Endocrine, behavioral and autonomic effects of neuropeptide AF

The actions of neuropeptide AF (NPAF), on the hypothalamic-pituitary-adrenal (HPA) axis, behavior and autonomic functions were investigated. NPAF (0.25, 0.5, 1, 2 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open-field (OF) observations and elevated plus-maze (EPM) tests. The temperature and heart rate were recorded by telemetry, and the plasma ACTH and corticosterone levels were used as indices of the HPA activation. The dopamine release from striatal and amygdala slices after peptide treatment (100 nM and 1 μM) was measured with a superfusion apparatus. To establish the transmission of the HPA response, animals were pretreated with the corticotrophin-releasing hormone (CRH) receptor antagonist antalarmin or astressin 2B (0.5 nmol). In the OF test, the animals were pretreated with antalarmin or haloperidol (10 μg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). NPAF stimulated ACTH and corticosterone release, which was inhibited by antalarmin. It activated exploratory locomotion (square crossings and rearings) and grooming in OF observations, and decreased the entries to and the time spent in the open arms during the EPM tests. The antagonists inhibited the locomotor responses, and also attenuated grooming and the EPM responses. NPAF also increased spontaneous locomotion, and tended to decrease the core temperature and the heart rate in telemetry, while it augmented the dopamine release from striatal and amygdala slices. These results demonstrate, that acute administration of exogenous NPAF stimulates the HPA axis and behavioral paradigms through CRH and dopamine release.     

Reduced anxiety-like and depression-related behavior in neuropeptide Y Y4 receptor knockout mice

Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4−/−) mice with control and germ line Y2−/− animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4−/− and Y2−/− mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4−/− but reduced in Y2−/− mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4−/− and Y2−/− mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4−/− and Y2−/− mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2−/− but not in Y4−/− mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.     

Cardiac function in neuropeptide Y Y4 receptor-knockout mice

Autonomic control of cardiovascular function in neuropeptide Y (NPY) Y4 receptor-knockout mice was investigated using pancreatic polypeptide (PP), NPY and specific agonists and antagonists for other NPY receptors well characterised in cardiovascular function. Y4 receptor-knockout mice, anaesthetised with sodium pentobarbitone, displayed slower heart rate, indicated by a higher pulse interval and lower blood pressure compared to control mice. After vagus nerves were cut heart rate increased but was still significantly slower than in control mice. PP had no effect on blood pressure or cardiac vagal activity in either group of mice, which was consistent with earlier studies in other species. Injection of NPY evoked an increase in blood pressure but the response was significantly reduced in Y4 receptor-knockout mice compared to the controls. The reduction in pressor activity was not Y1 mediated as the selective Y1 antagonist, BIBP 3226, was effective in blocking NPY pressor activity in knockout mice. In addition, cardiac vagal inhibitory activity evoked by low doses of NPY was also reduced when compared to control responses. As N-acetyl [Leu(28, 31)] NPY 24-36 inhibited vagal activity dose dependently in both groups of mice with no difference in response at any dose, it is unlikely that this effect also is receptor mediated. We propose that the reduced vasoconstrictor and vagal inhibitory activity evoked by NPY in Y4 receptor-knockout mice is due to a lack of adrenergic tone bought about by a proposed reduction in sympathetic activity, possibly resulting from altered NPY activity secondarily affecting adrenergic transmission. We conclude that Y4 receptor deletion disrupts autonomic balance within the cardiovascular system.     

New knockout model confirms a role for androgen receptors in regulating anxiety-like behaviors and HPA response in mice

Men are less likely than women to suffer from anxiety disorders. Because gonadal hormones play a crucial role in many behavioral sex differences, they may underlie sex differences in human anxiety. In rodents, testosterone (T) exerts anxiolytic effects via the androgen receptor (AR): we found that male mice with a naturally-occurring mutation rendering the AR dysfunctional, referred to as spontaneous testicular feminization mutation (sTfm), showed more anxiety-like behaviors than wildtype (WT) males. Here, we used Cre–lox recombination technology to create another dysfunctional allele for AR. These induced Tfm (iTfm) animals also displayed more anxiety-like behaviors than WTs. We further found that AR-modulation of these behaviors interacts with circadian phase. When tested in the resting phase, iTfms appeared more anxious than WTs in the open field, novel object and elevated plus maze tests, but not the light/dark box. However, when tested during the active phase (lights off), iTfms showed more anxiety-related behavior than WTs in all four tests. Finally, we confirmed a role of T acting via AR in regulating HPA axis activity, as WT males with T showed a lower baseline and overall corticosterone response, and a faster return to baseline following mild stress than did WT males without T or iTfms. These findings demonstrate that this recombined AR allele is a valuable model for studying androgenic modulation of anxiety, that the anxiolytic effects of AR in mice are more prominent in the active phase, and that HPA axis modulation by T is AR dependent.     

Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice

Which of Y1-Y5 receptors (Rs) mediate NPY's angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.     

Leptin prevents the fall in plasma osteocalcin during starvation in male mice

Plasma osteocalcin, a marker of osteoblastic activity, is reduced in starvation, malnutrition, and anorexia nervosa, resulting in low bone turnover osteoporosis. Contradictory findings about the role of leptin as a link between nutritional status and bone physiology have been reported. We demonstrate that leptin-deficient ob/ob and leptin-resistant db/db male mice have increased plasma osteocalcin, and that in male ob/ob mice osteocalcin is not decreased by starvation, unlike control mice. Intraperitoneal leptin administration increased plasma osteocalcin in male ob/ob mice, and prevented its fall during 24h fasting and 5 days of food restriction in normal male mice. This effect may be mediated via actions on the hypothalamic-pituitary-testicular or -growth hormone axes, or a direct action on osteoblasts. These studies support the hypothesis that the fall in leptin during starvation and weight loss is responsible for the associated reduction in osteoblast activity, and suggest a role for leptin in regulating bone turnover.     

5-羟色胺转运体敲除小鼠的分子和细胞改变

5-羟色胺转运体(5-HTT)在神经精神心理正常功能的维持及疾病的发生和发展中起重要作用。5-HTT的表达能力减低或消失的小鼠(称为:5-HTT敲除小鼠)表现出许多行为的改变,例如:焦虑类似行为增多、对应激更加敏感和攻击性行为减少。这些行为的改变有的与携带5-HTTLPR短等位基因的人很相似。因此5-HTT敲除小鼠被作为研究5-HTTLPR多态性导致情感性精神障碍发病机制的动物模型。本文主要就5-HTT敲除小鼠的5-HT浓度和代谢、下丘脑-垂体-肾上腺皮质轴以及对其他神经递质转运体影响的分子和细胞改变进行综述。     

雌性黑线仓鼠免疫功能的季节变化

温带地区动物的免疫功能常表现出季节性变化.本研究以捕获于2014年秋季、冬季和2015年春季、夏季的雌性黑线仓鼠为研究对象,通过注射植物血球凝集素(PHA)后足垫的肿胀程度反映细胞免疫能力;注射抗原匙孔血蓝蛋白(KLH)后,利用酶联免疫吸附(EHSA)方法检测血清免疫球蛋白IgG和IgM的浓度,反映体液免疫功能;检验冬...     

Endocrine status affects bladder size and postvoid residual urinary volume in mice

Urine is one of the major media for intraspecific chemical communication in mice. The urination pattern is dependent both on the mice's hormonal and social status. The urination pattern and the morphology of the urinary tract were examined in mice following hormonal manipulations. In the first experiment, we compared pairs of intact and castrated males: intact males urinated earlier when exposed to a new environment, with a greater number of drops that were smaller than those of castrated males. In the second experiment, groups of intact males, castrated, testosterone-supplemented castrated, and isolated intact males were compared. The micturition pattern of isolated intact males consisted of numerous small droplets of urine, with a high volume of urine retained in the bladder after voiding. This also applied to grouped intact males and testosterone-treated castrated mice, while castrated mice voided a larger fraction of bladder content. Bladder weight was higher in intact males and testosterone-treated castrated males, as compared to castrated males. In the third experiment, ovary-intact and testosterone-treated intact females were compared. Testosterone-treated ovary-intact females retained a larger quantity of urine in the bladder and also had a larger bladder compared to ovary-intact females. Testosterone thus induces the morphological modifications of the urinary tract necessary for the dominant male urination pattern, which is an increase in postvoid urinary residual volume and bladder weight. As evidenced from the comparison of histological sections from intact, castrated, and testosterone-treated castrated males, the increase in bladder weight was mainly due to the bladder muscular mass.     

Radioimmunoassay of neuropeptide Y

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.     

