Assessment of foot deformities in individuals with cerebral palsy using weight-bearing CT.

OBJECTIVE: The aims of this study were to visualize and quantify relative bone positions in the feet of individuals with cerebral palsy (CP) with a foot deformity and compare bone positions with those of typically developed (TD) controls. MATERIALS AND METHODS: Weight-bearing CT images of 14 individuals with CP scheduled for tendon transfer and/or bony surgery and of 20 TD controls were acquired on a Planmed Verity WBCT scanner. Centroids of the navicular and calcaneus with respect to the talus were used to quantify foot deformities. All taluses were aligned and the size and dimensions of the individuals' talus were scaled to correct for differences in bone sizes. In order to visualize and quantify variations in relative bone positions, 95% CI ellipsoids and standard deviations in its principle X-, Y-, and Z-directions were determined. RESULTS: In individuals with CP (age 11-17), a large variation in centroid positions was observed compared to data of TD controls. Radiuses of the ellipsoids, representing the standard deviations of the 95% CI in the principle X-, Y-, and Z-directions, were larger in individuals with CP compared to TD controls for both the calcaneus (3.16 vs 1.86 mm, 4.26 vs 2.60 mm, 9.19 vs 3.60 mm) and navicular (4.63 vs 1.55 mm, 5.18 vs 2.10 mm, 16.07 vs 4.16 mm). CONCLUSION: By determining centroids of the calcaneus and navicular with respect to the talus on WBCT images, normal and abnormal relative bone positions can be visualized and quantified in individuals with CP with various foot deformities.

A molecular blueprint of gene expression in hippocampal subregions CA1, CA3, and DG is conserved in the brain of the common marmoset.

Recent studies in rodents have shown that there are significant differences in gene expression profiles between the hippocampal subregions CA1, CA3, and DG. These differences in molecular make-up within the hippocampus most likely underlie the differences in morphology, physiology, and vulnerability to insults that exist between the subregions of the hippocampus and are as such part of the basic molecular architecture of the hippocampus. The aim of this study was to investigate at large scale whether these subregional differences in gene expression are conserved in the hippocampus of a nonhuman primate, the common marmoset. This study is very timely, given the recent development of the first marmoset-specific DNA microarray, exclusively containing sequences targeting transcripts derived from the marmoset hippocampus. Hippocampal subregions CA1, CA3, and DG were isolated by laser microdissection and RNA was isolated, amplified, and hybridized to the marmoset-specific microarray (EUMAMA) containing more than 1,500 transcripts expressed in the adult marmoset hippocampus. Large differences in expression were observed in particular between the DG region and both pyramidal subregions. Moreover, the subregion-specific patterns of gene expression showed a remarkable conservation with the rodent brain both in terms of individual genes and degree of differential expression. To our knowledge, this is the first study investigating large scale hippocampal gene expression in a nonhuman primate. The obtained expression profiles not only provide novel data on the expression of more than 1,500 transcripts per hippocampal subregion but also are of potential interest to neuroscientists interested in the role of the different subregions in learning and memory in the nonhuman primate brain.

Rapid glucocorticoid effects on the expression of hippocampal neurotransmission-related genes.

We previously assessed corticosterone mediated gene expression in acute explant hippocampal slices and found over 200 responsive genes 1, 3 and 5 h after glucocorticoid receptor (GR) activation by a brief corticosterone pulse. Interestingly, 1 h after GR activation all genes were downregulated, many of which are involved in hippocampal neurotransmission and plasticity. The aim of the current experiment was 1) to measure the expression of several of these neurotransmission-related genes that were corticosterone-responsive 1 h after GR-activation in an in vivo setting, 2) to elucidate in which hippocampal subregion these expression changes take place and 3) to assess the specificity of regulation by activated GRs. For this purpose, rats were subcutaneously injected with vehicle, corticosterone or corticosterone pretreated with GR-antagonist RU38486. One hour after the corticosterone injections, mRNA expression levels of 5 selected genes were measured using in situ hybridization. The mineralocorticoid receptor (MR), MAO-A, casein kinase 2 and voltage dependent potassium mRNA's, but not dynein mRNA, were rapidly downregulated in vivo after corticosterone administration in hippocampal subregions. Furthermore, RU38486 pretreatment reversed in all cases these effects, illustrating the GR-specificity of transcriptional regulation by corticosterone. The results are important for understanding the role of GR in pleiotropic control of hippocampal neurotransmission and plasticity, which is characterized by recovery of function transiently raised by excitatory input.

