Central RANK signalling in NPY neurons alters bone mass in male mice.

RANKL signalling known to be important for the control of bone mass, has recently also been implicated in the brain to control thermoregulation, however, it is not known which neuronal pathways are involved and whether other aspects of energy homeostasis are also affected. Here we show that selective deletion of RANK from NPY neurons down-regulated NPY mRNA expression in the hypothalamus. While comprehensive phenotyping of germline-induced NPY neuron specific RANK deficient mice revealed no significant changes in physical or metabolic parameters, adult onset deletion of RANK from NPY neurons led to a significant increase in fat mass and a decrease in whole body bone mineral content and bone mineral density. Intriguingly, when these conditional knockout mice were placed on a high fat diet, body weight and fat mass did not differ to control mice. However, they were able to significantly increase their bone mass to match their increased body weight, an ability that was lacking in control mice. Taken together, results from this study demonstrate that RANK signalling in NPY neurons is involved in modulating NPY levels and through that matching bone mass to body weight.

Hypothalamus Specific Re-Introduction of SNORD116 into Otherwise Snord116 Deficient Mice Increased Energy Expenditure.

The Snord116 gene cluster has been recognised as a critical contributor to the Prader-Willi syndrome (PWS), with mice lacking Snord116 displaying many classical PWS phenotypes, including low postnatal body weight, reduced bone mass and increased food intake. However, these mice do not develop obesity as a result of increased energy expenditure. To understand the physiological function of SNORD116 better and potentially rescue the altered metabolism of Snord116-/- mice, we used an adeno-associated viral (AAV) approach to reintroduce the product of the Snord116 gene into the hypothalamus in Snord116-/- mice at different ages. The results obtained show that mid-hypothalamic re-introduction of SNORD116 in 6-week-old Snord116-/- mice leads to significantly reduced body weight and weight gain, which is associated with elevated energy expenditure. Importantly, when the intervention targets other areas such as the anterior region of the hypothalamus or the reintroduction occurs in older mice, the positive effects on energy expenditure are diminished. These data indicate that the metabolic symptoms of PWS develop gradually and the Snord116 gene plays a critical role during this process. Furthermore, when we investigated the consequences of SNORD116 re-introduction under conditions of thermoneutrality where the mild cold stress influences are avoided, we also observed a significant increase in energy expenditure. In conclusion, the rescue of mid-hypothalamic Snord116 deficiency in young Snord116 germline deletion mice increases energy expenditure, providing fundamental information contributing to potential virus-mediated genetic therapy in PWS.

Ambient temperature modulates the effects of the Prader-Willi syndrome candidate gene Snord116 on energy homeostasis.

Germline deletion of the Prader-Willi syndrome (PWS) candidate gene Snord116 in mice leads to some classical symptoms of human PWS, notably reductions in body weight, linear growth and bone mass. However, Snord116 deficient mice (Snord116-/-) do not develop an obese phenotype despite their increased food intake and the underlying mechanism for that is unknown. We tested the phenotypes of germline Snord116-/- as well as neuropeptide Y (NPY) neuron specific Snord116lox/lox/NPYcre/+ mice at 30°C, the thermoneutral temperature of mice, and compared these to previous reports studies conducted at normal room temperature. Snord116-/- mice at 30°C still weighed less than wild type but had increased body weight gain. Importantly, food intake and energy expenditure were no longer different at 30°C, and the reduced bone mass and nasal-anal length observed in Snord116-/- mice at room temperature were also normalized. Mechanistically, the thermoneutral condition led to the correction of the mRNA expression of NPY and pro-opiomelanocortin (POMC), which were both previously observed to be significantly up-regulated at room temperature. Importantly, almost identical phenotypes and NPY/POMC mRNA expression alterations were also observed in Snord116lox/lox/NPYcre/+ mice, which lack the Snord116 gene only in NPY neurons. These data illustrate that mild cold stress is a critical factor preventing the development of obesity in Snord116-/- mice via the NPY system. Our study highlights that the function of Snord116 in the hypothalamus may be to enhance energy expenditure, likely via the NPY system, and also indicates that Snord116 function in mice is strongly dependent on environmental conditions such as cold exposure.

Chronic overproduction of ghrelin in the hypothalamus leads to temporal increase in food intake and body weight.

