Functional mapping of rat barrel activation following whisker stimulation using activity-induced manganese-dependent contrast

BACKGROUND: Cortical whisker barrels in the primary somatosensory cortex are a well-known example of brain function in rodents. The well-defined relationship between barrels and whiskers makes this system a unique model to study neuronal function and plasticity. In this study, we sought to establish a feasible working protocol of applying manganese-enhanced MRI (MEMRI) to map the cortical barrels following whisker stimulation. METHODS: The protocol was based on the principle of activity-induced manganese-dependent (AIM) contrast. Rats were prepared by sodium pentobarbital anesthetization, intraperitoneal manganese-chloride injection, right common carotid mannitol injection and temperature maintenance. Left whiskers were connected to a speaker through a cotton thread and were stimulated by a series of rectangular pulses. MEMRI was acquired with a 3T scanner 3 h after whisker stimulation. Before MR scanning, Wistar rats were euthanized to avoid motion artifacts. To improve the signal-to-noise ratio (SNR) and detection sensitivity, image coregistration, pixel intensity normalization, statistical mapping, group averaging and subtraction were performed. The AIM enhancement of the cortical barrels was quantified using volume of interest analysis on the acquired T1WI and R1 mapping. RESULTS: Both experimental and control groups showed greater enhancement in the right hemisphere, same side as mannitol injection. In the experimental group, however, activity-induced enhancement was more localized in the right barrel fields, whereas in the control group, the enhancement was uniform throughout the right cortex. In the right cortical barrels, the enhancement ratios and R1 values in the experimental group were significantly higher than those in the control group. In the left cortical barrels, there was no significant difference between the two groups. Subtracted images and voxel-based statistical t-value mapping between experimental and control groups showed additional enhancement concentrated in the right cortical barrels. CONCLUSIONS: We have mapped rat whisker barrels using the AIM method and have shown a clear relationship between manganese-enhanced cortical regions and whisker tactile-sense-evoked activity. It is possible that, with sufficient SNR, the AIM method may reach whisker barrel discrimination, potentially useful to study plasticity in surgically or genetically manipulated rat brains.     

胡须刺激通过调节神经可塑性 改善小鼠桶状皮质缺血性脑卒中后的神经功能缺损

目的：探讨规律胡须刺激是否可改善小鼠桶状皮质（Barrel Cortex）局灶性脑缺血后神经功能并从神经可塑性角度探讨其机制。方法：60只雄性C57BL/6随机分成假手术组、脑梗死组和胡须刺激组,每组20只。胡须刺激组在小鼠桶状皮质局灶性脑缺血模型建立后第3天开始给予规律胡须刺激（15 min/次,3次/d,共12 d）。3组小鼠均在造模后3、7、14 d进行贴纸去除试验;造模后14 d进行HomeCage行为学检测、采用Western blot技术检测脑梗死灶周围突触素（SPY）-1、生长相关蛋白43(GAP 43)、脑源性神经营养因子（BDNF）和神经丝（NF）蛋白表达水平,免疫荧光染色检测NF纤维。结果：脑梗死造模后3、7、14 d,脑梗死组小鼠的贴纸感知时间和贴纸去除时间均高于假手术组（均P<0.05）;造模后7 d,胡须刺激组小鼠的贴纸去除时间短于脑梗死组（P<0.05）;造模后14 d,胡须刺激组小鼠的贴纸感知时间和贴纸去除时间均短于脑梗死组（均P<0.05）。HomeCage行为学分析结果显示,与假手术组相比,脑梗死组和胡须刺激组小鼠活跃性指标均有明显下...     

Enhancement of repopulation haemopoiesis by heterozygous connexin 43 stem cells seeded on wild-type connexin 43 stroma

The present paper analyses the results of competitive blood-cell repopulation experiments in which Cx43-WT (connexin 43 wild-type) host mice, whose own HSCs (haemopoietic stem cells) were deleted, were grafted with fetal liver cells: 50% Gpi-1a (glucose phosphate isomerase-1a)/Cx43-WT cells competing with 50% Gpi-1b/Cx43-WT, 50% Gpi-1b/Cx43-HZ (heterozygous) or 50% Gpi-1b/Cx43-KO (knock-out) cells. The percentages of platelets, granulocytes, red cells, B-cells and T-cells containing Gpi-1b in blood samples obtained from 22 to 186 days after grafting, and the percentages of high-proliferation-potential colony-forming cells containing Gpi-1b at 255 days after grafting, were measured. The results show that, if we wait 4 months so that we measure the percentages of Gpi-1b end-cells formed by initially resting stem cells in the graft, values in HZ mice are greater than those in WT and KO mice by 10% or more. We propose a bipolar influence model for blood formation by grafted HSCs to explain this difference and other features of the data. Influence A is a direct one: for individual HSCs, the combined effect on HSC niching and HSC proliferation of Cx43 is superior to that of the KO allele. Influence B is a demographic one: HZ foundation mice compensate by having more HSCs than WT mice. The net outcome of influences A and B is that HZ is the winner.     

