Megan C. Holmes

A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.

Rett syndrome (RTT) is an inherited neurodevelopmental disorder of females that occurs once in 10,000–15,000 births. Affected females develop normally for 6–18 months, but then lose voluntary movements, including speech and hand skills. Most RTT patients are heterozygous for mutations in the X-linked gene MECP2 (refs. 3–12), encoding a protein that binds to methylated sites in genomic DNA and facilitates gene silencing. Previous work with Mecp2-null embryonic stem cells indicated that MeCP2 is essential for mouse embryogenesis. Here we generate mice lacking Mecp2 using Cre-loxP technology. Both Mecp2-null mice and mice in which Mecp2 was deleted in brain showed severe neurological symptoms at approximately six weeks of age. Compensation for absence of MeCP2 in other tissues by MeCP1 (refs. 19,20) was not apparent in genetic or biochemical tests. After several months, heterozygous female mice also showed behavioral symptoms. The overlapping delay before symptom onset in humans and mice, despite their profoundly different rates of development, raises the possibility that stability of brain function, not brain development per se, is compromised by the absence of MeCP2.

Prenatal glucocorticoid programming of brain corticosteroid receptors and corticotrophin-releasing hormone: possible implications for behaviour.

Glucocorticoids may underlie the association between low birth weight and adult disorders such as hypertension, type 2 diabetes and affective dysfunction. We investigated the behavioural and molecular consequences of two paradigms of prenatal dexamethasone administration in rats. Rats received dexamethasone (100 microg/kg per day) throughout pregnancy (DEX1-3), in the last third of pregnancy only (DEX3) or vehicle. Both dexamethasone treatments reduced birth weight, only DEX1-3 offspring had reduced body weight in adulthood. In adult offspring, both prenatal dexamethasone paradigms reduced exploratory behaviour in an open field. In contrast, only DEX3 reduced exploration in an elevated plus-maze and impaired behavioural responses and learning in a forced-swim test. This behavioural inhibition may reflect increased baseline corticotrophin-releasing hormone mRNA levels (30% higher) in the central nucleus of the amygdala in both dexamethasone-exposed groups. Adult DEX3 offspring also showed increased corticotrophin-releasing hormone mRNA with unaltered glucocorticoid receptor mRNA in the hypothalamic paraventricular nucleus and reduced hippocampal glucocorticoid- and mineralocorticoid receptor mRNA expression, suggesting reduced hippocampal sensitivity to glucocorticoid suppression of the stress axis. In contrast, DEX1-3 rats had no changes in hippocampal corticosteroid receptors, but showed increased mRNA levels for both receptors in the basolateral nucleus of the amygdala. From this data we suggest that prenatal glucocorticoid exposure programs behavioural inhibition perhaps via increased amygdalar corticotrophin-releasing hormone levels, while DEX3 also impairs coping and learning in aversive situations, possibly via altered hippocampal corticosteroid receptor levels. Overexposure to glucocorticoids, especially late in gestation, may explain the link between reduced early growth and adult affective dysfunction.

Inhibition of 11β‐hydroxysteroid dehydrogenase, the foeto‐placental barrier to maternal glucocorticoids, permanently programs amygdala GR mRNA expression and anxiety‐like behaviour in the offspring