Increased brain neuropeptide Y1 and Y2 receptor binding in NPY knock out mice does not result in increased receptor function

The brain neuropeptide Neuropeptide Y (NPY) is an important modulator of a number of centrally mediated processes including feeding, anxiety-like behaviors, blood pressure and others. NPY produces its effects through at least four functional G-protein coupled receptors termed Y1, Y2, Y4 and Y5. In the brain, the Y1 and Y2 receptor subtypes are the predominant receptor population. To better understand the roles of NPY, genetically modified mice lacking NPY were produced but lacked the expected phenotypes. These mice have previously been reported to have a marked increase in Y2 receptor binding. In the present study, we found an upregulation of both Y1 and Y2 receptor binding and extended these findings to the female. These increases were as large as 10-fold or greater in many brain regions. To assess functional coupling of the receptors, we performed agonist-induced [(35)S]GTPgammaS autoradiography. In the mouse brain, the Y1/Y4/Y5 agonist Leu(31),Pro(34)-NPY increased [(35)S]GTPgammaS binding with a regional distribution consistent with that produced when labeling adjacent sections with [(125)I]-Leu(31),Pro(34)-PYY. In a few brain regions, minor increases were noted in the agonist-induced binding when comparing knock out mice to wild type. The Y2 agonist C2-NPY stimulated [(35)S]GTPgammaS binding in numerous brain areas with a regional distribution similar to the binding observed with [(125)I]-PYY3-36. Again, no major increases were noted in the functional activation of Y2 receptors between knock out and wild type mice. Therefore, the increased Y1 and Y2 binding observed in the NPY knock out mice does not represent an increase in NPY receptor mediated signaling and is likely due to an increase in spare (uncoupled) receptors.     

The adrenocortical stress-response of Black-legged Kittiwake chicks in relation to dietary restrictions

In this study we examined hormonal responses of Black-legged Kittiwake (Rissatridactyla) chicks to experimental variations in energy content and nutritional quality (low or high lipid to protein ratio, LPR) of their food. Starting at the age of 10 days, chicks were fed either high or low LPR fish at 30, 50, 70 and 100% of ad libitum energy intake. After 20 days of treatment, chicks were exposed to a standardized acute handling and restraint stress protocol, where a baseline sample was taken immediately after taking a chick from the nest, and three additional blood samples were taken at intervals up to 50 min. Testosterone and corticosterone titres in plasma were measured via radioimmunoassay. We found that baseline testosterone levels were not significantly affected by the experimental treatments. Food-restricted chicks had elevated baseline and acute stress-induced levels of corticosterone compared to chicks fed ad libitum. An elevation of circulating levels of corticosterone in energetically stressed individuals was further magnified by low nutritional quality of food. Baseline and acute stress-induced corticosterone levels of chicks were negatively correlated with their fat reserves. We conclude that the physiological condition of Black-legged Kittiwake chicks can be assessed reliably by measuring circulating levels of corticosterone. We discuss short- and long-term effects of elevated corticosterone secretion in food-stressed nest-bound chicks. Accepted: 13 April 1999     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Differential regulation of neuropeptide Y receptors in the brains of NPY knock-out mice

To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using (125)I-[Leu(31),Pro(34)]PYY and (125)I-PYY(3-36) as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60-400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.     

Thermoneutral conditions correct the obese phenotype in female,but not male,Kiss1r knockout mice

Kisspeptin, a neuropeptide that activates gonadotropin-releasing hormone (GnRH) neurons, has also been implicated as a regulator of energy balance. Kisspeptin receptor (Kiss1r) knockout (KO) mice display an obese phenotype in adulthood compared to wild-type (WT) controls due to reduced energy expenditure. Additionally, experimental evidence shows that the temperature of typical rodent housing conditions (22 °C) increases the metabolism of mice above basal levels. Female Kiss1r KO mice show reduced core temperature and impaired temperature adaptation to an acute cold challenge, suggesting their temperature homeostasis processes are altered. The present study examined the phenotype of gonadectomised Kiss1r KO mice at both sub-thermoneutral and thermoneutral temperature (22 °C and 30 °C). Our results confirmed the obese phenotype in Kiss1r KO mice at 22 °C, and revealed a sexually dimorphic effect of thermal neutrality on the phenotype. In female KO mice, the obesity observed at 22 °C was attenuated at 30 °C. Plasma leptin levels were higher in KO than WT female mice at 22 °C (P < 0.001) but not at 30 °C. Importantly, the expression of Ucp1 mRNA in brown adipose tissue was lower in KO mice compared to WT mice at 22 °C (P < 0.05), but not different from WT at 30 °C. In male KO mice, a metabolic phenotype was observed at 22 °C and 30 °C. These results provide further evidence for kisspeptin-mediated regulation of adiposity via altered energy expenditure. Moreover, thermoneutral housing alleviated the obese phenotype in female Kiss1r KO mice, compared to WT, indicating the impairment in these mice may relate to an inability to adapt to the chronic cold stress that is experienced at 22 °C.     