Ghrelin is known to be a critical stimulator of feeding behavior mainly via actions in the hypothalamus. However, its functional contribution to the control of energy homeostasis under chronic elevated conditions is unknown. Here we show that overproduction of ghrelin via an AAV viral delivery system in the hypothalamus leads to an increase in food intake associated with increases in body weight. However, this increase in food intake is only temporary and is diminished and no longer significant after 3 weeks. Analysis of brain sections of mice 6 weeks after AAV-ghrelin virus injection demonstrates unaltered neuropeptide Y levels but strongly up-regulated pro-opiomelanocortin levels indicating that a compensatory mechanism has been activated to counter regulate the feeding stimulatory actions of ghrelin. This demonstrates that control mechanism exists that is activated under conditions of prolonged high ghrelin levels, which could potentially be utilized to control feeding and the development of obesity.

Neuropeptide Y mediates the short-term hypometabolic effect of estrogen deficiency in mice.

BACKGROUND: Estrogen deficiency increases body weight or total and central adiposity and decreases energy expenditure. Hypothalamic neuropeptide Y (NPY) expression is altered by estrogen deficiency in rodents, but the long-term consequences on energy homeostasis are unknown. OBJECTIVE: To investigate the role of NPY in the changes in energy expenditure and physical activity, as well as the associated changes in body weight and composition in response to short-term and long-term estrogen deficiency. DESIGN: Sham and ovariectomy (OVX) operations were performed at 8 weeks of age in wild-type (WT) and NPY(-/-) mice. Energy expenditure, physical activity, body composition and weight, as well as food intake were measured at 10-18 days (short-term) and 46-54 days (long-term) after OVX. RESULTS: OVX influences energy homeostasis differently at early compared with later time-points. At the early but not the late time point, OVX in WT mice reduced oxygen consumption and energy expenditure and tended to reduce resting metabolic rate. Interestingly, these effects of short-term estrogen deficiency were ablated by NPY deletion, with NPY(-/-) mice exhibiting significant increases in energy expenditure and resting metabolic rate. In addition to these hypermetabolic effects, OVX NPY(-/-) mice exhibited significantly lower body weight and whole-body fat mass relative to OVX WT controls at the short-term but not the long-term time point. Food intake and physical activity were unaltered by OVX, but NPY(-/-) mice exhibited significant reductions in these parameters relative to WT. CONCLUSION: The effects of estrogen deficiency to reduce energy metabolism are transient, and NPY is critical to this effect as well as the early OVX-induced obesity.

Optimization of magnetic neurostimulation waveforms for minimum power loss.

Magnetic stimulation is a key tool in experimental brain research and several clinical applications. Whereas coil designs and the spatial field properties have been intensively studied in the literature, the temporal dynamics of the field has received little attention. The available pulse shapes are typically determined by the relatively limited capabilities of commercial stimulation devices instead of efficiency or optimality. Furthermore, magnetic stimulation is relatively inefficient with respect to the required energy compared to other neurostimulation techniques. We therefore analyze and optimize the waveform dynamics with a nonlinear model of a mammalian motor axon for the first time, without any pre-definition of waveform candidates. We implemented an unbiased and stable numerical algorithm using variational calculus in combination with a global optimization method. This approach yields very stable results with comprehensible characteristic properties, such as a first phase which reduces ohmic losses in the subsequent pulse phase. We compare the energy loss of these optimal waveforms with the waveforms generated by existing magnetic stimulation devices.

Increased novelty-induced motor activity and reduced depression-like behavior in neuropeptide Y (NPY)-Y4 receptor knockout mice.

There is growing evidence that neuropeptide Y (NPY) acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y-receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble burying test and in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared with Y4 and Y2 knockout mice, respectively. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of pancreatic polypeptide (PP) induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion.

Compensatory changes in [125I]-PYY binding in Y receptor knockout mice suggest the potential existence of further Y receptor(s).

Gene knockout approaches have helped to better understand the functions of the different Y receptors. However, some results obtained from these knockout mice are unexpected and differ from the results of pharmacological intervention experiments. One possible explanation for this is that germ-line gene deletion of a particular Y receptor can influence expression and function of the remaining Y receptors. Here we show that such compensation in mRNA and protein expression does occur in Y receptor single, double and triple knockout models. Radio-ligand binding experiments using [(125)I]-PYY revealed significant up- and down-regulation of remaining Y receptor binding sites in various Y receptor knockout models compared to results from control mice employing Y receptor preferring agonist or antagonists for displacement of the radio-ligand. The most obvious change can be seen in the hippocampus of Y(1) knockout mice, where the level of the remaining Y receptors is strongly down-regulated. In Y(2) knockout mice no such trend can be seen, however, the expression pattern is significantly changed with a strong up-regulation of [(125)I]-PYY specific binding in the dentate gyrus. Interestingly, this pattern was also seen in Y(1)Y(2)Y(4) triple knockout mice. Y(5) receptor mRNA was approximately 20% higher in the hippocampus and dentate gyrus in the triple knockout mice compared to wild-type controls, while Y(6) mRNA expression could not be detected. However, competition binding experiments in Y(1)Y(2)Y(4) triple knockout mice with the Y(5) receptor preferring ligands [Leu(31), Pro(34)] NPY and [A(31), Aib(32)] NPY were able to replace only approximately 50% of [(125)I]-PYY binding in the dentate gyrus suggesting the existence of further yet unidentified Y receptor(s).

