Oxytocin and vasopressin systems in genetic syndromes and neurodevelopmental disorders.

Oxytocin (OT) and arginine vasopressin (AVP) are two small, related neuropeptide hormones found in many mammalian species, including humans. Dysregulation of these neuropeptides have been associated with changes in behavior, especially social interactions. We review how the OT and AVP systems have been investigated in Autism Spectrum Disorder (ASD), Prader-Willi Syndrome (PWS), Williams Syndrome (WS) and Fragile X syndrome (FXS). All of these neurodevelopmental disorders (NDD) are marked by social deficits. While PWS, WS and FXS have identified genetic mutations, ASD stems from multiple genes with complex interactions. Animal models of NDD are invaluable for studying the role and relatedness of OT and AVP in the developing brain. We present data from a FXS mouse model affecting the fragile X mental retardation 1 (Fmr1) gene, resulting in decreased OT and AVP staining cells in some brain regions. Reviewing the research about OT and AVP in these NDD suggests that altered OT pathways may be downstream from different etiological factors and perturbations in development. This has implications for ongoing studies of the therapeutic application of OT in NDD. This article is part of a Special Issue entitled Oxytocin and Social Behav.

Parental broader autism subphenotypes in ASD affected families: relationship to gender, child's symptoms, SSRI treatment, and platelet serotonin.

Relationships between parental broader autism phenotype (BAP) scores, gender, selective serotonin reuptake inhibitor (SSRI) treatment, serotonin (5HT) levels, and the child's symptoms were investigated in a family study of autism spectrum disorder (ASD). The Broader Autism Phenotype Questionnaire (BAPQ) was used to measure the BAP of 275 parents. Fathers not taking SSRIs (F-SSRI; n = 115) scored significantly higher on BAP Total and Aloof subscales compared to mothers not receiving treatment (M-SSRI; n = 136.) However, mothers taking SSRIs (M + SSRI; n = 19) scored higher than those not taking medication on BAP Total and Rigid subscales, and they were more likely to be BAPQ Total, Aloof, and Rigid positive. Significant correlations were noted between proband autism symptoms and parental BAPQ scores such that Total, Aloof, and Rigid subscale scores of F-SSRI correlated with proband restricted repetitive behavior (RRB) measures on the ADOS, CRI, and RBS-R. However, only the Aloof subscale score of M + SSRI correlated with proband RRB on the ADOS. The correlation between the BAPQ scores of mothers taking SSRIs and child scores, as well as the increase in BAPQ scores of this group of mothers, requires careful interpretation and further study because correlations would not withstand multiple corrections. As expected by previous research, significant parent-child correlations were observed for 5HT levels. However, 5HT levels were not correlated with behavioral measures. Study results suggest that the expression of the BAP varies not only across parental gender, but also across individuals using psychotropic medication and those who do not.

Rare inherited A2BP1 deletion in a proband with autism and developmental hemiparesis.

Ataxin 2 binding protein 1 (A2BP1 aka FOX1, RBFOX1) is an RNA binding protein responsible for regulation of pre-mRNA splicing events in a number of critical developmental genes expressed in muscle, heart and neuronal cells [Shibata et al. (2000); Mamm Genome 12:595-601; Jin et al. (2003); EMBO J 22:905-912; Underwood et al. (2005); Mol Cell Biol 25:10005-10016]. Rare copy number abnormalities of A2BP1 have been previously associated with cognitive impairment, attention deficit disorder and autism [Martin et al. (2007); Am J Med Gen Part B 144B:869-876; Elia et al. (2010); Mol Psychiatry 15:637-646.]. Using a 1M Illumina SNP microarray, we identified a 1.3 kb deletion in A2BP1, which was subsequently validated by quantitative PCR. Here we present an in depth case study of an individual with autism and mild developmental hemiparesis in whom the deletion was detected. This study provides further support for the possible role of rare copy number variants in A2BP1 in the development of autism and associated motor asymmetries.

Sonohysterography for evaluation of the endometrium in women treated with tamoxifen.