P27 knockout mice: reduced myostatin in muscle and altered adipogenesis

Knockout of the P27(kip) gene, which encodes a cyclin-dependent kinase inhibitor involved in cell proliferation regulation, results in growth enhancement in mice. To investigate how p27 deficiency affected adipogenesis and myogenesis, levels of PPARgamma, C/EBPalpha, and the myogenesis inhibitor, myostatin, were measured in p27(-/-) (n=14), p27(+/-) (n=18), and p27(+/+) mice (n=11). Body weight and gastrocnemius muscle (GC) mass were increased in p27(-/-) mice (P<0.05), but there were no differences in fat depot weights, percent body fat or serum leptin concentrations among genotypes. PPARgamma, but not C/EBPalpha, was markedly increased in p27(-/-) mice (P<0.05). There was also a higher incidence of inguinal fat apoptosis (P<0.01) in p27(-/-) mice. Myostatin levels were reduced in GC muscle of p27(-/-) mice (P<0.05). These findings suggest that in p27 deficient mice, increased skeletal muscle mass is mediated in part through decreased myostatin. Although total adiposity was not changed, increased PPARgamma levels suggest an alteration in adipogenesis.     

Influence of neuropeptide Y and pancreatic polypeptide on islet function and beta-cell survival

BackgroundIn the present study we assessed the impact of neuropeptide Y receptor (NPYR) modulators, neuropeptide Y (NPY) and pancreatic polypeptide (PP), on islet function and beta-cell survival.MethodsThe effects of NPY and PP on beta-cell function were examined in BRIN BD11 and 1.1B4 beta-cells, as well as isolated mouse islets. Involvement of both peptides in pancreatic islet adaptations to streptozotocin and hydrocortisone, as well as effects on beta-cell proliferation and apoptosis was also evaluated.ResultsNeither NPY nor PP affected in vivo glucose disposal or insulin secretion in mice. However, both peptides inhibited (p < 0.05 to p < 0.001) glucose stimulated insulin secretion from rat and human beta-cells. NPY exerted similar insulinostatic effects in isolated mouse islets. NPY and PP inhibited alanine-induced changes in BRIN BD11 cell membrane potential and (Ca^2 +)_i. Streptozotocin treatment decreased and hydrocortisone treatment increased beta-cell mass in mice. In addition, streptozotocin, but not hydrocortisone, increased PP cell area. Streptozotocin also shifted the normal co-localisation of NPY with PP, towards more pronounced co-expression with somatostatin in delta-cells. Both streptozotocin and hydrocortisone increased pancreatic exocrine expression of NPY. More detailed in vitro investigations revealed that NPY, but not PP, augmented (p < 0.01) BRIN BD11 beta-cell proliferation. In addition, both peptides exerted protective effects against streptozotocin-induced DNA damage in beta-cells.ConclusionThese data emphasise the involvement of PP, and particularly NPY, in the regulation of beta-cell mass and function.General significanceModulation of PP and NPY signalling is suitable for further evaluation and possible clinical development for the treatment of diabetes.     

Attenuated feeding responses to circadian and palatability cues in mice lacking neuropeptide Y

Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy−/−) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, we assessed food intake during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy−/− mice was reduced by 33% during this period (0.6 ± 0.1 g versus 0.9 ± 0.1 g; p ≤ 0.05). In contrast, intake did not differ between genotypes when measured over a 24-h period (3.7 ± 0.2 g versus 3.5 ± 0.3 g; p = ns). Furthermore, reduced dark cycle 4 h food intake in Npy−/− mice was not evident after a 24-h fast (1.4 ± 0.1 g for both genotypes; p = ns), despite a pronounced delay in the initiation of feeding (636 ± 133 s versus 162 ± 29 s; p ≤ 0.05). To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1 h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source (41 ± 3%). By comparison, HP intake was markedly reduced in Npy−/− mice during the first week (24 ± 7% of daily energy intake, p ≤ 0.05 versus Npy+/+), although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction.