Monkey Expression Research Articles

MO138: The Level of Microrna Expression in Urine of Cynomolgus Monkeys, Who Received Diet Rich in Common Salt

Abstract BACKGROUND AND AIMS High consumption of NaCl is traditionally considered the cardiovascular risk factor and the blood pressure (BP) growth factor. The considerable sodium content in the diet is associated with myocardium remodelling. Recently, attention has been also paid to studying the impact of high NaCl consumption at the condition of kidneys. However, no mechanisms of potential negative impact of high salt diets on kidneys were established. The microRNA may act as one of the important links in this process. Track changes in microRNA-21, 203 and 133 expression in urine of cynomolgus monkeys at high sodium chloride consumption with and without inclusion of isolated soy proteins into the diet. METHOD A total of 18 cynomolgus male monkeys with age 6–8 years were studied. The animals were divided into three groups (six in each group). The first (control) group received the standard diet. The second group received the diet rich in common salt (8 g of NaCl/kg), and the third, the high salt diet in combination with soy protein SUPRO 760 (200 g/kg; Protеin Tеchnology Intеrnational, USA). The observation period was 4 months. Determination of the relative level of microRNA expression in urine was performed by isolating total RNA with phenol reagent (Trirеagеnt-LS) and its subsequent extraction with chloroform. The reverse transcription reaction for ‘complementary’ DNA (cDNA) preparation was carried out under the Stеm Loop technology separately for the studied microRNA. The polymerase chain reaction (PCR) was carried out in the presence of ЕvaGrееn intercalating dye. The following primers were used in PCR: microRNA-21–5`-GCCCGCTAGCTTATCAGACTGATG-3′, microRNA-133–5′-GCCCGCAGCTGGTAAAATGGAAC-3′, microRNA-203–5′- GCCGGTGAAATGTTTAGGACC-3′ and U6–5′-GCGCGTCGTGAAGCGTTC-3′, and common reverse 5′-GTGCAGGGTCCGAGGT-3′. Reaction mixtures were prepared individually for each cDNA. The semi-qualitative evaluation of the microRNA expression level (in relative units, RU) under the 2–ΔΔCt protocol at the laboratory referent (0.09) was used in calculations. RESULTS No significant changes in microRNA-21 expression during the observation period (the baseline point and 4 months of observation) in the control group (P < .05). The significant growth approximately to equal extent of this parameter versus baseline values was observed in two other samples in 4 months after receiving the high salt diet. Changes in microRNA-133 or microRNA-203 in studied groups of monkeys were similar to those revealed for microRNA-21. Both high salt diets caused the significant growth in the relative level of expression of these microRNAs in urine versus basal values. At this, the median of the relative level of microRNA-133 expression in monkeys that consumed large amounts of salt for 4 months was significantly higher than in animals at the high salt diet, complemented with soybean isolate (P = .008; Mann–Whitney test). The similar situation has come about in relation to microRNA-203 (P = .0065; Mann–Whitney test). CONCLUSION The data received allow the conclusion that the potentially negative effect of high salt diets on the condition of kidneys can be mediated by epigenomic mechanisms, in particular, changes in the expression of the certain microRNAs. There is a good chance that activation of microRNA-21 expression (which at this term of study is not inhibited by administration of soy proteins) is responsible for renal impairment to the certain extent. It can also be assumed that soy proteins can interfere into this processes and change expression of some microRNAs.

Cannabinoid Type 1 Receptor is Undetectable in Rodent and Primate Cerebral Neural Stem Cells but Participates in Radial Neuronal Migration.