Glucocorticoids may underlie the association between prenatal stress, low birth weight and adult stress‐associated disorders, e.g. hypertension and type 2 diabetes, increased hypothalamic–pituitary–adrenal (HPA) activity and affective dysfunction. Normally, 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) rapidly inactivates glucocorticoids in placenta and many foetal tissues, thus acting as a ‘barrier’ to maternal steroids. We investigated the effect of inhibiting foeto‐placental 11β‐HSD in rats, using carbenoxolone (CBX), on subsequent HPA activity and regulation and stress‐induced behaviour in adult offspring. Pregnant Wistar rats were injected with CBX (12.5u2003mg s.c.) or vehicle daily throughout pregnancy. CBX treatment reduced birth weight. Adult offspring of CBX‐treated dams had persistently reduced body weight, increased basal corticosterone (CORT) levels, increased corticotropin‐releasing hormone (CRH) and reduced glucocorticoid receptor (GR) mRNA in the hypothalamic paraventricular nucleus, though hippocampal GR and mineralocorticoid receptor (MR) mRNA expression were unaltered. In addition, these animals showed less grooming and rearing in an open field and reduced immobility in a forced swim test, and had increased GR mRNA expression in the basolateral (BLA), central (CEA) and medial (MEA) nuclei of the amygdala, with unaltered MR mRNA. These data suggest that disturbance of the foeto‐placental enzymatic barrier to maternal glucocorticoids reduces birth and body weight, and produces permanent alterations of the HPA axis and anxiety‐like behaviour in aversive situations. The behavioural and HPA effects may reflect GR gene programming in amygdala and hypothalamus, respectively. Foetal overexposure to endogenous glucocorticoids (prenatal stress or reduced activity of foeto‐placental 11β‐HSD) may represent a common link between the prenatal environment, foetal growth and adult neuroendocrine and affective disorders.

Environmental enrichment selectively increases 5-HT1A receptor mRNA expression and binding in the rat hippocampus

Environmental enrichment augments neuronal plasticity and cognitive function and possible mediators of these changes are of considerable interest. In this study, male rats were exposed to environmental enrichment or single housing for 30 days. Rats from the enriched group had significantly higher 5-HT1A receptor mRNA expression in the dorsal hippocampus (62%, 59% and 44% increase in the CA1, CA2 and CA3 subfields, respectively). This was associated with significantly higher [3H]8-OH-DPAT binding in the inferior part of CA1. No changes were seen for 5-HT2A or 5-HT2C receptor mRNAs. The neuronal plasticity detected after environmental change may be mediated, in part, through 5-HT1A receptors.

Modulation of serotonin and corticosteroid receptor gene expression in the rat hippocampus with circadian rhythm and stress

Glucocorticoids and serotonin (5-HT) modulate behaviour and hypothalamic-pituitary-adrenal (HPA) axis responses. The two systems interact prominently in the hippocampus, where these effects may occur. We have previously shown that hippocampal 5-HT2C receptor mRNA expression is increased by adrenalectomy or central 5-HT lesions. We have now determined expression of corticosteroid and 5-HT receptor subtype genes in the hippocampus across the diurnal cycle, when there are changes both in plasma corticosterone and hippocampal 5-HT levels, as well as the responses of these transcripts to acute and chronic stress, using in situ hybridisation histochemistry. Expression of both glucocorticoid (GR) and mineralocorticoid (MR) receptor mRNAs was significantly higher (131-153%) in the hippocampus at 08.00 h (corticosterone nadir) than at 20.00 h (corticosterone peak). 5-HT2C receptor mRNA expression also showed circadian variation (106-184% higher in CA1-CA3 in the morning). Hippocampal 5-HT1A and 5-HT2A receptor mRNA expression had no diurnal variation. Chronic (15 day) adjuvant arthritis stress, abolished the circadian corticosterone nadir, maintaining plasma corticosterone around diurnal peak values. Chronic arthritis stress suppressed hippocampal 5-HT2C receptor mRNA expression at 08.00 h to levels comparable to 20.00 h controls. By contrast to chronic stress, 6 h after acute laparotomy stress, plasma corticosterone was elevated above control (20.00 h) and 5-HT2C receptor mRNA expression was increased (CA2). Neither acute nor chronic stress altered MR, GR, 5-HT1A or 5-HT2A receptor mRNA expression in any hippocampal subfield. These results show that hippocampal expression of the 5-HT2C receptor gene, but not other subtypes, is sensitive to a variety of manipulations.(ABSTRACT TRUNCATED AT 250 WORDS)

