A radiation hybrid transcript map of the mouse genome.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid-based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation-placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

A mutation in KCNJ11 causing human hyperinsulinism (Y12X) results in a glucose-intolerant phenotype in the mouse.

AIMS/HYPOTHESIS: We identified a mouse with a point mutation (Y12STOP) in the Kcnj11 subunit of the K(ATP) channel. This point mutation is identical to that found in a patient with congenital hyperinsulinism of infancy (HI). We aimed to characterise the phenotype arising from this loss-of-function mutation and to compare it with that of other mouse models and patients with HI. METHODS: We phenotyped an N-ethyl-N-nitrosourea-induced mutation on a C3H/HeH background (Kcnj11 ( Y12STOP )) using intraperitoneal glucose tolerance testing to measure glucose and insulin plasma concentrations. Insulin secretion and response to incretins were measured on isolated islets. RESULTS: Homozygous male and female adult Kcnj11 ( Y12STOP ) mice exhibited impaired glucose tolerance and a defect in insulin secretion as measured in vivo and in vitro. Islets had an impaired incretin response and reduced insulin content. CONCLUSIONS/INTERPRETATION: The phenotype of homozygous Kcnj11 ( Y12STOP ) mice is consistent with that of other Kcnj11-knockout mouse models. In contrast to the patient carrying this mutation homozygously, the mice studied did not have hyperinsulinaemia or hypoglycaemia. It has been reported that HI patients may develop diabetes and our mouse model may reflect this clinical feature. The Kcnj11 ( Y12STOP ) model may thus be useful in further studies of K(ATP) channel function in various cell types and in investigation of the development of hyperglycaemia in HI patients.

Unique Genetic and Histological Signatures of Mouse Pericardial Adipose Tissue.

Obesity is a major risk factor for a plethora of metabolic disturbances including diabetes and cardiovascular disease. Accumulating evidence is showing that there is an adipose tissue depot-dependent relationship with obesity-induced metabolic dysfunction. While some adipose depots, such as subcutaneous fat, are generally metabolically innocuous, others such as visceral fat, are directly deleterious. A lesser known visceral adipose depot is the pericardial adipose tissue depot. We therefore set out to examine its transcriptional and morphological signature under chow and high-fat fed conditions, in comparison with other adipose depots, using a mouse model. Our results revealed that under chow conditions pericardial adipose tissue has uncoupling-protein 1 gene expression levels which are significantly higher than classical subcutaneous and visceral adipose depots. We also observed that under high-fat diet conditions, the pericardial adipose depot exhibits greatly upregulated transcript levels of inflammatory cytokines. Our results collectively indicate, for the first time, that the pericardial adipose tissue possesses a unique transcriptional and histological signature which has features of both a beige (brown fat-like) but also pro-inflammatory depot, such as visceral fat. This unique profile may be involved in metabolic dysfunction associated with obesity.

Reduction of fibrillar strain-rate sensitivity in steroid-induced osteoporosis linked to changes in mineralized fibrillar nanostructure.

As bone is used in a dynamic mechanical environment, understanding the structural origins of its time-dependent mechanical behaviour - and the alterations in metabolic bone disease - is of interest. However, at the scale of the mineralized fibrillar matrix (nanometre-level), the nature of the strain-rate dependent mechanics is incompletely understood. Here, we investigate the fibrillar- and mineral-deformation behaviour in a murine model of Cushing's syndrome, used to understand steroid induced osteoporosis, using synchrotron small- and wide-angle scattering/diffraction combined with in situ tensile testing at three strain rates ranging from 10-4 to 10-1 s-1. We find that the effective fibril- and mineral-modulus and fibrillar-reorientation show no significant increase with strain-rate in osteoporotic bone, but increase significantly in normal (wild-type) bone. By applying a fibril-lamellar two-level structural model of bone matrix deformation to fit the results, we obtain indications that altered collagen-mineral interactions at the nanoscale - along with altered fibrillar orientation distributions - may be the underlying reason for this altered strain-rate sensitivity. Our results suggest that an altered strain-rate sensitivity of the bone matrix in osteoporosis may be one of the contributing factors to reduced mechanical competence in such metabolic bone disorders, and that increasing this sensitivity may improve biomechanical performance.

A missense mutation in the non-neural G-protein alpha-subunit isoforms modulates susceptibility to obesity.