Conscious and subconscious sensorimotor synchronization--prefrontal cortex and the influence of awareness.

One of the most compelling challenges for modern neuroscience is the influence of awareness on behavior. We studied prefrontal correlates of conscious and subconscious motor adjustments to changing auditory rhythms using regional cerebral blood flow measurements. At a subconscious level, movement adjustments were performed employing bilateral ventral mediofrontal cortex. Awareness of change without explicit knowledge of the nature of change led to additional ventral prefrontal and premotor but not dorsolateral prefrontal activations. Only fully conscious motor adaptations to a changing rhythmic pattern showed prominent involvement of anterior cingulate and dorsolateral prefrontal cortex. These results demonstrate that while ventral prefrontal areas may be engaged in motor adaptations performed subconsciously, only fully conscious motor control which includes motor planning will involve dorsolateral prefrontal cortex.

The peptide YY-preferring receptor mediating inhibition of small intestinal secretion is a peripheral Y(2) receptor: pharmacological evidence and molecular cloning.

A peptide YY (PYY)-preferring receptor [PYY > neuropeptide Y (NPY)] was previously characterized in rat small intestinal crypt cells, where it mediates inhibition of fluid secretion. Here, we investigated the possible status of this receptor as a peripheral Y(2) receptor in rats. Typical Y(2) agonists (PYY(3-36), NPY(3-36), NPY(13-36), C2-NPY) and very short PYY analogs (N-alpha-Ac-PYY(22-36) and N-alpha-Ac-PYY(25-36)) acting at the intestinal PYY receptor were tested for their ability to inhibit the binding of (125)I-PYY to membranes of rat intestinal crypt cells and of CHO cells stably transfected with the rat hippocampal Y(2) receptor cDNA. Similar PYY preference was observed and all analogs exhibited comparable high affinity in both binding assays. The same held true for the specific Y(2) antagonist BIIE0246 with a K(i) value of 6.5 and 9.0 nM, respectively. BIIE0246 completely abolished the inhibition of cAMP production by PYY in crypt cells and transfected CHO cells. Moreover, the antagonist 1) considerably reversed the PYY-induced reduction of short-circuit current in rat jejunum mucosa in Ussing chamber and 2) completely abolished the antisecretory action of PYY on vasoactive intestinal peptide (VIP)-induced fluid secretion in rat jejunum in vivo. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that Y(2) receptor transcripts were present in intestinal crypt cells (3 x 10(2) molecules/100 ng RNA(T)) with no expression in villus cells, in complete agreement with the exclusive binding of PYY in crypt cells. Finally, a full-length Y(2) receptor was cloned by RT-PCR from rat intestinal crypt cells and also from human small intestine. We conclude that the so-called PYY-preferring receptor mediating inhibition of intestinal secretion is a peripheral Y(2) receptor.

Distribution and utilization of 15N in cowpeas injected into the stem under influence of water deficit.

Investigations were carried out on Vigna unguiculata L. Walp. to estimate the distribution and utilization of 15N in different organs after stem injection during vegetative, flowering and pod filling stage. During flowering effects of water deficit were also examined. In well watered plants, within 4 days after injection, 65% of 15N accumulated in leaves. This was drastically reduced to 42% by water deficit. 15N accumulation in stems increased under water deficit. The translocation of 15N from the stem base to roots were not altered by water deficit. During pod filling 62% of recovered 15N in the plants had accumulated in seeds, 24% in leaves and 11% in stems within 4 days, whereas the uptake of nitrogen in pod walls and roots remained low (2%). These results demonstrate that the method of injecting very small quantities (1 mg/plant) of 15N into the stem base allows an exact and detailed quantitative assessment of N translocation/distribution with regard to different organs, different growth stages and different treatments.

Conservation of expression of neuropeptide Y5 receptor between human and rat hypothalamus and limbic regions suggests an integral role in central neuroendocrine control.

