesion. Which components of the enriched environment that are of greatest importance for recovery after brain ischemia is uncertain. We have previously found that enriched environment and social interaction alone both improve functional recovery after focal cerebral ischemia, compared with isolated housing with voluntary wheel-running. In this study, the aim was to separate components of the enriched environment and investigate the effects on some potential mediators of improved functional recovery; such as the inducible transcription factors nerve growth factor-induced gene A (NGFI-A) and NGFI-B, and the glucocorticoid and serotonin systems. After permanent middle cerebral artery occlusion, rats were divided into four groups: individually housed with no equipment (deprived group), individually housed with free access to a running wheel (running group), housed together in a large cage with no equipment (social group) or in a large cage furnished with exchangeable bars, chains and other objects (enriched group). mRNA expression of inducible transcription factors, serotonin and glucocorticoid receptors was determined with in situ hybridisation 1 month after cerebral ischemia. Rats housed in enriched or social environments showed significantly higher mRNA expression of NGFI-A and NGFI-B in cortical regions outside the lesion and in the CA1 (cornu ammonis region of the hippocampus), compared with isolated rats with or without a running wheel. NGFI-A and NGFI-B mRNA expression in cortex and in CA1 was significantly correlated to functional outcome. 5-Hydroxytryptamine receptor 1A (5-HT 1A) mRNA expression and binding, as well as 5-HT 2A receptor mRNA expression were decreased in the hippocampus (CA4 region) of the running wheel rats. Mineralocorticoid receptor gene expression was increased in the dentate gyrus amongst wheel-running rats. No group differences were found in plasma corticosterone levels or mRNA levels of glucocorticoid receptor, corticotropin-releasing hormone, 5-HT 2C or c-fos. In conclusion, we have found that social interaction is a major component of the enriched environment regarding the effects on NGFI-A and NGFI-B expression. These transcription factors may be important mediators of improved functional recovery after brain infarctions, induced by environmental enrichment.

Neonatal ventral hippocampus lesion leads to reductions in nerve growth factor inducible-B mRNA in the prefrontal cortex and increased amphetamine response in the nucleus accumbens and dorsal striatum

Converging evidence in schizophrenia suggests prefrontal cortical neuronal deficits that correlate with exaggerated subcortical dopamine (DA) functions: Excitotoxic lesion of the ventral hippocampus (VH) in neonatal rats is widely considered a putative animal model of schizophrenia as they lead to characteristic post-pubertal emergence of behavioral and cognitive abnormalities suggesting a developmental change in the neural circuits comprising the prefrontal cortex (PFC) and subcortical DA. Nerve growth factor inducible-B (NGFI-B, also known as Nur77), an orphan nuclear receptor and transcriptional regulator, is constitutively expressed in the target structures of DA pathways. It acts as an immediate early gene with rapid modulation of its mRNA expression by stress, DA and antipsychotic drugs. The present study assessed the effects of neonatal VH (nVH) lesion and amphetamine treatment on the expression of NGFI-B mRNA in pre- and post-pubertal rats. Sprague–Dawley rat pups received bilateral injection of ibotenic acid or phosphate buffered saline in VH at postnatal (PD) 7. At PD35 and PD56, groups of sham and lesioned animals were administered with d-amphetamine (1.5 mg/kg) or saline and killed 20 min later. In situ hybridization analyses showed that the basal level of NGFI-B mRNA in saline-treated lesioned rats was significantly reduced in the medial PFC (mPFC) and cingulate cortex (CC) only at post-pubertal (PD56) age. No significant difference in NGFI-B mRNA levels was seen in the dorsal striatum or nucleus accumbens (NAcc). Amphetamine treatment increased the expression of NGFI-B mRNA in the mPFC, CC, striatum and NAcc in both control and lesioned animals of both ages. Interestingly, however, striatal and NAcc regions of lesioned rats showed a significantly greater effect of amphetamine at PD56. The data suggest that nVH lesions lead to delayed changes in PFC gene expression along with functional DAergic hyperactivity in subcortical regions.

