Amplitude modulation detection as a function of modulation frequency and stimulus duration: comparisons between macaques and humans.

Previous observations show that humans outperform non-human primates on some temporally-based auditory discrimination tasks, suggesting there are species differences in the proficiency of auditory temporal processing among primates. To further resolve these differences we compared the abilities of rhesus macaques and humans to detect sine-amplitude modulation (AM) of a broad-band noise carrier as a function of both AM frequency (2.5 Hz-2 kHz) and signal duration (50-800 ms), under similar testing conditions. Using a go/no-go AM detection task, we found that macaques were less sensitive than humans at the lower frequencies and shorter durations tested but were as, or slightly more, sensitive at higher frequencies and longer durations. Humans had broader AM tuning functions, with lower frequency regions of peak sensitivity (10-60 Hz) than macaques (30-120 Hz). These results support the notion that there are species differences in temporal processing among primates, and underscore the importance of stimulus duration when making cross-species comparisons for temporally-based tasks.

Influence of age and 17beta-estradiol on kisspeptin, neurokinin B, and prodynorphin gene expression in the arcuate-median eminence of female rhesus macaques.

The neuropeptides kisspeptin, neurokinin B, and dynorphin A (collectively abbreviated as KNDy) are, respectively, encoded by KiSS-1, NKB, and PDYN and are coexpressed by neurons of the hypothalamic arcuate nucleus (ARC). Here, using quantitative real-time PCR, we examined age-related changes in the expression of genes encoding KNDy and associated receptors G protein-coupled receptor 54 (encoded by GPR54), neurokinin 3 receptor (encoded by NK3), and kappa-opioid receptor (encoded by KOR), in the female rhesus macaque ARC-median eminence (ARC-ME). Expression of KiSS-1 and NKB was highly elevated in old perimenopausal compared with young or middle-aged premenopausal animals. To test whether these age-related changes could be attributed to perimenopausal loss of sex steroids, we then examined KNDy, GPR54, NK3, and KOR expression changes in response to ovariectomy (OVX) and exposure to 17beta-estradiol (E(2)). Short-term (7 months) OVX (with or without 1 month of estrogen replacement) failed to modulate the expression of any of the KNDy-related genes. In contrast, long-term ( approximately 4 yr) OVX significantly increased KiSS-1 and NKB expression, and this was reversed by E(2) administration. Finally, we examined the expression of KNDy-related genes in young adult females during the early follicular, late follicular, or midluteal phases of their menstrual cycle but found no difference. Together, the results suggest that short-term alterations in circulating E(2) levels, such as those occurring during the menstrual cycle, may have little effect on the ARC-ME expression of KNDy and associated receptors. Nevertheless, they clearly demonstrate that loss of ovarian steroid negative feedback that occurs during perimenopause plays a major role in modulating the activity of KNDy circuits of the aging primate ARC-ME.

Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain.

BACKGROUND: Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix GeneChip rhesus Macaque Genome Array.Having shown that qRT-PCR and Affymetrix GeneChip data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages. RESULTS: Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip facilitated more targeted analysis than could be accomplished using the rhesus GeneChip. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed. CONCLUSIONS: Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.

Influence of 17beta-estradiol and progesterone on GABAergic gene expression in the arcuate nucleus, amygdala and hippocampus of the rhesus macaque.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and the responsiveness of neurons to GABA can be modulated by sex steroids. To better understand how ovarian steroids influence the GABAergic system in the primate brain, we evaluated the expression of genes encoding GABA receptor subunits, glutamic acid decarboxylase (GAD) and a GABA transporter in the brains of female rhesus macaques. Ovariectomized adults were subjected to a hormone replacement paradigm involving either 17beta-estradiol (E), or E plus progesterone (E+P). Untreated animals served as controls. Using GeneChip microarray analysis and real-time RT-PCR (qPCR), we examined gene expression differences within and between the amygdala (AMD), hippocampus (HPC) and arcuate nuclei of the medial basal hypothalamus (MBH). The results from PCR corresponded with results from representative GeneChip probesets, and showed similar effects of sex steroids on GABA receptor subunit gene expression in the AMD and HPC, and a more pronounced expression than in the MBH. Exposure to E+P attenuated GAD1, GAD2 and SLC32A1 gene expression in the AMD and HPC, but not in the MBH. GABA receptor subunit gene expression was generally higher in the AMD and HPC than in the MBH, with the exception of receptor subunits epsilon and gamma 2. Taken together, the data demonstrate differential regulation of GABA receptor subunits and GABAergic system components in the MBH compared to the AMD and HPC of rhesus macaques. Elevated epsilon and reduced delta subunit expression in the MBH supports the hypothesis that the hypothalamic GABAergic system is resistant to the modulatory effects of sex steroids.

Pubertal administration of DEHP delays puberty, suppresses testosterone production, and inhibits reproductive tract development in male Sprague-Dawley and Long-Evans rats.

Although is clear that exposure to high dosage levels of some phthalates delays the onset of puberty in the male rat, it has been hypothesized that low levels of di(2-ethylhexyl) phthalate (DEHP) accelerate puberty by enhancing testicular androgen synthesis. The current study was designed to determine if the dose response to DEHP was nonmonotonic, as hypothesized. Pubertal administration of DEHP delayed the onset of puberty and reduced androgen-dependent tissue weights in both Long-Evans (LE) and Sprague-Dawley (SD) male rats 300 and 900 mg DEHP/kg/day. These effects were generally of greater magnitude in LE than SD rats. By contrast, alterations in testis histopathology (300 and 900 mg/kg/day) were more severe in SD than in LE rats. Taken together, these results suggest that DEHP may be acting on the pubertal male rat testis via two modes of action; one via the Leydig cells and the other via the Sertoli cells. Treatment with DEHP generally reduced serum testosterone and increased serum luteinizing hormone (LH) levels, demonstrating that the reduction in testosterone was due to the effect of DEHP on the testis and not via an inhibition of LH from hypothalamic-pituitary axis. Testosterone production ex vivo (with and without human chorionic gonadotropin stimulation) was consistently reduced in males at the time of puberty and shortly thereafter. DEHP treatment did not accelerate the age at puberty or enhance testosterone levels at 10 or 100 mg/kg/day in either LE or SD rats, as some have hypothesized. Taken together, these results do not provide any evidence of a nonmonotonic dose response to DEHP during puberty.