Cannabinoid type 1 receptor (CB1R) is expressed and participates in several aspects of cerebral cortex embryonic development as demonstrated with whole-transcriptome mRNA sequencing and other contemporary methods. However, the cellular location of CB1R, which helps to specify molecular mechanisms, remains to be documented. Using three-dimensional (3D) electron microscopic reconstruction, we examined CB1R immunolabeling in proliferating neural stem cells (NSCs) and migrating neurons in the embryonic mouse (Mus musculus) and rhesus macaque (Macaca mulatta) cerebral cortex. We found that the mitotic and postmitotic ventricular and subventricular zone (VZ and SVZ) cells are immunonegative in both species while radially migrating neurons in the intermediate zone (IZ) and cortical plate (CP) contain CB1R-positive intracellular vesicles. CB1R immunolabeling was more numerous and more extensive in monkeys compared to mice. In CB1R-knock out mice, projection neurons in the IZ show migration abnormalities such as an increased number of lateral processes. Thus, in radially migrating neurons CB1R provides a molecular substrate for the regulation of cell movement. Undetectable level of CB1R in VZ/SVZ cells indicates that previously suggested direct CB1R-transmitted regulation of cellular proliferation and fate determination demands rigorous re-examination. More abundant CB1R expression in monkey compared to mouse suggests that therapeutic or recreational cannabis use may be more distressing for immature primate neurons than inferred from experiments with rodents.

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

Cell versus Chemokine Therapy in a Nonhuman Primate Model of Chronic Intrinsic Urinary Sphincter Deficiency

Mixed efficacy results of autologous skeletal muscle precursor cell therapy in women with chronic intrinsic urinary sphincter deficiency have increased interest in the therapeutic value of alternative regenerative medicine approaches. The goal of this study was to compare the effects of the cell homing chemokine CXCL12 (C-X-C motif chemokine 12) and skeletal muscle precursor cells on chronic urinary sphincter regeneration in chronic intrinsic urinary sphincter deficiency. Five million autologous skeletal muscle precursor cells or 100 ng CXCL12 were injected in the urinary sphincter complex of adult female cynomolgus monkeys with chronic (6-month history) intrinsic urinary sphincter deficiency. These treatment groups of 3 monkeys per group were compared to a group of 3 with no intrinsic urinary sphincter deficiency and no injection, and a group of 3 with intrinsic urinary sphincter deficiency plus vehicle injection. Maximal urethral pressure was measured at rest, during stimulation of the urinary sphincter pudendal nerves at baseline and again 6 months after treatment. The monkeys were then necropsied. The urinary sphincters were collected for tissue analysis of muscle and collagen content, vascularization and motor endplates. CXCL12 but not skeletal muscle precursor cells increased resting maximal urethral pressure in nonhuman primates with chronic intrinsic urinary sphincter deficiency compared to that in monkeys with intrinsic urinary sphincter plus vehicle injection (p >0.05). Skeletal muscle precursor cells and CXCL12 only partially restored pudendal nerve stimulated increases in maximal urethral pressure (p >0.05), sphincter vascularization and motor endplate expression in monkeys with chronic intrinsic urinary sphincter deficiency. Additionally, CXCL12 but not skeletal muscle precursor cell injections decreased collagen and increased the muscle content of urinary sphincter complex in monkeys with chronic intrinsic urinary sphincter deficiency compared to those with intrinsic urinary sphincter plus vehicle injection and no intrinsic urinary sphincter plus no injection (p <0.05 and >0.05, respectively). These results raise questions about cell therapy for chronic intrinsic urinary sphincter deficiency and identify a chemokine treatment (CXCL12) as a potential alternative treatment of chronic intrinsic urinary sphincter deficiency.