11β-Hydroxysteroid dehydrogenase type 2 in the postnatal and adult rat brain

Abstract 11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. The recently discovered type 2 isozyme (11β-HSD-2) is a high affinity, NAD-dependent, exclusive 11β-dehydrogenase, which rapidly inactivates glucocorticoids. Thus the enzyme generates aldosterone-selectivity for intrinsically non-selective mineralocorticoid receptors in vivo as well as excluding glucocorticoids from glucocorticoid receptors, the latter being particularly important during development. Aldosterone exerts selective central effects upon salt appetite and blood pressure whilst glucocorticoids have potent effects upon postnatal neurogenesis and brain remodelling. We examined 11β-HSD-2 expression during postnatal ontogeny and in adult rat brain. High 11β-HSD-2 mRNA expression was found specifically in the postnatal thalamus and the external granule cell layer of the cerebellum. Expression peaked at the end of the first postnatal week and declined rapidly thereafter. Postnatal brain showed considerable activity of high affinity 11β-HSD-2 which paralleled expression of 11β-HSD-2 messenger ribonucleic acid (mRNA). Adult brain showed high 11β-HSD-2 mRNA expression limited to the subcommissural organ, with lower expression in the ventromedial nucleus of the hypothalamus, amygdala, locus coeruleus and nucleus tractus solitarius. These discrete areas are compatible with proposed selective central actions of aldosterone on blood pressure (subcommissural organ, nucleus tractus solitarius) and salt appetite (ventromedial nucleus, amygdala). In contrast, early postnatal 11β-HSD-2 coincides with glucocorticoid receptor rather than mineralocorticoid receptor expression, and areas of expression are among the regions where glucocorticoids have been demonstrated to have profound effects upon neuronal division, growth and maturation.

THE EFFECT OF ADRENALECTOMY ON 5-HYDROXYTRYPTAMINE AND CORTICOSTEROID RECEPTOR SUBTYPE MESSENGER-RNA EXPRESSION IN RAT HIPPOCAMPUS

Both central serotonergic dysfunction and glucocorticoid hypersecretion have been separately implicated in the aetiology of affective disorders. The hippocampus highly expresses receptors for 5-hydroxytryptamine and glucocorticoids, and adrenalectomy alters the responsivity of hippocampal neurons to 5-hydroxytryptamine. The hippocampus thus represents a prime locus for interactions between the two systems. In this study we examined the effects of glucocorticoid manipulations on neuronal expression of messenger RNA encoding corticosteroid receptor and 5-hydroxytryptamine receptor subtypes in the hippocampus and 5-hydroxytryptamine1A messenger RNA expression in the dorsal raphe, in the rat. Interestingly, there was no effect of adrenalectomy on 5-hydroxytryptamine1A or 5-hydroxytryptamine2A receptor messenger RNA expression in the dorsal or ventral hippocampus at any time point measured. Furthermore, no changes in 5-hydroxytryptamine1A receptor gene expression were seen in the dorsal raphe (encoding autoreceptors) after adrenalectomy. However, 5-hydroxytryptamine2C (5-hydroxytryptamine1C) receptor messenger RNA expression was increased specifically in posterior CA1 and CA3 neurons following adrenalectomy, an effect that was reversed by glucocorticoid replacement. Following adrenalectomy, glucocorticoid and mineralocorticoid receptor messenger RNA expression increased in the dentate gyrus, CA1 and CA3 subfields of the hippocampus. These increases were apparent 6 h after adrenalectomy, were maintained at two days, but 14 days after adrenalectomy hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression had returned to control levels. These effects of adrenalectomy were abolished by dexamethasone, but not aldosterone administration, suggesting mediation by autoregulatory glucocorticoid receptors. Our results show that adrenalectomy only transiently increases corticosteroid receptor gene expression in the hippocampus, and selectively increases hippocampal 5-hydroxytryptamine2C receptor messenger RNA expression. The resulting change in 5-hydroxytryptamine2C receptor-mediated responses may produce the alterations in hippocampal neuronal activity in response to 5-hydroxytryptamine observed after adrenalectomy.