Gene expression profiling in the rhesus macaque: methodology, annotation and data interpretation.

Gene microarray analyses represent potentially effective means for high-throughput gene expression profiling in non-human primates. In the companion article, we emphasize effective experimental design based on the in vivo physiology of the rhesus macaque, whereas this article emphasizes considerations for gene annotation and data interpretation using gene microarray platforms from Affymetrix. Initial annotation of the rhesus genome array was based on Affymetrix human GeneChips. However, annotation revisions improve the precision with which rhesus transcripts are identified. Annotation of the rhesus GeneChip is under continuous revision with large percentages of probesets under multiple annotation systems having undergone multiple reassignments between March 2007 and November 2008. It is also important to consider that quantitation and comparison of gene expression levels across multiple chips requires appropriate normalization. External corroboration of microarray results using PCR-based methodology also requires validation of appropriate internal reference genes for normalization of expression values. Many tools are now freely available to aid investigators with microarray normalization and selection of internal reference genes to be used for independent corroboration of microarray results.

Gene expression profiling in the rhesus macaque: experimental design considerations.

The development of species-specific gene microarrays has greatly facilitated gene expression profiling in nonhuman primates. However, to obtain accurate and physiologically meaningful data from these microarrays, one needs to consider several factors when designing the studies. This article focuses on effective experimental design while the companion article focuses on methodology and data analysis. Biological cycles have a major influence on gene expression, and at least 10% of the expressed genes are likely to show a 24-h expression pattern. Consequently, the time of day when RNA samples are collected can influence detection of significant changes in gene expression levels. Similarly, when photoperiodic species such as the rhesus macaque are housed outdoors, some of their genes show differential expression according to the time of year. In addition, the sex-steroid environment of humans and many nonhuman primates changes markedly across the menstrual cycle, and so phase of the cycle needs to be considered when studying gene expression in adult females.

Glutamate receptor subunit expression in the rhesus macaque locus coeruleus.

The locus coeruleus (LC) is a major noradrenergic brain nucleus that regulates states of arousal, optimizes task-oriented decision making, and may also play an important role in modulating the activity of the reproductive neuroendocrine axis. Rodent studies have shown that the LC is responsive to glutamate receptor agonists, and that it expresses various glutamate receptor subunits. However, glutamate receptor subunit expression has not been extensively examined in the primate LC. We previously demonstrated expression of the NR1 NMDA glutamate receptor subunit in the rhesus macaque LC and now extend this work by also examining the expression of non-NMDA (AMPA and kainate) ionotropic glutamate receptor subunits. Using in situ hybridization histochemistry and immunohistochemistry, we confirmed the presence of the obligatory NR1 subunit in the LC. In addition, we demonstrated expression of the AMPA glutamate receptor subunits GluR1, GluR2, and GluR3. More extensive receptor profiling, using rhesus monkey gene microarrays (Affymetrix GeneChip), further corroborated the histological findings and showed expression of mRNA encoding ionotropic glutamate receptor subunits NR2A, NR2D, GluR4, and GluR6, as well as the metabotropic glutamate receptor subunits mGluR1, mGluR3, mGluR4, mGluR5, and mGluR7. These data provide a foundation for future examination of how changes in glutamate receptor composition contribute to the control of primate physiology.

Late gestational exposure to the fungicide prochloraz delays the onset of parturition and causes reproductive malformations in male but not female rat offspring.

Prochloraz (PZ) is an imidazole fungicide that displays multiple endocrine activities. It inhibits steroid synthesis via P450 modulation and acts as an androgen receptor (AR) antagonist, but its effects on male sexual differentiation have not been described. The purpose of the current study was to expand in vitro observations and to determine whether PZ affected sexual differentiation. PZ effects on AR-mediated gene expression were tested using a cell line (MDA-kb2) containing endogenous AR and stably transfected with an MMTV-luc reporter. PZ concentrations greater than 1 microM caused a dose-dependent inhibition of dihydrotestosterone-induced gene expression. PZ also inhibited R1881 binding to the rat AR (IC50 approximately 60 microM). In vivo, pregnant rats received PZ by gavage from Gestational Day 14 to 18 at doses of 31.25, 62.5, 125, and 250 mg/kg of body weight per day. PZ delayed delivery in a dose-dependent manner and resulted in pup mortalities at the two highest doses. In male offspring, anogenital distance and body weight were slightly reduced at 3 days of age. Additionally, female-like areolas were observed at 13 days of age at frequencies of 31%, 43%, 41%, and 71% in the lowest-dose to highest-dose groups, respectively. Weights of androgen-dependent tissues showed dose-dependent reductions. Hypospadias and vaginal pouches were noted in all males treated with 250 mg/kg, whereas those defects were observed in 12.5% and 6.25%, respectively, of males treated with 125 mg/kg. Treatment did not affect age of preputial separation in animals without penile malformations. Despite severe malformations in males, no malformations were noted in females. Together, these results indicate that PZ alters sexual differentiation in an antiandrogenic manner.