微管相关蛋白1B和突触素I在FMR1基因敲除小鼠脑组织中的表达及相关性

目的 了解FMR1基因敲除小鼠脑组织中微管相关蛋白1B的改变是否与突触的异常有关，MAP1B在脆性X综合征的发病机制中是否通过调节突触数量的改变起作用，以探讨脆性X综合征的发病机制。方法 将小鼠分为成年KO（KO6W）组、成年WT(WT6W)组、新生KO(KO1W)组、新生WT(WT1W)组4组，每组10只，用免疫组化法检测MAP1B及SYN的分布和表达。用图象分析仪分别对大脑皮质、海马及小脑DAB显色的平均光密度值（MOD）进行采集，分析不同基因型及不同年龄组MAP1B和SYN在各脑区MOD值的差异，以及二者之间的相关性。结果 KO6W组及KO1W组小鼠各个脑区中MAP1B与SYN的改变均无明显相关（p>0.05）。结论 FMR1基因敲除小鼠MAP1B与突触素Ⅰ的表达之间并无显著相关关系，提示MAP1B在脆性X综合征的发病机制中不是通过调节突触数量的改变起作用的。     

Experience-dependent formation of activity propagation patterns at the somatosensory S1 and S2 boundary in rat cortical slices

Somatosensory information is serially processed by the primary (S1) and secondary (S2) cortices, which can be identified in fresh cortical slices. We visualized activity propagation between S1 and S2 in rat cortical slices using flavoprotein fluorescence imaging. When S1 was stimulated, fluorescence responses extended into S2, while responses hardly propagated to S1 following S2 stimulation. The dominant activity propagation pattern from S1 to S2 was not affected by antagonists of glutamate or GABA(A) receptors. Ca(2+) imaging and electrophysiological recordings confirmed the anisotropic activity propagation pattern. This pattern could be formed as a result of serial information processing in S1 and S2. To test this hypothesis, activity propagation was investigated in cortical slices prepared 2 weeks or 3 days after trimming contralateral whiskers that provide massive inputs to S1. Supragranular activities in the barrel cortex were clearly suppressed. Furthermore, activities elicited in the rostral small vibrissae/mouth area of S1 near the border between S1 and S2 spread into the adjacent barrel cortex rather than into S2. Behavioral effects of whisker trimming were evaluated using a test, in which rats chose one of two bridges that had a wall on the right or left side only. Immediately after hemilateral whisker trimming, rats preferred to use the bridge with a wall close to the intact side. However, this preference disappeared 3 days after trimming. Modified activities observed in cortical slices after whisker trimming might be mechanisms for this behavioral compensation. These findings suggest experience-dependent formation of activity propagation patterns in the somatosensory cortex.     

Angiogenesis and angiotensin II: differential postnatal angiogenic activities unmasked by gene-engineered mouse models of angiotensin AT1 and AT2 receptor subtypes

We tested angiogenic activities of angiotensin II(Ang II) in ischemic hindlimbs using AT1 receptor(AT1R)-knock out(KO), AT2R-KO, wild-type(WT) mice. METHODS AND RESULTS: Angiogenesis was evaluated three weeks after unilateral hindlimb ischemia by laser Doppler perfusion(LDP) and capillary density. The ischemia/normal LDP ratio was markedly(p < 0.001) decreased in AT1R-KO(54 +/- 5% recovery) and AngII infusion-AT1R-KO(43 +/- 3%) than in WT(71 +/- 6%). In contrast, ischemia/normal LDP ratio was significantly(p < 0.01) increased in AT2R-KO(82 +/- 5%) and AngII infusion-AT2R-KO(96 +/- 6%) than in WT(71 +/- 6%). AT1R-KO and AngII infusion -AT1R-KO mice displayed lower capillary densities than WT(15 +/- 3, 11 +/- 3 vs 24 +/- 3 per field; p < 0.001). CONCLUSION: Ischemia in skeletal muscle causes upregulation of AT1R and AT2R expression, which positively and negatively modulates VEGF expression. This VEGF regulation via AngII receptor subtypes is closely involved in postnatal angiogenesis in ischemic limbs.     

Transcranial Functional Ultrasound Imaging in Freely Moving Awake Mice and Anesthetized Young Rats without Contrast Agent

Functional ultrasound (fUS) imaging by ultrasensitive Doppler detection of blood volume was previously reported to measure adult rat brain activation and functional connectivity with unmatched spatiotemporal sampling (100 μm, 1 ms), but skull-induced attenuation of ultrasonic waves imposed skull surgery or contrast agent use. Also, fUS feasibility remains to be validated in mice, a major pre-clinical model organism. In the study described here, we performed full-depth ultrasensitive Doppler imaging and 3-D Doppler tomography of the entire mouse brain under anesthesia, non-invasively through the intact skull and skin, without contrast agents. Similar results were obtained in anesthetized young rats up to postnatal day 35, thus enabling longitudinal studies on postnatal brain development. Using a newly developed ultralight ultrasonic probe and an optimized ultrasonic sequence, we also performed minimally invasive full-transcranial fUS imaging of brain vasculature and whisker stimulation-induced barrel cortex activation in awake and freely moving mice, validating transcranial fUS for brain imaging, without anesthesia-induced bias, for behavioral studies.     