Learning to cope with stress modulates anterior cingulate cortex stargazin expression in monkeys and mice

Intermittent mildly stressful situations provide opportunities to learn, practice, and improve coping with gains in subsequent emotion regulation. Here we investigate the effects of learning to cope with stress on anterior cingulate cortex gene expression in monkeys and mice. Anterior cingulate cortex is involved in learning, memory, cognitive control, and emotion regulation. Monkeys and mice were randomized to either stress coping or no-stress treatment conditions. Profiles of gene expression were acquired with HumanHT-12v4.0 Expression BeadChip arrays adapted for monkeys. Three genes identified in monkeys by arrays were then assessed in mice by quantitative real-time polymerase chain reaction. Expression of a key gene (PEMT) involved in acetylcholine biosynthesis was increased in monkeys by coping but this result was not verified in mice. Another gene (SPRY2) that encodes a negative regulator of neurotrophic factor signaling was decreased in monkeys by coping but this result was only partly verified in mice. The CACNG2 gene that encodes stargazin (also called TARP gamma-2) was increased by coping in monkeys as well as mice randomized to coping with or without subsequent behavioral tests of emotionality. As evidence of coping effects distinct from repeated stress exposures per se, increased stargazin expression induced by coping correlated with diminished emotionality in mice. Stargazin modulates glutamate receptor signaling and plays a role in synaptic plasticity. Molecular mechanisms of synaptic plasticity that mediate learning and memory in the context of coping with stress may provide novel targets for new treatments of disorders in human mental health.

Changes in hippocampal ultrastructure and vimentin expression in rhesus monkeys following selective deep hypothermia and blood occlusion.

Previous studies have shown that selective cerebral profound hypothermia combined with antegrade cerebral perfusion can improve resistance to cerebral hypoxia-ischemia in monkeys. The aim of this study was to observe the effect of selective cerebral profound hypothermia on the ultrastructure and vimentin expression in monkey hippocampi after severe cerebral ischemia. Eight healthy adult rhesus monkeys were randomly divided into two groups: profound hypothermia (N = 5) and normothermia (N = 3). Monkeys in the profound hypothermia group underwent bilateral carotid artery and jugular vein occlusion for 10 minutes at room temperature. Ringer's solution at 4°C was then perfused through the right internal carotid artery and out of the right jugular vein, maintaining the brain temperature below 18°C. Sixty minutes later, cerebral blood flow was restored. The normothermia group underwent all procedures with the exception that the Ringer's solution was 37°C during perfusion. All animals in the profound hypothermia group were successfully resuscitated. No significant abnormalities of hippocampal morphology or ultrastructure were observed. In contrast, no monkeys were alive after perfusion in the normothermia group and they had abnormal hippocampal morphology and ultrastructure to different extents. Vimentin expression in the hippocampus was significantly lower in the profound hypothermia group (47.88% ± 1.66) than the normothermia group (79.51% ± 1.00; P < 0.01). We conclude that selective cerebral profound hypothermia following 10-min occlusion of the bilateral common carotid arteries was able to downregulate vimentin expression in the hippocampus and protect it from severe cerebral ischemia.

The effect of short-term stress on serotonin gene expression in high and low resilient macaques.

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. When stressed with a paradigm of relocation and diet for 60 days, or 2 menstrual cycles, highly stress resilient monkeys continue to ovulate during both stress cycles (HSR); medium stress resilient monkeys ovulate once (MSR) and stress sensitive monkeys do not ovulate for the entire 60 days (SS). This study examines serotonin-related gene expression in monkeys with different sensitivity to stress and exposed to 5 days of moderate stress. Monkeys were first characterized as HSR, MSR or SS. After resumption of menstrual cycles, each monkey was re-stressed for 5 days in the early follicular phase. The expression of 3 genes pivotal to serotonin neural function was assessed in the 3 groups of monkeys (n=4-5/group). Tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor mRNAs expression were determined at 4 morphological levels of the dorsal raphe nucleus with in situ hybridization (ISH) using digoxigenin-incorporated riboprobes. In addition, cFos was examined with immunohistochemistry. Positive pixel area and/or cell number were measured. All data were analyzed with ANOVA (3 groups) and with a t-test (2 groups). After 5 days of stress, TPH2, SERT, 5HT1A and cFos were significantly lower in the SS group than the HSR group (p<0.05, all). This pattern of expression was the same as the pattern observed in the absence of stress in previous studies. Therefore, the ratio of the HSR/SS expression of each serotonergic gene was calculated in the presence and absence of stress. There was little or no difference in the ratio of HSR/SS gene expression in the presence or absence of stress. Moreover, cFos expression indicates that overall, cell activation in the dorsal raphe nucleus and periaquaductal gray is lower in SS than HSR animals. These data suggest that the serotonin system may set the sensitivity or resilience of the individual, but serotonin-related gene expression may not rapidly respond to moderate stress in nonhuman primates.