Reduced Adrenal Activation in a Rat Line Selected for High Alcohol Sensitivity

Background A rat line developed by selective breeding for high alcohol sensitivity has blunted corticosterone responses to alcohol and stress. In the present study, we determined possible differences in adrenal activation after alcohol and motor performance testing between the alcohol-sensitive alcohol-nontolerant and alcohol-insensitive alcohol-tolerant rats. Methods The animals received ethanol (2 g/kg, intraperitoneally), and 30 min later they were subjected to a motor function test (i.e., normal selection test used in the breeding of the lines); the control animals for both rat lines received no treatment and minimal handling. Blood corticosterone and ACTH levels at the single time point were determined by radioimmunoassay, and adrenal activation was determined by in situ hybridization of the immediate early gene c-fos, nor1, nurr1, and NGFI-B mRNA expression. Results The alcohol nontolerant rats had lower corticosterone but normal ACTH levels after ethanol and motor testing. Adrenal early gene expression of all of the genes studied was strongly induced by the treatment in both rat lines, but the inductions of c-fos, nor1, and nurr1 were significantly lower in the alcohol-sensitive animals. Acute treatment with a high dose of ACTH also induced less adrenal gene expression in the alcohol-sensitive animals. Conclusions The results suggest that the reduced adrenal activation is associated with high alcohol sensitivity in a genetic animal model, which is in agreement with the human findings of alcohol insensitivity during glucocorticoid treatment.

Comparisons of Behavioral and Neurochemical Characteristics between WKY, WKHA, and Wistar Rat Strains

WKHA rats constitute a recombinant inbred rat strain derived by phenotypic selection of the progeny of hybrid F2 crosses between SHR and WKY rats. WKHA are normotensive and show some features of hyperactivity and of hyper-reactivity to stress, but their utility as model of attention deficit/hyperactivity disorder (ADHD) has not yet been settled. To address these questions, we performed behavioral and neurochemical evaluations of WKHA, and compared them to both WKY and Wistar (WIS) rats. In locomotor activity tests, the respective scores for each strain were WKY<WKHA<WIS. The relative amplitudes of the inhibition of the startle response by preexposure of the animals to a low-level acoustic cue (prepulse inhibition (PPI) stimulus filtering test) were WKY< WKHA = WIS. In nontreated rats, prepulse inhibition was significantly lower in WKY than in WIS and WKHA rats at low prepulse intensity. Methylphenidate did not decrease locomotor activity in any of the strains, but rather increased locomotion, with the effect being of higher amplitude in WKY. Methylphenidate also impaired prepulse inhibition in a dose-dependent manner. In situ hybridization for NGFI-B, a transcription factor related to the dopaminergic system, revealed that methylphenidate increased the expression of NGFI-B mRNA in the accumbens (core/shell) and striatum only in WKY rats. We conclude that: (1) the behavioral differences between WKY and WKHA can be more readily explained by deficits present in WKY than by unusual characteristics of WKHA, (2) the WKHA does not appear to represent a useful model of ADHD, and (3) the use of WKY rats as the only control for behavioral studies is potentially misleading, because they show a combination of behavioral and neurochemical (dopamine-related) characteristics that make them very different from other common rat strains. Although these characteristics may make them interesting models in their own right, it also follows that the conclusions of any study based on comparisons of one particular strain to just WKY should be interpreted very cautiously.

Changes in rat frontal cortex gene expression following chronic cocaine

Alterations in gene expression caused by repeated cocaine administration have been implicated in the long-term behavioral aspects of cocaine abuse. The frontal cortex mediates reinforcement, sensory, associative, and executive functions and plays an important role in the mesocortical dopamine reinforcement system. Repeated cocaine administration causes changes in frontal cortex gene expression that may lead to changes in the behaviors subserved by this brain region. Rats treated non-contingently with a binge model of cocaine (45 mg/kg/day, i.p.) for 14 days were screened for changes in relative mRNA abundance in the frontal cortex by cDNA hybridization arrays. To confirm changes, immunoreactive protein was measured (via protein-specific immunoblots) in a second group of identically-treated animals. Protein levels of protein tyrosine kinase 2 (PYK2), activity-regulated cytoskeletal protein (ARC), as well as an antigen related to nerve growth factor I-B (NGFI-B-RA) were shown to be significantly induced after cocaine administration. Levels of NGFI-B mRNA were confirmed by real-time RT–PCR to be increased with cocaine administration. These observations are similar to previously reported cocaine-responsive changes in gene expression but novel to the frontal cortex. This study also validates the use of hybridization arrays for screening of neuronal gene expression changes and the utility of relative protein quantification as a post-hoc confirmation tool.

Human aldolase C gene expression is regulated by adenosine 3′,5′-cyclic monophosphate (cAMP) in PC12 cells

We have examined the effects of an adenosine 3′,5′-cyclic monophosphate (cAMP) analog on human aldolase C gene expression in the rat pheochromocytoma cell line PC12. Incubation for 4 h with 500 μM 8-Br-cAMP increased aldolase C mRNA expression 2.5-fold and the expression was still above basal level 24 h later. Using transient transfection experiments we demonstrate that the distal element D in the promoter region of the human aldolase C gene, which binds a transcriptional activator (NGFI-B), is involved in this regulation. NGFI-B mRNA and protein expression were promptly (15 min) increased after 8-Br-cAMP treatment and precedes aldolase C mRNA increase (30 min). After 4 h of 8-Br-cAMP treatment, the binding of NGFI-B protein to the distal element D in the distal promoter region was increased twofold and this correlates with the increased expression of the clone that contains distal element D. These results indicate that the distal element D in the promoter region of the human aldolase C gene is the target of a cAMP-dependent regulation pathway.