Neuropeptide Y receptors belong to the G-protein-coupled receptor superfamily and mediate a wide variety of physiological functions, including blood pressure regulation, hormone release, appetite control, seizure propensity, cognition, and emotion. The recent description of a new neuropeptide Y receptor, Y5, expressed in hypothalamic nuclei in rat brain, raised the possibility that Y5 was the receptor mediating the feeding and appetite-related functions of neuropeptide Y. This was supported by subsequent data showing a downregulation of this "feeding" receptor in the brain of the obese Zucker rat (Widdowson, 1997). We have performed a detailed analysis of Y5 expression in rat brain using in situ hybridization histochemistry with digoxygenin-labeled riboprobes and compared this to expression of Y5 in human brain regions. mRNA for the human Y5 receptor was highly expressed in human hypothalamic and thalamic nuclei. In particular, the arcuate and paraventricular nuclei of the hypothalamus, midline thalamic nuclei, and amygdala showed very high levels of expression with high levels in hippocampus. The striking conservation of expression of the rat and human Y5 receptors in relevant hypothalamic and other nuclei implies sharing of a major neuroendocrine functional role by this receptor.

Functional anatomy of intrinsic alertness: evidence for a fronto-parietal-thalamic-brainstem network in the right hemisphere.

Alertness, the most basic intensity aspect of attention, probably is a prerequisite for the more complex and capacity demanding domains of attention selectivity. Behaviorally, intrinsic alertness represents the internal (cognitive) control of wakefulness and arousal; typical tasks to assess optimal levels of intrinsic alertness are simple reaction time measurements without preceding warning stimuli. Up until now only parts of the cerebral network subserving alertness have been revealed in animal, lesion, and functional imaging studies. Here, in a 15O-butanol PET activation study in 15 right-handed young healthy male volunteers for this basic attention function we found an extended right hemisphere network including frontal (anterior cingulate-dorsolateral cortical)-inferior parietal-thalamic (pulvinar and possibly the reticular nucleus) and brainstem (ponto-mesencephalic tegmentum, possibly involving the locus coeruleus) structures, when subjects waited for and rapidly responded to a centrally presented white dot by pressing a response key with the right-hand thumb.

Episodic retrieval activates the precuneus irrespective of the imagery content of word pair associates. A PET study.

The aim of this study was to evaluate further the role of the precuneus in episodic memory retrieval. The specific hypothesis addressed was that the precuneus is involved in episodic memory retrieval irrespective of the imagery content. Two groups of six right-handed normal male volunteers took part in the study. Each subject underwent six [15O]butanol-PET scans. In each of the six trials, the memory task began with the injection of a bolus of 1500 MBq of [15O]butanol. For Group 1, 12 word pair associates were presented visually, for Group 2 auditorily. The subjects of each group had to learn and retrieve two sets of 12 word pairs each. One set consisted of highly imaginable words and another one of abstract words. Words of both sets were not related semantically, representing 'hard' associations. The presentations of nonsense words served as reference conditions. We demonstrate that the precuneus shows consistent activation during episodic memory retrieval. Precuneus activation occurred in visual and auditory presentation modalities and for both highly imaginable and abstract words. The present study therefore provides further evidence that the precuneus has a specific function in episodic memory retrieval as a multimodal association area.

GPR56, a novel secretin-like human G-protein-coupled receptor gene.

A novel gene product, GPR56, with homology to the seven transmembrane-domain receptor superfamily, has been cloned by PCR amplification using degenerate oligonucleotide primers and subsequent screening of a human heart cDNA library. The isolated 2.8-kb cDNA clone encodes a protein of 693 amino acids that shows highest identity (32%) to HE6, a member of a subclass of the class B secretin-like G-protein-coupled receptors. Northern analysis of various human tissues revealed a wide distribution of the transcript with highest levels found in thyroid gland, brain, and heart. In situ hybridization analysis of human thyroid gland as well as rat heart and brain tissue confirms these results and identifies the hippocampus and hypothalamic nuclei as brain areas with particularly high expression of GPR56 mRNA. The high level of mRNA expression, its wide distribution, and the mucin-like extracellular domain of the receptor protein suggest a possible role for this receptor in cell-cell interaction processes. The human gene for GPR56 has been isolated and its exon-intron structure determined. The total length of the human GPR56 gene is approximately 15 kb, and it consists of 13 exons. Fluorescence in situ hybridization, PCR analysis of somatic cell hybrids, and interspecific mouse backcross mapping have localized the genes to human chromosome 16q13 and mouse chromosome 8.