Antiangiogenic Activity of Aganirsen in Nonhuman Primate and Rodent Models of Retinal Neovascular Disease after Topical Administration

Aganirsen, an antisense oligonucleotide inhibiting insulin receptor substrate (IRS)-1 expression, has been shown to promote the regression of pathologic corneal neovascularization in patients. In this study, the authors aimed to demonstrate the antiangiogenic activity of aganirsen in animal models of retinal neovascularization. Eyedrops of aganirsen were applied daily in nonhuman primates after laser-induced choroidal neovascularization (CNV; model of wet age-related macular degeneration [AMD]) and in newborn rats after oxygen-induced retinopathy (OIR; model of ischemic retinopathy). Retinal aganirsen concentrations were assessed in rabbits and monkeys after topical delivery (21.5, 43, or 86 μg). Clinical significance was further evaluated by determination of IRS-1 expression in monkey and human retinal biopsy specimens. Topical corneal application of aganirsen attenuated neovascular lesion development dose dependently in African green monkeys. The incidence of high-grade CNV lesions (grade IV) decreased from 20.5% in vehicle-treated animals to 1.7% (P < 0.05) at the 86-μg dose. Topical aganirsen inhibited retinal neovascularization after OIR in rats (P < 0.05); furthermore, a single intravitreal injection of aganirsen reduced OIR as effectively as ranibizumab, and their effects were additive. Significantly, topical applications of aganirsen did not interfere with physiological retinal vessel development in newborn rats. Retinal delivery after topical administration was confirmed, and retinal expression of IRS-1 was demonstrated to be elevated in patients with subretinal neovascularization and AMD. Topical application of aganirsen offers a safe and effective therapy for both choroidal and retinal neovascularization without preventing its normal vascularization. Together, these findings support the clinical testing of aganirsen for human retinal neovascular diseases.

Systemic Administration of AAV8-α-galactosidase A Induces Humoral Tolerance in Nonhuman Primates Despite Low Hepatic Expression

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.

Characterization of the Expression and Activity of Carboxylesterases 1 and 2 from the Beagle Dog, Cynomolgus Monkey, and Human

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

Cynomolgus monkey has been used as a model for the prediction of drug disposition in human brain. The purpose of this study was to clarify protein expression levels of membrane proteins affecting drug distribution to brain, such as transporters, receptors, and junctional proteins, in cynomolgus monkey brain microvessels by using liquid chromatography tandem mass spectrometory. In adult monkeys, three ATP-binding cassette transporters (multidrug resistance 1 (MDR1), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4)), six solute carrier transporters (glucose transporter 1 (GLUT1), GLUT3/14, monocarboxylate transporter 1 (MCT1), MCT8, organic anion transporting polypeptide 1A2, and equilibrative nucleoside tranporter 1), two junctional proteins (claudin-5 and vascular endothelial cadherin), and two receptors (insulin receptor and low-density lipoprotein receptor-related protein 1) were detected. Comparison of the expression levels with those in mouse, which we reported previously, revealed a pronounced species difference. BCRP expression in monkey was greater by 3.52-fold than that in mouse, whereas MDR1 and MRP4 expression levels in monkey were lower by 0.304- and 0.180-fold, respectively, than that in mouse. This study also investigated the developmental changes in expression of membrane proteins in neonate and child monkeys. Expression of MDR1 was similar in neonate and adult monkeys, whereas in rat, P-glycoprotein expression was reported to be significantly lower in brain microvessels of neonate as compared with adult rat. These results will be helpful to understand and predict brain concentrations of drugs in different species and at different ages of primates. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3939–3950, 2011