Acute methamphetamine administration upregulates NGFI-B mRNA expression in the striatum: co-localization with c-Fos immunoreactivity.

In this study, the effects of acute methamphetamine administration on expression of the nuclear transcription factor NGFI-B mRNA and its co-localization with c-Fos immunoreactivity in the striatum were evaluated in animals receiving a single dose of methamphetamine (4 mg/kg) given at 2 or 6 h prior to perfusion. All animals received a daily saline injection for 6 days prior to methamphetamine treatment. We have found that, similar to c-fos activation, NGFI-B mRNA levels were significantly increased 2 h after methamphetamine treatment and returned to basal levels 6 h later. Induction of NGFI-B mRNA levels by methamphetamine was highest in central striatum as compared to the dorsomedial distribution pattern observed in control animals. After acute methamphetamine treatment, the distribution pattern of NGFI-B mRNA upregulation was very similar to that of methamphetamine induced c-Fos immunoreactivity. However, co-localization studies with c-Fos immunoreactivity showed that not all NGFI-B-positive cells contained c-Fos after methamphetamine treatment. Forty-five percent of all NGFI-B mRNA expressing neurons contained c-Fos immunoreactivity in the dorsomedial striatum as compared to 60% in central and 35% in ventrolateral striatum. This study provides a detailed description of the differential spatial and temporal modulation of NGFI-B and c-Fos expression in the striatum by acute methamphetamine treatment over time.

Induction of nuclear orphan receptor NGFI-B gene and apoptosis in rat vascular smooth muscle cells treated with pyrrolidinedithiocarbamate.

NGFI-B is one of the orphan nuclear receptors, and its gene is implicated in the apoptosis of T cells. The aim of this study was to investigate the expression and the role of NGFI-B in vascular smooth muscle cells (VSMCs). Pyrrolidinedithiocarbamate (PDTC) is a modulator of an oxidative state and is reported to induce apoptosis only when the density of VSMCs is low. Under low VSMC density (10 000 cells/cm(2)), addition of PDTC (0.1 to 10 micromol/L) caused apoptosis of VSMCs, which was confirmed by Hoechst 33258 staining under fluorescence microscopy. At low VSMC density, expression of NGFI-B mRNA was induced 1 hour after the addition of PDTC, peaking at 6 hours, and persisted for up to 12 hours. The protein level of NGFI-B was increased 4 hours after PDTC addition and persisted for up to 12 hours. Under low VSMC density, PDTC-induced expression of NGFI-B mRNA was correlated with the magnitude of apoptosis, which was quantified by enzyme immunoassay for histone-associated DNA fragments. In contrast, when the density of VSMCs was high (50 000 cells/cm(2)), PDTC did not induce apoptosis, and the expression of NGFI-B was only transient. This transient expression pattern was also seen when VSMCs were treated with phorbol ester, calcium ionophore, hydrogen peroxide, or angiotensin II, even at low cell density. We next investigated whether the NGFI-B gene may act as a transcription factor under treatment with PDTC by measuring the promoter activity of luciferase reporter plasmids that contained typical NGFI-B-responsive elements. The PDTC-induced transcriptional activity of NGFI-B was 2-fold higher at low cell density than at high cell density. These data demonstrate that NGFI-B can be induced in VSMCs and suggest that NGFI-B may play a role in PDTC-induced VSMC apoptosis.

Impact of antipsychotic drug administration on the expression of nuclear receptors in the neocortex and striatum of the rat brain

We have recently shown that the expression of the nerve growth factor-inducible gene B (NGFI-B, or Nur77), a transcription factor belonging to the large ligand-activated nuclear receptor family, is modulated by antipsychotic drugs in the rat forebrain. In the present work, we have investigated the impact of antipsychotic drugs on a series of transcription factors also belonging to the nuclear receptor family. The receptors investigated include retinoid X receptor (RXR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), RAR-related orphan receptor (RZR) and Rev- erb receptor isoforms in addition to the NGFI-B transcript. We have used in situ hybridization to monitor their mRNA levels after acute and chronic antipsychotic drug administration. RZRβ and NGFI-B mRNA levels are down-regulated after chronic haloperidol or clozapine treatment in the primary somatosensory cortex. The TRβ1 isoform mainly expressed in the cingulate cortex is modulated only after chronic clozapine treatment, whereas TRα isoform mRNAs are modulated by both antipsychotics in the cingulate cortex and nucleus accumbens shell; two brain areas associated with limbic functions. The RXRγ1 isoform, mostly expressed in the dorsolateral portion of the striatum is modestly affected by antipsychotics. Modulation of the expression of transcription factors belonging to the ligand-activated nuclear receptor family by antipsychotics represents an additional molecular event in the mechanism of action of these drugs. We suggest that modification of the pattern of transcription factor expression may play a role in long-term cellular responses to these drugs.