Steroid receptor coregulator diversity: what can it mean for the stressed brain?

Glucocorticoid hormones modulate brain function and as such are crucial for responding and adjusting to physical and psychological stressors. Their effects are mediated via mineralo- and glucocorticoid receptors, which in large measure act as transcription factors to modulate transcription of target genes, in a receptor-, cell-, and state-specific manner. The nature and magnitude of these transcriptional effects depend on the presence and activity of downstream proteins, such as steroid receptor coactivators and corepressors (together: coregulators), many of which are expressed in the brain. We address the role of coregulators for mineralo- and glucocorticoid receptor-mediated modulation of gene transcription. We first address evidence from cell lines for the importance of coregulator stoichiometry for steroid signaling. The in vivo importance of coregulators-when possible specifically for glucocorticoid signaling in the brain-is discussed based on knockout mice, transient knockdown of steroid receptor coactivators, and distribution and regulation of coactivator expression in the brain. We conclude that for a better understanding of modulation of brain function by glucocorticoids, it is necessary to take into account the role of coregulators, and to assess their importance relative to changes in hormone levels and receptor expression.

Acute activation of hippocampal glucocorticoid receptors results in different waves of gene expression throughout time.

Several aspects of hippocampal cell function are influenced by adrenal-secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)-induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR-mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR-activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down-regulated genes 1 h after GR-activation to both up and down regulated genes 3 h after GR-activation. After 5 h, the response was almost back to baseline. Additionally, real-time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR-responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid-mediated effects on hippocampal cell function.

Correlations between hypothalamus-pituitary-adrenal axis parameters depend on age and learning capacity.

Glucocorticoid hormones are released after activation of the hypothalamus-pituitary-adrenal (HPA) axis and in the brain can modulate synaptic plasticity and memory formation. Clear individual differences in spatial learning and memory in the water maze allowed classification of groups of young (3 months) and aged (24 months) male Wistar rats as superior and inferior learners. We tested 1) whether measures of HPA activity are associated with cognitive functions and aging and 2) whether correlations of these measures depend on age and learning performance. Basal ACTH, but not corticosterone, was increased in aged rats, with the stress-induced ACTH response exaggerated in aged-inferior learners. Aged-superior learners had lower expression of glucocorticoid receptor and CRH mRNA in the parvocellular paraventricular nucleus of the hypothalamus compared with all other groups. Hippocampal mineralocorticoid receptor and glucocorticoid receptor mRNAs differed modestly between groups, but steroid receptor coactivator and heat-shock-protein 90 mRNAs were not different. Strikingly, correlations between HPA axis markers were dependent on grouping animals according to learning performance or age. CRH mRNA correlated with ACTH only in aged animals. Parvocellular arginine vasopressin mRNA was negatively correlated to basal corticosterone, except in aged-inferior learners. Corticosteroid receptor mRNA expression showed a number of correlations with other HPA axis regulators specifically in superior learners. In summary, the relationships between HPA axis markers differ for subgroups of animals. These distinct interdependencies may reflect adjusted set-points of the HPA axis, resulting in adaptation (or maladaptation) to the environment and, possibly, an age-independent determination of learning ability.

Steroid receptor coactivator-1 splice variants differentially affect corticosteroid receptor signaling.