Paraquat Exposure Reduces Nicotinic Receptor-Evoked Dopamine Release in Monkey Striatum

Paraquat, an herbicide widely used in the agricultural industry, has been associated with lung, liver, and kidney toxicity in humans. In addition, it is linked to an increased risk of Parkinson's disease. For this reason, we had previously investigated the effects of paraquat in mice and showed that it influenced striatal nicotinic receptor (nAChR) expression but not nAChR-mediated dopaminergic function. Because nonhuman primates are evolutionarily closer to humans and may better model the effects of pesticide exposure in man, we examined the effects of paraquat on striatal nAChR function and expression in monkeys. Monkeys were administered saline or paraquat once weekly for 6 weeks, after which nAChR levels and receptor-evoked [(3)H]dopamine ([(3)H]DA) release were measured in the striatum. The functional studies showed that paraquat exposure attenuated dopamine (DA) release evoked by alpha3/alpha6beta2(*) (nAChR that is composed of the alpha3 or alpha6 subunits, and beta2; the asterisk indicates the possible presence of additional subunits) nAChRs, a subtype present only on striatal dopaminergic terminals, with no decline in release mediated by alpha4beta2(*) (nAChR containing alpha4 and beta2 subunits, but not alpha3 or alpha6) nAChRs, present on both DA terminals and striatal neurons. Paraquat treatment decreased alpha4beta2(*) but not alpha3/alpha6beta2(*) nAChR expression. The differential effects of paraquat on nAChR expression and receptor-evoked [(3)H]DA release emphasize the importance of evaluating changes in functional measures. The finding that paraquat treatment has a negative impact on striatal nAChR-mediated dopaminergic activity in monkeys but not mice indicates the need for determining the effects of pesticides in higher species.

Heat Treatment Induces Liver Receptor Homolog-1 Expression in Monkey and Rat Sertoli Cells

We demonstrated in this study that liver receptor homolog-1 (LRH-1) was expressed in the round spermatids in normal monkey testis, and no LRH-1 signal was observed in the Sertoli cells. After local warming (43 C) the monkey testis, however, LRH-1 expression was induced in the Sertoli cells in coincidence with activation of cytokeratin 18 (CK-18), a Sertoli cell dedifferentiated marker. Furthermore, we isolated rat primary Sertoli cells from testes at various stages of development and treated with 43 C water in vitro. The changes in LRH-1 as well as CK-18 expression were analyzed by confocal immunohistochemistry and Western blot. The results showed that LRH-1 was stage-dependently expressed in the Sertoli cells; no LRH-1-positive signal was detected in the cells obtained from the testes of adult rat on d 60 after birth when mature spermatozoa in the testis was completed. However, the mature Sertoli cells were warmed at the 43 C water bath for 15 min, and the LRH-1 signal was remarkably induced in a time-dependent manner, just like the changes of CK-18 expression in the Sertoli cells, suggesting that the heat-induced dedifferentiation of the mature Sertoli cells might be related to LRH-1 regulation. LRH-1 expression induced by the heat treatment was completely inhibited by the addition of ERK inhibitor U0126 in the culture, indicating that the heat-induced LRH-1 expression in the Sertoli cells may be regulated via ERK1/2 activation pathway. Testosterone was found to have no such effect on LRH-1 expression in the monkey and rat Sertoli cells.