Representations of graphomotor trajectories in the human parietal cortex: evidence for controlled processing and automatic performance.

The aim of this study was to identify the cerebral areas activated during kinematic processing of movement trajectories. We measured regional cerebral blood flow (rCBF) during learning, performance and imagery of right-hand writing in eight right-handed volunteers. Compared with viewing the writing space, increases in rCBF were observed in the left motor, premotor and frontomesial cortex, and in the right anterior cerebellum in all movement conditions, and the increases were related to mean tangential writing velocity. No rCBF increases occurred in these areas during imagery. Early learning of new ideomotor trajectories and deliberately exact writing of letters both induced rCBF increases in the cortex lining the right intraparietal sulcus. In contrast, during fast writing of overlearned trajectories and in the later phase of learning new ideograms the rCBF increased bilaterally in the posterior parietal cortex. Imagery of ideograms that had not been practised previously activated the anterior and posterior parietal areas simultaneously. Our results provide evidence suggesting that the kinematic representations of graphomotor trajectories are multiply represented in the human parietal cortex. It is concluded that different parietal subsystems may subserve attentive sensory movement control and whole-field visuospatial processing during automatic performance.

The influence of image resolution on the positron emission tomographic measurement of caudate glucose consumption.

The aim of this study was to investigate the influence of image resolution on (a) relative and absolute values of caudate glucose consumption (rCMRGlc) determined by positron emission tomography (PET), and (b) the detection of significant differences in these metabolic values between groups of subjects. For this purpose, raw data of cerebral accumulation of fluorine-18 fluorodeoxyglucose (FDG) obtained in 11 normal subjects and in nine patients with unilateral thalamic infarction were reconstructed using filtered backprojection with four different cut-off frequencies (CFs), yielding images with a transaxial resolution of 5.7, 7.1, 8.9 and 11 mm (full-width at half-maximum; FWHM). Absolute values of caudate rCMRGlc decreased significantly by more than 30% over the range of image resolutions studied. Bilateral ratios of caudate rCMRGlc were insensitive to variations in image resolution. Levels of significance assessing the differences in mean metabolic values between patients and controls were all below 0.01. They were, however, slightly better at image resolutions of 7.1 and 8.9 mm than at a resolution of 5.7 mm. These data indicate (a) that relative values of rCMRGlc are better suited to compare quantitative results from different PET cameras than are absolute values, and (b) that the CF used for the filtered back-projection exerts a small but not negligible influence on levels of significance assessing differences in metabolic values between groups of subjects.

Cortical activation in profoundly deaf patients during cochlear implant stimulation demonstrated by H2(15)O PET.

Cochlear implants (CIs) are used to provide sensations of sound to profoundly deaf patients. The performance of the CI is assessed mainly by the subjective reports of patients. The aim of this study was to look for objective cortical responses to the stimulation of the CI. Two postlingually and two prelingually deaf patients were investigated by positron emission tomography (PET) using 15O-labeled water (H2(15)O) to determine the regional cerebral blood flow (rCBF). Instead of quantifying rCBF in absolute terms, it was estimated by referring the regional tissue concentration of H2(15)O to the mean whole brain concentration. CI stimulation encoded from white noise and sequential words led to an increased rCBF in the primary and secondary (Wernicke) auditory cortex. Relative elevations of up to 33% were observed bilaterally, although they were higher contralateral to the CI. These results were obtained not only in the postlingually deaf patients but also in two patients who had never been able to hear. Thus, it could be demonstrated that PET measurements of cerebral H2(15)O distribution yield objective responses of the central auditory system during electrical stimulation by CIs in profoundly deaf patients.

Changed pattern of regional glucose metabolism during yoga meditative relaxation.

Using positron emission tomography (PET), measurements of the regional cerebral metabolic rate of glucose (rCMRGlc) are able to delineate cerebral metabolic responses to external or mental stimulation. In order to examine possible changes of brain metabolism due to Yoga meditation PET scans were performed in 8 members of a Yoga meditation group during the normal control state (C) and Yoga meditative relaxation (YMR). Whereas there were intraindividual changes of the total CMRGlc, the alterations were not significant for intergroup comparison; specific focal changes or changes in the interhemispheric differences in metabolism were also not seen; however the ratios of frontal vs. occipital rCMRGlc were significantly elevated (p less than 0.05) during YMR. These altered ratios were caused by a slight increase of frontal rCMRGlc and a more pronounced reduction in primary and secondary visual centers. These data indicate a holistic behavior of the brain metabolism during the time of altered state of consciousness during YMR.