The mechanisms of receptor- and cell-specific effects of the adrenal corticosteroid hormones via mineralo- (MRs) and glucocorticoid receptors (GRs) are still poorly understood. Because the expression levels of two splice variants of the steroid receptor coactivator-1 (SRC-1) 1a and 1e, can differ significantly in certain cell populations, we tested the hypothesis that their relative abundance could determine cell- and receptor-specific effects of corticosteroid receptor-mediated transcription. In transient transfections, we demonstrate three novel types of SRC-1a- and SRC-1e-specific effects for corticosteroid receptors. One is promoter dependence: SRC-1e much more potently coactivated transcription from several multiple response element-containing promoters. Mammalian 1-hydrid studies indicated that this likely does not involve promoter-specific coactivator recruitment. Endogenous phenylethanolamine-N-methyltransferase mRNA induction via GRs was also differentially affected by the splice variants. Another type is receptor specificity: responses mediated by the N-terminal part of the MR, but not the GR, were augmented by SRC-1e at synergizing response elements. SRC fragment SRC(988-1240) by the MR but not the GR N-terminal fragment in a 1-hybrid assay. The last type, for GRs, is ligand dependence. Due to effects on partial agonism of RU486-activated GRs, different ratios of SRC-1a and 1e can lead to large differences in the extent of antagonism of RU486 on GR-mediated transcription. Furthermore, we show that SRC-1e but not SRC-1a mRNA expression was regulated in the pituitary by corticosterone. We conclude that the cellular differences in SRC-1a to SRC-1e ratio demonstrated in vivo might be involved in cell-specific responses to corticosteroids in a promoter- and ligand-dependent way.

Differential expression and regional distribution of steroid receptor coactivators SRC-1 and SRC-2 in brain and pituitary.

Members of the p160 family of steroid receptor coactivator proteins mediate the stimulatory effects on gene transcription brought about by nuclear receptors, which comprise all steroid receptors. Using in situ hybridization we have examined the neuroanatomical distribution of the messenger RNAs (mRNAs) for two functionally distinct splice variants of Steroid Receptor Coactivator 1 (SRC-1/NCoA-1) and of Steroid Receptor Coactivator 2 (SRC-2/NCoA-2/GRIP-1/TIF-2). Transcripts encoding these coactivators show highly differential expression patterns. SRC-2 mRNA is expressed at very low levels in brain, but shows expression in the anterior pituitary. SRC-la and le mRNA are expressed in many brain areas, including hippocampus, amygdala, hypothalamus, basal ganglia, and isocortex. Striking differences between SRC-1a and le expression were observed in several brain nuclei. Relative levels of SRC-1a mRNA were much higher in anterior pituitary, and the arcuate, paraventricular and ventromedial nucleus of the hypothalamus, the locus coeruleus and the trigeminal motor nucleus, all important targets of steroid hormones in the brain. SRC-le mRNA showed modest elevation of relative expression in the caudal nucleus accumbens (shell), basolateral amygdala, and some thalamic nuclei. The differential and uneven neuroanatomical distribution of these coactivators may underlie diversity and cell-specificity of steroid receptor mediated signals in the brain.

Chronic unpredictable stress causes attenuation of serotonin responses in cornu ammonis 1 pyramidal neurons.

In the present study, serotonin (5-HT) responses of hippocampal pyramidal cornu ammonis 1 (CA1) neurons were studied in rats subjected twice daily for 21 days to unpredictable stressors. In hippocampal tissue from thus stressed rats mRNA expression of the 5-HT(1A) receptor and mineralo- as well as glucocorticoid receptors were examined with in situ hybridization. On average, stressed rats displayed increased adrenal weight and attenuated body weight gain compared with controls, supporting that the animals had experienced increased corticosterone levels due to the stress exposure. One day after the last stressor, under conditions that corticosterone levels were low (predominant mineralocorticoid receptor activation), the 5-HT(1A) receptor mediated hyperpolarization of CA1 neurons in response to 10 microM 5-HT was significantly reduced compared with controls. Basal membrane properties of CA1 cells in stressed rats were comparable to those of controls. The 5-HT(1A) receptor mRNA expression was not changed after chronic stress exposure, in any of the hippocampal areas. A small but significant increase in mineralocorticoid receptor mRNA expression was observed after stress in the dentate gyrus, while glucocorticoid receptor expression was unchanged. The data indicate that unpredictable stress exposure for 3 weeks results in suppression of 5-HT(1A) receptor-mediated responses, possibly due to posttranslational modification of the receptor.