Transcription profiling of early responses to hemorrhagic fever in rhesus macaque

Monkeys infected intravenously with a lethal dose of LCMV-WE provide a model for Lassa fever virus infection of man. Like Lassa fever in man, disease begins with flu-like symptoms and progresses rapidly to death within two weeks of infection. It is essential to correctly diagnose hemorrhagic fever rather than more benign viral diseases to provide the proper treatment. Microarray analyses were conducted on blood of macaques infected with virulent WE virus, and compared to gene expression in monkeys infected with non-virulent LCMV-ARM. We observed gene expression changes that occur before the viremic stage of the disease, and could potentially serve as biomarkers that discriminate between exposure to a hemorrhagic fever virus and exposure to a benign virus. In particular, transcription analysis revealed an early and severe suppression of the COX2 pathways indicating that disease treatments should avoid COX-inhibitors, like aspirin, and favor pathway stimulators.

Effect of Fasting on Cocaine-Amphetamine-Regulated Transcript, Neuropeptide Y, and Leptin Receptor Expression in the Non-Human Primate Hypothalamus

Leptin is a cytokine produced by white adipose tissue that circulates in direct proportion to adiposity and is an important signal of energy balance. Leptin inhibits food intake in rodents by inhibiting the orexigenic neuropetides neuropeptide Y (NPY) and agouti regulated peptide (AgRP) and stimulating the anorexigenic neuropeptides α-melanocyte-stimulating hormone (α-MSH) and cocaine-amphetamine-regulated transcript (CART). In order to extend our understanding of neuroendocrine regulation of appetite in the primate, we determined the effect of a metabolic challenge on CART, NPY, and leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) in the nonhuman primate (NHP) hypothalamus. Ten adult female rhesus monkeys were either maintained on a regular diet or fasted for two days before euthanasia. CART, NPY, and Ob-R mRNA were measured by in situ hybridization histochemistry (ISHH). A 2-day fast decreased CART expression in the ARC, increased NPY gene expression in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and increased Ob-R expression in the ventromedial nucleus (VMN). This is the first report that fasting inhibits CART expression and stimulates Ob-R expression in monkeys. Increased NPY expression in the SON and PVN, but not the ARC of fasted monkeys also is novel. With some exceptions, our observations are confirmatory of findings in rodent studies. Similarities in the neuroendocrine responses to a metabolic challenge in monkeys and rodents support extending existing hypotheses of neuroendocrine control of energy homeostasis to primates.

Categorical signals in a single-trial neuron activity of the inferotemporal cortex

We developed an algorithm that decodes categorical signals from the single-trial activity of a neuronal population in the monkey inferotemporal cortex. We defined a global category (i.e. human faces vs. monkey faces vs. shape) and fine categories (i.e. human identity, monkey expression, and shape form) from the single-trial activity. The accuracy of estimation for the trials was roughly 100% for the global category and 88.1% for the fine categories. The accuracy of stimulus identification for the trials was 70.4%. These results suggest that signals concerning global and fine categories as well as object identification can be decoded using the single-trial activity of a neuronal population in the inferotemporal cortex.

Population Dynamics of Face-responsive Neurons in the Inferior Temporal Cortex

Neurons in the inferior temporal (IT) cortex of monkeys respond selectively to complex visual stimuli, such as faces. Single neurons in the IT cortex encode different kinds of information about visual stimuli in their temporal firing patterns. To understand the temporal aspects of the information encoded at a population level in the IT cortex, we applied principal component analysis (PCA) to the responses of a population of neurons. The responses of each neuron were recorded while visual stimuli that consisted of geometric shapes and faces of humans and monkeys were presented. We found that global categorization, i.e. human faces versus monkey faces versus shapes, occurred in the earlier part of the population response, and that fine categorization occurred within each member of the global category in the later part of the population response. A cluster analysis, a mixture of Gaussians analysis, confirmed that the clusters in the earlier part of the responses represented the global category. Moreover, the clusters in the earlier part separated into sub-clusters corresponding to either human identity or monkey expression in the later part of the responses, and the global categorization was maintained even after the appearance of the sub-clusters. The results suggest that a hierarchical relationship of the test stimuli is represented temporally by the population response of IT neurons.