OBJECTIVE: This study was performed to evaluate sonohysterography for the diagnosis of endometrial abnormalities in women treated with tamoxifen. MATERIALS AND METHODS: Fifty sonohysterograms were obtained in 48 consecutive tamoxifen-treated women. All women were postmenopausal and had been undergoing tamoxifen therapy for a mean of 2.6 years. Forty-six sonohysterograms (92%) were completed and four were unsuccessful. Sonohysterogram findings were correlated with prior endometrial biopsy results for 23 sonohysterograms (46%) that were preceded by endometrial biopsy. Sonohysterogram findings were also compared with histopathology results, available for 38 sonohysterograms (76%) that were followed by hysteroscopy with dilatation and curettage. RESULTS: Sonohysterography revealed 31 endometrial polyps (62%), six thickened endometria (12%), five normal endometria (10%), and four subendometrial cysts (8%). Surgery was avoided when seven sonohysterograms (14%) revealed normal endometria or subendometrial cysts. In the group with histopathologic correlation, 23 of 28 polyps were confirmed and two of five thickened endometria were shown to represent endometrial hyperplasia. Twelve (63%) of 19 sonohysterograms with prior normal endometrial biopsy findings had abnormalities on sonohysterography, including 10 polyps and two thickened endometria. CONCLUSION: Sonohysterography aids the diagnosis of endometrial abnormalities in tamoxifen-treated women even if prior endometrial biopsies have negative findings. In 14% of cases, visualization of a normal endometrium on sonohysterography obviated surgery.

Differences in pituitary cell number but not cell type between genetically lean and fat Coopworth sheep.

Coopworth sheep selected for low backfat (lean genotype) have been shown to have heavier pituitary glands than those selected for high backfat (fat genotype). This paper investigated whether this difference was due to an increase in pituitary cell number or cell size and whether the relative proportions of different pituitary cell types differed between the genotypes. In three separate trials, ram lambs aged 6 to 8 months were slaughtered and the pituitary glands were processed for stereological or immunocytochemical studies. The pituitary glands of lean genotype sheep were between 30 and 60% heavier than those of the fat sheep. Lean sheep had a significantly (P<0.05) larger cross-sectional area of the pituitary fossa (96.6 vs. 81.7 mm(2)) than fat genotype sheep. The pituitaries from lean sheep contained significantly more total cells than fat sheep (Trial 1: 290 vs. 183 million cells, P<0.01; Trial 2: 353 vs. 239 million cells, P <0.05). The volume of individual cells did not differ between the genotypes. Trial 3 showed that there was no difference between lean and fat sheep in the percentage of cells staining positive for the five pituitary hormones studied. It is concluded that the larger pituitary glands of lean compared to fat genotype sheep are a result of a nonspecific increase in the size of the whole gland through increased cell numbers, with no change in cell size or the relative proportion of different cell types.

Plasma glucose and insulin levels in genetically lean and fat sheep.

This study investigated whether genetically lean and fat sheep displayed differences in insulin and glucose statuses. Lean genotype sheep had significantly (P < 0.05) greater basal glucose concentrations than fat genotype sheep (4.78 versus 4.52, SED = 0. 104 mmol/l), although basal plasma insulin was not significantly different (mean 304, SEM = 37.3 pmol/l) between the genotypes. During glucose tolerance tests (GTT), carried out at 4 levels of injection: 0, 0.28, 1.39 or 2.78 mmol glucose/kg liveweight, the area under the plasma insulin curve was significantly (P < 0.05) greater for fat than lean genotype sheep, although there were no differences in any glucose parameters. There were no significant differences between genotypes in insulin or glucose concentrations during or following glucose infusion (GINF) experiments at 0, 0.09, 0.46 or 0.93 mmol glucose/kg live-weight/h over 3 hours. Elevated plasma insulin concentrations after a glucose tolerance test are concluded to be associated with increased fatness in this genetically selected line of sheep. However, the differences in insulin and glucose levels between the lean and fat genotype sheep are minor, relative to the differences in carcass composition.

Expression of obese mRNA in genetically lean and fat selection lines of sheep.

Genetically separate lines of Coopworth sheep have been bred by selecting for (fat genotype) or against (lean genotype) backfat depth. Typically, the total fat content, adjusted for carcass weight, is 21.2 and 29.3% for the lean and fat lines, respectively. As a homologue of the obese gene, which shows altered expression in several forms of obesity, is also expressed in sheep, it was decided to determine whether the obese gene was differentially expressed in each line of sheep. The relative level of expression of obese mRNA was approximately twofold higher in the fat line compared with the lean line in back, omental and perirenal fat depots of ram lambs fed ad libitum or fasted for 48 h. This elevation in the fat line is most likely a secondary consequence of obesity rather than a cause. Fasting for 48 h decreased obese mRNA levels by 8.9-, 8.5-, and 4.2-fold in back, omental and perirenal fat, respectively, in the lean line, and by 8.3-, 5.7-, and 3.5-fold in back, omental and perirenal fat, respectively, in the fat line. The lean and fat lines of sheep, therefore, responded in a similar way to fasting.