OBJECTIVE: The Gnas transcription unit located within an imprinting region encodes several proteins, including the G-protein alpha-subunit, Gsalpha, its isoform XLalphas and their variant truncated neural forms GsalphaN1 and XLN1. Gsalpha and GsalphaN1 are expressed predominantly from the maternally derived allele in some tissues, whereas XLalphas and XLN1 are expressed exclusively from the paternally derived allele. The relative contribution of full-length Gsalpha and XLalphas, and truncated forms GsalphaN1 and XLN1 to phenotype is unknown. The edematous-small point mutation (Oed-Sml) in exon 6 of Gnas lies downstream of GsalphaN1 and XLN1, but affects full-length Gsalpha and XLalphas, allowing us to address the role of full-length Gsalpha and XLalphas. The aim of this study was therefore to determine the metabolic phenotypes of Oed and Sml mice, and to correlate phenotypes with affected transcripts. METHODS: Mice were fed standard or high-fat diets and weighed regularly. Fat mass was determined by DEXA analysis. Indirect calorimetry was used to measure metabolic rate. Glucose was measured in tolerance tests and biochemical parameters in fasted plasma samples. Histological analysis of fat and liver was carried out post mortem. RESULTS: Oed mice are obese on either diet and have a reduced metabolic rate. Sml mice are lean and are resistant to a high-fat diet and have an increased metabolic rate. CONCLUSION: Adult Oed and Sml mice have opposite metabolic phenotypes. On maternal inheritance, the obese Oed phenotype can be attributed to non-functional full-length Gsalpha. In contrast, on paternal inheritance, Sml mice were small and resistant to the development of obesity on a high-fat diet, effects that can be attributed to mutant XLalphas. Thus, the neural isoforms, GsalphaN1 and XLN1, do not appear to play a role in these metabolic phenotypes.

Bone matrix development in steroid-induced osteoporosis is associated with a consistently reduced fibrillar stiffness linked to altered bone mineral quality.

Glucocorticoid-induced osteoporosis (GIOP) is a major secondary form of osteoporosis, with the fracture risk significantly elevated - at similar levels of bone mineral density - in patients taking glucocorticoids compared with non-users. The adverse bone structural changes at multiple hierarchical levels in GIOP, and their mechanistic consequences leading to reduced load-bearing capacity, are not clearly understood. Here we combine experimental X-ray nanoscale mechanical imaging with analytical modelling of the bone matrix mechanics to determine mechanisms causing bone material quality deterioration during development of GIOP. In situ synchrotron small-angle X-ray diffraction combined with tensile testing was used to measure nanoscale deformation mechanisms in a murine model of GIOP, due to a corticotrophin-releasing hormone promoter mutation, at multiple ages (8-, 12-, 24- and 36 weeks), complemented by quantitative micro-computed tomography and backscattered electron imaging to determine mineral concentrations. We develop a two-level hierarchical model of the bone matrix (mineralized fibril and lamella) to predict fibrillar mechanical response as a function of architectural parameters of the mineralized matrix. The fibrillar elastic modulus of GIOP-bone is lower than healthy bone throughout development, and nearly constant in time, in contrast to the progressively increasing stiffness in healthy bone. The lower mineral platelet aspect ratio value for GIOP compared to healthy bone in the multiscale model can explain the fibrillar deformation. Consistent with this result, independent measurement of mineral platelet lengths from wide-angle X-ray diffraction finds a shorter mineral platelet length in GIOP. Our results show how lowered mineralization combined with altered mineral nanostructure in GIOP leads to lowered mechanical competence. SIGNIFICANCE STATEMENT: Increased fragility in musculoskeletal disorders like osteoporosis are believed to arise due to alterations in bone structure at multiple length-scales from the organ down to the supramolecular-level, where collagen molecules and elongated mineral nanoparticles form stiff fibrils. However, the nature of these molecular-level alterations are not known. Here we used X-ray scattering to determine both how bone fibrils deform in secondary osteoporosis, as well as how the fibril orientation and mineral nanoparticle structure changes. We found that osteoporotic fibrils become less stiff both because the mineral nanoparticles became shorter and less efficient at transferring load from collagen, and because the fibrils are more randomly oriented. These results will help in the design of new composite musculoskeletal implants for bone repair.

A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis.

A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh(-120/+)) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh(-120/+) mice. We also find a much larger fibril strain/tissue strain ratio in Crh(-120/+) mice (~1.5) compared to the wild-type mice (~0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.

Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses.

EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85%) of the 34 probands: in 22 of these (76%), the mutation was in EXT1; seven patients (24%) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95% and 93% respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP.

Changes in morphine self-administration after brainstem lesions in rats.