Measuring thigmotaxis in larval zebrafish.

One of the most commonly used behavioral endpoints measured in preclinical studies using rodent models is thigmotaxis (or "wall-hugging"). Thigmotaxis is a well-validated index of anxiety in animals and humans. While assays measuring thigmotaxis in adult zebrafish have been developed, a thigmotaxis assay has not yet been validated in larval zebrafish. Here we present a novel assay for measurement of thigmotaxis in zebrafish larvae that is triggered by a sudden change in illumination (i.e. sudden light-to-darkness transition) and performed in a standard 24-well plate. We show that zebrafish larvae as young as 5 days post fertilization respond to this challenge by engaging in thigmotaxis. Thigmotaxis was significantly attenuated by anxiolytic (diazepam) and significantly enhanced by anxiogenic (caffeine) drugs, thus representing the first validated thigmotaxis assay for larval zebrafish. We also show that exposure to sudden darkness per se may represent an anxiogenic situation for larval zebrafish since less contrasting light-to-darkness transitions (achieved by lowering darkness degrees) significantly decreased thigmotaxis levels in a manner similar to what was achieved with diazepam. These findings suggest that stimuli such as exposure to sudden darkness could be used proficiently to trigger the expression of anxiety-like behaviors in laboratory settings. In sum, this is a versatile protocol allowing testing of both anxiolytic and anxiogenic drugs in a cost-effective manner (only 10 min). This assay is also amenable to medium to high-throughput capacity while constituting a valuable tool for stress and central nervous system research as well as for preclinical drug screening and discovery.

Strain differences in the effects of adrenalectomy on the midbrain dopamine system: implication for behavioral sensitization to cocaine.

Adrenalectomy (ADX) abolishes behavioral sensitization to cocaine in DBA/2, but not C57BL/6 inbred mice. The present study tests the hypothesis that this ADX effect on behavioral sensitization in the DBA/2 strain involves changes in midbrain dopamine systems that do not occur in the C57BL/6 strain. For that purpose, we have measured tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA and D1- and D2-like receptor binding in C57BL/6 and DBA/2 mice that were i) unoperated, ii) ADX or sham (SHAM) operated, or iii) ADX or SHAM operated and subjected to a cocaine sensitization regimen (15.0 mg/kg cocaine on nine consecutive days, followed by a 7.5 mg/kg challenge after a 5-day withdrawal). ADX prevented behavioral sensitization to cocaine in the DBA/2, but not the C57BL/6 strain [de Jong IEM, Oitzl MS, de Kloet ER (2007) Adrenalectomy prevents behavioural sensitisation of mice to cocaine in a genotype-dependent manner. Behav Brain Res 177:329-339]. Mice were killed under basal conditions, in the latter case 24 h after the cocaine challenge. ADX did not affect the dopaminergic markers in drug naïve mice. By contrast, strain-dependent neuroadaptations were found in the midbrain dopamine system of mice subjected to the sensitization regimen. In the DBA/2 strain, sensitization-resistant ADX mice were characterized by reduced D2 binding in the nucleus accumbens core and rostral caudate putamen. Furthermore, ADX prevented the increase in TH and DAT mRNA expression in the substantia nigra, and the decrease in D2 binding in the dorsomedial subdivision of the caudal caudate putamen associated with sensitization in SHAM mice. In the C57BL/6 strain ADX only marginally affected dopaminergic adaptations. These data suggest that adrenal hormones modulate behavioral sensitization to cocaine in a genotype-dependent fashion possibly through adaptations in pre- and post-synaptic components of the midbrain dopamine system. During cocaine sensitization, the DBA/2, but not the C57BL/6 strain, was susceptible to ADX in the dopamine system with respect to presynaptic TH and DAT and terminal D2 receptor expression.

Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression.

Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.