Cellular mRNA expression of the transcription factor NGFI-B suggests a gene regulatory role in striatal opiate-peptide neurons

Previous studies have shown that NGFI-B mRNA is highly expressed in the adult striatum. In the present study we analyzed the anatomical distribution of NGFI-B mRNA within this brain region as well as the degree of co-existence of NGFI-B with different striatal markers in the adult brain. NGFI-B mRNA levels were found to be significantly higher within the dorsomedial portion of the striatum as compared to the ventrolateral striatum. This distribution pattern was maintained throughout the rostro–caudal axis of the striatum. Double in situ hybridization studies showed that striatal NGFI-B mRNA colocalized with a subset of preproenkephalin and prodynorphin positive spiny neurons within the dorsomedial striatum; 22–28% of all opiate-peptide positive cells co-expressed NGFI-B mRNA. NGFI-B did not colocalize with striatal aspiny interneurons expressing choline acetyl transferase mRNA or those containing the calcium-binding protein parvalbumin. The pattern of NGFI-B mRNA expression within different striatal spiny projecting neurons suggests that this transcription factor may have a direct effect on the function of different striatal efferent pathways.

Selective effects on NGFI-A, MR, GR and NGFI-B hippocampal mRNA expression after chronic treatment with different subclasses of antidepressants in the rat

There is a latency period of several weeks before the onset of clinical effect of antidepressant drugs. The detailed mechanisms underlying drug-induced adaptive neuronal changes are not known. To elucidate the involvement of changes in gene expression of candidate transcription factors, we treated rats for 21 days with buspirone, fluoxetine, 8-OH-DPAT and moclobemide. In situ hybridization was used to study mRNAs encoding NGFI-A, NGFI-B and the glucocorticoid receptors, MR and GR. NGFI-A mRNA expression increased profoundly in the hippocampal formation and the cerebral cortex after all drug treatments, especially after moclobemide treatment (77-122% increase); with the exception of buspirone. MR mRNA expression was induced in hippocampal CA1/CA2 subregions (27-37%) by all antidepressants, while moclobemide and 8-OH-DPAT significantly increased GR gene expression mainly in the CA1 region (31-44%). NGFI-B mRNA was significantly decreased in the hippocampal CA3 subfield (23%) and restrosplenial granular cortex (38%) by moclobemide treatment. There are selective effects of antidepressant drugs on specific transcription factors. These may be important for adaptive neuronal and neuroendocrine changes after antidepressant treatment including HPA axis negative feedback regulation.

Differential patterns of induction of NGFI-B, Nor1 and c-fos mRNAs in striatal subregions by haloperidol and clozapine

Disturbances of retinoid activated transcription mechanisms have recently been implicated as risk factors for schizophrenia. In this study we have compared the regulation of mRNAs for the nuclear orphan receptor NGFI-B, which forms a functional heterodimer with the retinoid x receptor and the related orphan nuclear receptor Nor1 with c-fos mRNA after acute and chronic treatments with haloperidol and clozapine. The antipsychotic drugs haloperidol and clozapine have different clinical profiles. Haloperidol is a typical neuroleptic giving extrapyramidal side effects (EPS), whereas the atypical compound clozapine does not. Acute haloperidol treatment increased NGFI-B, Nor1 and c-fos mRNAs in nucleus accumbens shell and core as well as medial and lateral caudate putamen. In contrast, clozapine lead to an increase of NGFI-B, Nor1 and c-fos only in the accumbens shell. No haloperidol or clozapine effect on these mRNAs was detected in cingulate, sensory or motor cortex. Chronic haloperidol lead to an increase of NGFI-B mRNA in the accumbens core. Acutely, it is possible that the increased levels of NGFI-B, Nor1 and c-fos mRNA levels in striatum and accumbens might indicate a neural activation which possibly can be used when screening for drugs that do not produce EPS. Also, the increased levels of NGFI-B, which is an important component in retinoid signaling, both after acute and chronic treatments of haloperidol suggests altered sensitivity to retinoids which could be an important component for the beneficial antipsychotic effect.