Rats were trained to bar press for intravenous infusions of morphine sulfate during 1-h daily test regions. Rates of morphine self-administration were reduced by bilateral lesions of the substantia nigra and enhanced by lesions of the medial raphe nucleus. Dose-response studies indicated that sensitivity to morphine's rewarding property was increased by substantia nigra lesions and decreased by medial raphe lesions. Lesions of the dorsal raphe nucleus and of the locus coeruleus had no effect on self-administration behavior. An interaction between ascending dopaminergic and serotonergic pathways appears to be involved in the mechanism of morphine reinforcement.

Changes in morphine self-administration after tel-diencephalic lesions in rats.

Rats were trained to bar-press for intravenous infusions of morphine sulfate during 1-h daily test sessions. Rates of morphine self-administration were enhanced by lesions of the frontal cortex and hippocampus and transiently reduced by lesions of the medial forebrain bundle and medial thalamus. Dose-response studies indicated that sensitivity to morphine's rewarding property was decreased by frontal cortical and hippocampal lesions. Lesions of the posterior cortex, the tuberculum olfactorium, and the nucleus accumbens had no effect on self-administration behavior. The results are discussed in relation to previous findings with caudate and brainstem lesions. A neuroanatomical substrate for morphine reinforcement is suggested.

Nocturnal rotation in normal rats: correlation with amphetamine-induced rotation and effects of nigrostriatal lesions.

Normal unoperated rats were observed to rotate (turn in circles) at night. For most (91.7%) rats, the preferred direction of rotation was consistent across hours and days and was the same as the direction of rotation elicited by D-amphetamine (1.0 mg/kg) during the day. The magnitudes of nocturnal and D-amphetamine-induced rotation were also highly correlated. After rats showed stable diurnal patterns of rotation, unilateral lesions were made in either the substantia nigra, the nigrostriatal bundle or the caudate nucleus. All lesions produced transient contralateral rotation within the first 24-48 h after surgery. The time-course of this contralateral rotation was more prolonged after nigral lesions than after nigrostriatal bundle lesions and least after caudate lesions, suggesting that the duration of a degeneration release of dopamine is proportional to the length of the degenerating axon. Lesion size was correlated with the intensity of contralateral rotation but not with the time-course. At each rostralcaudal level, the magnitude of contralateral rotation was greater if the lesion was in the side of the brain opposite to the preoperative direction of rotation than if in the same side. By three days after surgery, all rats returned to a mostly normal diurnal cycle with the direction of rotation now being ipsilateral to the lesion. D-Amphetamine potentiated the ipsilateral rotation, though rats with lesions in the same side of the brain as the preoperative direction of rotation had larger drug responses than rats with similar lesions in the opposite side of the brain. By one month after surgery, the direction of spontaneous rotation of most rats had returned to the preoperative direction. As at all other times, the magnitude of rotation was, in part, dependent on the side of the lesion with respect to the preoperative bias. It is suggested that, following a unilateral lesion, compensatory processes occur to a greater extent if the lesion is in the normally more active side of the brain.

Phencyclidine-induced rotation and hippocampal modulation of nigrostriatal asymmetry.

Phencyclidine (PCP) elicited dose-related rotation in naive rats. The effect of PCP was consistent in direction and magnitude from one week to the next but was dissimilar to the rotatory effects of dopaminergic (D-amphetamine, apomorphine) or anticholinergic (scopolamine) drugs. Study of the effects of PCP on regional brain uptake of labeled 2-deoxy-D-glucose suggested that PCP-induced rotation is at least in part mediated by an action in the hippocampus. PCP elicited ipsilateral rotation following unilateral hippocampal lesions whereas such lesions did not alter the direction of either nocturnal or D-amphetamine-induced rotation. PCP appears to activate a hippocampal mechanism that normally only modulates the intensity of rotation.

The ontogeny of hippocampal energy metabolism.

The postnatal functional development of the hippocampus has been studied with the use of the 2-deoxyglucose method. Metabolic changes occurred along three different axes with increasing age. High metabolic activity appeared first in the pyramidal cell layer and shifted to the molecular layer. Activity in the regio inferior preceded the regio superior at posterior and ventral levels. All of these changes occurred first in the dorsal hippocampus and progressed along the septal-temporal axis. The data suggest that maturational changes occur at different hippocampal regions and that critical periods in development may ultimately be specified with respect to different regions in the same structure.

Intraperitoneal administration and other modifications of the 2-deoxy-D-glucose technique.