Detection of growth factors and proto-oncogene mRNA in the growing tip of red deer (Cervus elaphus) antler using reverse-transcriptase polymerase chain reaction (RT-PCR).

Deer antler is a unique mammalian organ that has an annual cycle of regeneration. The antler grows very rapidly from the tip at up to 1 cm/day in red deer for a 90- to 120-day period. It is hypothesised that locally produced growth factors are required to control and stimulate this growth. The tip of the growing antler from animals whose antlers had been growing for 30, 60, or 90 days was dissected into four zones: epidermis/dermis, reserve mesenchyme, precartilaginous, and cartilaginous. Total RNA was extracted, and the presence of various growth factors and proto-oncogenes was detected using RT-PCR, IGF-I, IGF-II, TGF beta 1, TGF beta 2, c-fos, c-myc, and beta-actin were all present as single bands of the expected molecular weight in the four zones of the antler at each stage of growth. There were higher levels of IGF-I, TGF beta 2, and c-myc relative to beta-actin in the epidermis/dermis layer than in the other three zones. There were no differences in the expression of any of the genes between the three stages of growth. The presence of TGF beta 3 cannot be confirmed since multiple bands were seen in all antler tissues. A single band of the expected size for TGF alpha was seen only in the epidermal/dermal layer of the antler, with multiple bands of different molecular weight being detected in the other zones of the antler. This work has demonstrated the presence of multiple growth factors in the growing deer antler and supports the hypothesis that paracrine/autocrine stimulation is important for regulating antler growth.

Expression of neurotrophin-3 in the growing velvet antler of the red deer Cervus elaphus.

Antlers are organs of bone which regenerate each year from the heads of male deer. In addition to bone, support tissues such as nerves also regenerate. Nerves must grow at up to 1 cm/day. The control of this rapid growth of nerves is unknown. We examined the relative expression of neurotrophin-3 (NT-3) mRNA in the different tissues of the growing antler tip and along the epidermal/dermal layer of the antler shaft of the red deer Cervus elaphus, using semi-quantitative reverse transcription-polymerase chain reaction. Expression in the tip was found to be highest in the epidermal/dermal layer and lowest in the cartilaginous layer in all developmental stages examined. These data correlate well with the density and pattern of innervation of these tissues. Along the epidermal/dermal layer of the antler shaft, expression was highest in the segments subjacent to the tip and lowest near the base, arguing for differences in the temporal expression of NT-3 in these segments. The expression of NT-3 in cells isolated from the different layers of 60-day antlers did not mirror that observed when whole tissues were used and may suggest regional specificity of NT-3 expression within antler tissues.

The insulin status of sheep with genetic differences in glucose clearance.

Lines of sheep have been selected for Slow or Fast glucose clearance after a glucose tolerance test. The aim of this work was to establish what characteristics of the insulin status were altered by the breeding program. Six animals from each line with consistently Slow (T-half > 70 min) or Fast (T-half < 60 min) decreases in plasma glucose concentration were studied in three different experiments. After the injection of [125I]insulin, blood was sampled for 300 min. The change in radioactivity with time was used in a three-compartment series model to estimate theoretical insulin pool sizes and flow rates between pools. All three pools were significantly (P < 0.05) larger in the Slow (61, 115, and 191 mU) than in the Fast glucose clearance animals (45, 82, and 112 mU). Flow rates between the pools were not significantly different. A euglycemic clamp experiment was performed at two insulin infusion rates, each for 4 hr. A significantly higher glucose infusion rate was required to maintain blood glucose at basal levels in the Slow (3 and 9 mg of glucose/kg liveweight [lwt]0.75 per min) than in the Fast glucose clearance animals (1 and 5 mg/kg lwt0.75 per min). The increase in glucose infusion rate when the insulin infusion rate was increased from 0.63 to 3.46 mU/kg lwt0.75 per min (insulin sensitivity index) was significantly greater in Slow than in Fast glucose clearance animals (0.68 vs. 0.35 mU of insulin/kg lwt0.75 per min). There was no difference between the lines in insulin binding to membranes isolated from muscle or adipose tissue. It is concluded that selection for Slow or Fast glucose clearance has altered several aspects of insulin status, but further work is required to identify the primary difference between the lines.

Growth hormone (GH) regulation of a rat serine protease inhibitor fusion gene in cells transfected with GH receptor cDNA.

The mechanism by which GH transmits a signal to the nucleus via its membrane-bound receptor is unknown. To study this process, Buffalo rat liver (BRL), rat hepatoma (FAO), human hepatoma (HepG2) and Chinese hamster ovary (CHO) cell lines were transfected with GH receptor cDNA, and stable clones expressing GH receptor mRNA and protein were selected. From previous in vivo studies it is known that GH regulates the expression of the rat hepatic serine protease inhibitor (SPI) 2.1 gene at the transcriptional level. However, in all the cell lines tested, SPI gene expression was less than 0.2% of that measured in rat liver, and GH did not affect the expression of the endogenous SPI gene in GH receptor-expressing cells. A 45 bp GH-responsive element (GHRE) has previously been defined in the SPI 2.1 gene. A construct containing six repeats of this GHRE was assembled with the thymidine kinase promoter and a chloramphenicol acetyl transferase (CAT) reporter gene. Transient transfection of this reporter gene resulted in GH stimulation of CAT activity in all GH receptor-transfected cell lines. A 33-fold induction was measured in the GH receptor-expressing BRL cells. Induction of CAT activity was observed after 8 h of GH treatment in the BRL-GHR638 cell line. Stable BRL cell lines expressing GH receptors with carboxy-terminal truncations (GHR380 and GHR454) did not show increased CAT activity on GH stimulation. This suggests that more than half of the intracellular domain of the GH receptor is required to activate transcription of the SPI 2.1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons.

In the basal ganglia, centrally active suicide transport agents produce apparently selective lesions of the striatopallidal and striatonigral pathways based on receptor binding and neuropeptide mRNA studies. In the present study we sought to determine the selectivity of suicide transport lesions for specific subsets of striatal neurons. Using immunohistochemical methods, the neostriata of adult rats were examined 10 days after an injection of volkensin into the substantia nigra or an injection of OX7-saporin into the globus pallidus. Ricin, a suicide transport agent active in the peripheral but not the central nervous system, was injected into each target as a control. Adjacent sections were processed for (1) Nissl stain to assess neuronal density, both overall and for large interneurons, (2) NADPH-diaphorase (NADPH-d) histochemistry, to mark medium-sized aspiny interneurons, (3) enkephalin immunocytochemistry, to label striatopallidal neurons, or (4) substance P immunocytochemistry, to label striatonigral neurons. Ricin injections produced no change in the densities of these subsets of striatal cells. In animals receiving volkensin or OX7-saporin injections, analyses of Nissl material revealed that the striata ipsilateral to the toxin injections appeared normal and did not exhibit shrinkage or gliosis; however, a quantitation analysis revealed a moderate decrease in cell density (12-16% loss, P < 0.01). The densities of both large and NADPH-d-containing striatal interneurons were unchanged after lesions in either target. Following nigral injections with volkensin, the density of striatal substance P-labeled cells decreased (26% loss, P < 0.01), while the density of enkephalin-labeled cells did not decrease significantly (11% decrease, P > 0.1). After pallidal injections with OX7-saporin, the density of striatal enkephalin-labeled cells decreased (20% loss, P < 0.01), while that of substance P-labeled cells remained unchanged. These data show that nigral volkensin and pallidal OX7-saporin injections differentially lesion striatonigral and striatopallidal projection neurons and spare striatal interneurons. This study provides further evidence for the selectivity, specificity, and utility of suicide transport agents to study brain structure and function.

A novel in vitro model for studying signal transduction and gene regulation via the growth hormone receptor.

Buffalo rat liver cells were stably transfected with an expression vector containing rat GH (rGH) receptor cDNA. Transfected cells expressed rGH receptor mRNA and specifically bound GH with high affinity. When transfected cells were stimulated with GH, levels of lipoprotein lipase (LPL) mRNA were increased in a time- and dose-dependent fashion, while glyceraldehyde-3-phosphate-dehydrogenase mRNA levels were unaffected. No GH binding or LPL mRNA could be detected in untransfected cells. Treatment of transfected cells with actinomycin D inhibited the GH-stimulated increase in LPL mRNA, indicating that GH acts at a transcriptional level. When protein synthesis was inhibited using cycloheximide, basal levels of LPL mRNA were increased, and there was no GH stimulation. This suggests that LPL gene expression is constantly repressed by a labile protein. Chloramphenicol acetyltransferase constructs containing the human LPL promoter could be regulated by GH. In conclusion, stimulation of the rGH receptor in stably transfected Buffalo rat liver cells results in specific induction of LPL gene expression. This provides a novel model to study the mechanism of GH action, particularly in relation to gene regulation.