Molecular mechanisms underlying dexamethasone inhibition of iNOS expression and activity in C6 glioma cells.

The synthetic glucocorticoid dexamethasone is routinely used to stabilize patients with malignant gliomas. One putative target for glucocorticoid action is inducible nitric oxide synthase (iNOS), which is produced by the tumor cells as well as the host immune cells. In this study, we characterize the stimulatory effects of lipopolysaccharide (LPS) and the cytokine, tumor necrosis factor‐α (TNFα), as well as the inhibitory effect of glucocorticoids, on iNOS gene expression and activity in C6 glioma cells cultured in vitro. LPS significantly increased iNOS mRNA expression, peaking at 6 h, while nitrite formation increased with time up to 72 h. Although TNFα alone induced neither iNOS mRNA expression nor nitrite formation, it significantly potentiated the effect of LPS on both. iNOS activity induced by LPS with or without TNFα was dose‐dependently inhibited by dexamethasone, reaching a maximum of approximately 83% inhibition. This was completely reversed by the addition of RU38486, an antagonist of glucocorticoid receptors (GR). Dexamethasone inhibited iNOS mRNA expression; however, the maximum inhibition obtained was only 10%. These results suggest that as for induction of iNOS activity in C6 cells in vitro, the stimulatory effect of LPS is mainly due to an action at the transcriptional level. TNFα does not have intrinsic inducing activity, but has potentiating effects at the transcriptional and possibly at the posttranscriptional levels in the presence of LPS. The inhibitory effect of dexamethasone is GR‐mediated and is mainly due to action at the posttranscriptional level. GLIA 42:68–76, 2003.

Glioma tumourgenicity is decreased by iNOS knockout: experimental studies using the C6 striatal implantation glioma model

Nitric oxide synthase (NOS) has recently been shown to be an important pathophysiological regulator in experimental implantation glioma since manipulation of NOS can significantly alter tumoural blood flow and inhibit tumour growth. In this study we investigated the role of iNOS (inducible NOS) in glioma tumourogenisis using the rodent C6 striatal implantation model. We produced genetically engineered C6 clones that do not express iNOS activity even after stimulation with a mixture of lipopolysaccaride (LPS) and tumour necrosis factor (TNF)-α. These iNOS knockout cells showed a similar growth rate to control cells in vivo at 5 days. We then performed an in vivo implantation glioma study using either the iNOS knockout clone or two genetically engineered control C6 clones. There was a significant reduction (p < 0.01) of tumour mass with the iNOS knockout clone 28 days after the implantation. Immunocytochemistry indicated infiltrates of CD3 positive T cells and macrophages in the controls and the iNOS knockout group. These studies indicate that iNOS expression by tumour parenchymal cells is a critical factor for tumour growth with this model. The mechanisms that cause failure of tumour growth need clarification prior to considering that specific iNOS inhibitors might be candidates for adjuvant treatment of malignant glioma.

Expression of interleukin-6 messenger RNA in a rat model of diffuse axonal injury

Having demonstrated a transcranial gradient of the cytokine interleukin-6 (IL-6) in patients with either traumatic brain injury or spontaneous subarachnoid haemorrhage we have employed in situ hybridisation for IL-6 messenger RNA (mRNA) to determine the site of this IL-6 production within the central nervous system (CNS). A rodent weight drop model of traumatic brain injury was used. IL-6 mRNA levels in brains were determined 6 h after injury. Sham animals had normal constitutive expression for IL6 mRNA. In traumatised animals an intense area of IL-6 mRNA labelling was found below the hippocampus. Cells strongly expressing IL-6 mRNA were also seen in the dentate gyrus. This inflammatory cytokine is clearly implicated in the response to CNS injury, but whether this response is neuroprotective or pathological is uncertain.