Experiments were conducted to determine the optimal conditions necessary for implementing modifications of the 2-deoxyglucose (2-DDG) technique. Substitution of tritium-labeled 2-DDG with subsequent microdissection of selected brain regions and liquid scintillation counting produced results that were highly correlated with both [14C]radioautograms and glucose utilization values as obtained by Sokoloff et al. The route of administration of isotope was also varied. Whole brain uptake at maximal levels of incorporation was the same for both intravenously and intraperitoneally injected animals. Radioautograms from i.p. and i.v. injected animals were indistinguishable. Densitometric analyses of the i.p. radioautograms were highly correlated with glucose utilization values. Thus, relative indices of functional activity may be obtained when experimental circumstances preclude arteriovenous cannulations and restraint. The use of naive, unrestrained animals, therefore, makes the 2-DDG technique applicable to a broader range of studies.

Human N-CAM gene: mapping to chromosome 11 by analysis of somatic cell hybrids with mouse and human cDNA probes.

We have used mouse and human cDNA probes to map the chromosomal position of the N-CAM gene in the human genome. Southern analysis of DNA isolated from a panel of mouse-human somatic cell hybrids has assigned the N-CAM gene to chromosome 11. This assignment was found with both mouse and human N-CAM cDNAs.

Changes in sensitivity to operant effects of dopaminergic and cholinergic agents following morphine withdrawal in rats.

Rats trained to bar-press on either a FI 15 sec or FR 30 schedule for water reinforcement were administered various doses of appomorphine, haloperidol, pilocarpine and scopolamine both before and 1--4 months after a 3-day period of continuous morphine administration. All drugs monotonically depressed response rates on both schedules with increasing dose. Chronic morphrine administration produced a persistent increase in sensitivity to apomorphine and pilocarpine and a persistent decrease in sensitivity to haloperidol and scopolamine. The results suggest that both dopaminergic and cholinergic supersensitivity are likely to be involved in protracted abstinence phenomena.

Relationship of rats' spatial preferences to effects of d-amphetamine on timing behavior.

Rats were trained to bar-press on a differential reinforcement of low rate 16 sec (DRL 16) schedule for water reinforcement. On alternate days, rats were tested in the presence or absence of a light which signaled the availability of reinforcement. Rats were allowed to bar-press on either of two levers (left and right). All rats show consistent side preferences. Doses (0.5-2.0 mg/kg) of damphetamine differentially affected performance under signaled and nonsignaled conditions. Performance during the nonsignaled condition was much more sensitive to a drug-induced rate increment and timing impairment than performance during the signaled condition. With increasing drug dosage, under both conditions, side preferences reliably increased, decreased or remained unchanged depending upon the particular pattern of paw usage and the relationship between paw and side preferences. For the nonsignaled condition but not for the signaled condition, baseline rates were related to the strength of side preferences; lower rates and better timing performance wwere significantly correlated with greater preferences. Observations of bar-pressing behavior suggested that stereotyped motor patterns associated with side preferences might be related to mechanisms involved in timing behavior and perhaps, in behavior controlled by internal stimuli generally.

Anticholinergic behavioral effect of phencyclidine.

Phencyclidine (PCP) impaired spatial alternation performance in rats. This effects was mimicked by antimuscarinic anticholinergics (scopolamine, atropine) and PCP derivatives (ketamine, cyclohexamine) but not by a variety of other agents. Muscarinic cholinergic agonists antagonized PCP. Impairment of spatial alteration performance by PCP appears to be mediated, at least in part, by and anticholinergic action; this is the first instance of a behavioral effect of PCP that can be largely attributed to a specific mechanism.

The neuroprotective effect of high-dose methylprednisolone in rat spinal cord hemisection.

Multiple studies support a neuroprotective effect for high-dose methylprednisolone (MP) in acute blunt spinal cord injury. We know of no study that addresses the role of MP in prophylaxis for surgical trauma to the spinal cord or for the treatment of non-missile penetrating injuries to the spinal cord. We examined the neuroprotective effect of MP as measured by the retrograde transport of the fluorescent tracer Fluoro-Gold in 20 rats undergoing C-2 hemisection. Mean cell counts of retrogradely labeled rubrospinal neurons were determined 1 week post-injury. The group receiving MP had a significantly higher (P < 0.0001) number of labeled cells (x = 594) compared to controls (x = 387). The highly significant increase in mean cell counts in rats receiving steroids suggests less secondary axonal injury in the MP group. These findings are the first report of a neuroprotective effect of MP in rat spinal cord hemisection. We suggest that MP may be beneficial as prophylaxis during planned or incidental surgical trauma to the spinal cord and after non-missile penetrating injuries to the spinal cord.