Genetic analysis of four novel peroxisome proliferator activated receptor- [formula omitted] splice variants in monkey macrophages

Peroxisome proliferator activated receptor-γ (PPAR-γ) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-γ1, PPAR-γ2, and PPAR-γ3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-γ exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5 ′ terminal region to generate four novel PPAR-γ subtypes, PPAR-γ4, -γ5, -γ6, and -γ7. PPAR-γ4 and γ5 were detected only in macrophages whereas γ6 and γ7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-γ exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-γ1, PPAR-γ4, and PPAR-γ5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-γ expression in monkeys. RT-PCR showed a significant increase in total PPAR-γ and ABCA1 mRNA levels in macrophages of fat-fed monkeys ( n=7) compared to those maintained on a normal diet ( n=2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-γ4 and -γ5 in CHO cells. In these cells, expression of PPAR-γ5 but not -γ4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-γ isoforms may be responsible for unique tissue-specific biological effects and that PPAR-γ4 and -γ5 may modulate macrophage function and atherogenesis.

Expression of Myocilin/TIGR in Normal and Glaucomatous Primate Optic Nerves

Myocilin/TIGR was the first molecule discovered to be linked with primary open angle glaucoma (POAG), a blinding disease characterized by progressive loss of retinal ganglion cells. Mutations in myocilin/TIGR have been associated with age of disease onset and severity. The function of myocilin/TIGR and its role in glaucoma is unknown. Myocilin/TIGR has been studied in the trabecular meshwork to determine a role in regulation of intraocular pressure. The site of damage to the axons of the retinal ganglion cells is the optic nerve head (ONH). The myocilin/TIGR expression was examined in fetal through adult human optic nerve as well as in POAG. Myocilin/TIGR was expressed in the myelinated optic nerve of children and normal adults but not in the fetal optic nerve before myelination. Also examined was the expression in monkeys with experimental glaucoma. The results demonstrate that optic nerve head astrocytes constitutively express myocilin/TIGR in vivo in primates. Nevertheless, myocilin/TIGR is apparently reduced in glaucomatous ONH. The colocalization of myocilin/TIGR to the myelin suggests a role of myocilin/TIGR in the myelinated optic nerve.

The ovulatory gonadotrophin surge stimulates cyclooxygenase expression and prostaglandin production by the monkey follicle.

Follicular prostaglandin concentrations increase following the gonadotrophin surge in domestic animals and rodents approximately 10 h before follicle rupture, suggesting a unifying role for prostaglandins in the timing of ovulation. However, little is known about prostaglandin production by the primate ovulatory follicle. In this study, adult female macaques received gonadotrophins to promote follicular development. Granulosa cells, follicular fluid, and ovaries were collected before (0 h) and 12, 24 or 36 h after administration of the ovulatory stimulus, human chorionic gonadotrophin (HCG). Cyclooxygenase (COX) isoform expression was assessed by reverse transcription-polymerase chain reaction and immunocytochemistry and follicular prostaglandin production was determined by enzyme immunoassay. COX-2 mRNA expression in granulosa cells was low at 0 h, rose 50-fold by 12 h, and remained elevated through to 36 h. COX-2 immunostaining was present in granulosa cells after, but not before, exposure to HCG. COX-1 mRNA levels did not change during the periovulatory interval, and COX-1 immunostaining of granulosa cells was not detected. Follicular fluid prostaglandin (PG) E (2) and PGF (2alpha) concentrations were low through to 24 h but increased 100-fold at 36 h. The elevated follicular prostaglandin concentrations 4-16 h before the expected time of ovulation support the hypothesis that the time between the LH surge and increased follicular prostaglandins determines the length of the periovulatory period. Differences between the localization and timing of COX-2 expression in monkey versus non-primate follicles suggest that the pattern of COX-2 expression and activity has aspects unique to primates.