Vasodilator mRNA levels are increased in the livers of portal hypertensive NO-synthase 3-deficient mice

BACKGROUND/AIMS: Nitric oxide synthase (NOS) 3-deficient (NOS-3 KO) mice have an increased systemic arterial pressure but develop portal hypertension to the same extent as wildtype (WT) mice. We hypothesized that other vasodilators in the portal circulation compensate for the lack in NOS-3 activity. We used quantitative PCR as a screening method to identify mediators that possibly compensate for NOS-3 in NOS-3 KO mice. METHODS: Mean arterial pressure (MAP) and portal venous pressure (PVP) were measured in the anaesthetized animal. mRNA levels in whole liver tissue were determined by quantitative RT-PCR. RESULTS: NOS-3 KO mice had a significantly higher mean arterial pressure than WT mice, but portal venous pressure did not differ. Bile duct ligation (BDL) induced a drop in MAP and a rise in PVP in both groups. Bile duct ligation induced a significant increase in mRNA levels of the cannabinoid receptor (CB)-1, adrenomedullin and NOS-2 in the liver of NOS-3 KO and WT mice. Nitric oxide synthase-1 and NOS-3 mRNA levels were elevated in BDL WT mice compared with sham-operated WT mice. Higher mRNA levels of CB-1, NOS-1 and the adrenomedullin receptor were found in sham-operated NOS-3 KO mice compared with sham-operated WT mice. CONCLUSIONS: We used quantitative PCR as a screening method to identify vasodilative mediators that might be involved in the compensation for the lack of NOS-3 activity in NOS-3 KO mice. Elevated mRNA levels in sham-operated NOS-3 KO mice compared with sham-operated WT mice were demonstrated for CB-1, NOS-1 and the adrenomedullin receptor.     

Mu-opioid receptors selectively regulate basal inhibitory transmission in the central amygdala: lack of ethanol interactions

Endogenous opioid systems are implicated in the actions of ethanol. For example, mu-opioid receptor (MOR) knockout (KO) mice self-administer less alcohol than the genetically intact counterpart wild-type (WT) mice (Roberts et al., 2000). MOR KO mice also exhibit less anxiety-like behavior than WT mice (Filliol et al., 2000). To investigate the neurobiological mechanisms underlying these behaviors, we examined the effect of ethanol in brain slices from MOR KO and WT mice using sharp-electrode and whole-cell patch recording techniques. We focused our study in the central nucleus of the amygdala (CeA) because it is implicated in alcohol drinking behavior and stress behavior. We found that the amplitudes of evoked inhibitory postsynaptic currents (IPSCs) or inhibitory postsynaptic potentials (IPSPs) were significantly greater in MOR KO mice than WT mice. In addition, the baseline frequencies of spontaneous and miniature GABA(A) receptor-mediated inhibitory postsynaptic currents were significantly greater in CeA neurons from MOR KO than WT mice. However, ethanol enhancements of evoked IPSP and IPSC amplitudes and the frequency of miniature IPSCs were comparable between WT and MOR KO mice. Baseline spontaneous and miniature excitatory postsynaptic currents (EPSCs) and ethanol effects on EPSCs were not significantly different between MOR KO and WT mice. Based on knowledge of CeA circuitry and projections, we hypothesize that the role of MOR- and GABA receptor-mediated mechanisms in CeA underlying reinforcing effects of ethanol operate independently, possibly through pathway-specific responses within CeA.     

A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis

We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.     

Connexin43-dependent mechanism modulates renin secretion and hypertension

To investigate the function of Cx43 during hypertension, we studied the mouse line Cx43KI32 (KI32), in which the coding region of Cx32 replaces that of Cx43. Within the kidneys of homozygous KI32 mice, Cx32 was expressed in cortical and medullary tubules, as well as in some extra- and intraglomerular vessels, i.e., at sites where Cx32 and Cx43 are found in WT mice. Under such conditions, renin expression was much reduced compared with that observed in the kidneys of WT and heterozygous KI32 littermates. After exposure to a high-salt diet, all mice retained a normal blood pressure. However, whereas the levels of renin were significantly reduced in the kidneys of WT and heterozygous KI32 mice, reaching levels comparable to those observed in homozygous littermates, they were not further affected in the latter animals. Four weeks after the clipping of a renal artery (the 2-kidney, 1-clip [2K1C] model), 2K1C WT and heterozygous mice showed an increase in blood pressure and in the circulating levels of renin, whereas 2K1C homozygous littermates remained normotensive and showed unchanged plasma renin activity. Hypertensive, but not normotensive, mice also developed cardiac hypertrophy. The data indicate that replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin, thus preventing the renin-dependent hypertension that is normally induced in the 2K1C model.     

H+-ATPase activity in selective disruption of H+-K+-ATPase alpha 1 gene of mice under normal and K-depleted conditions

The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by two luminal proton ATPases, H(+)-ATPase and H(+)-K(+)-ATPase (HKA), both of which are in the intercalated cells (ICs) of OMCD. We showed previously that HKAalpha1 (gastric H(+)-K(+)-ATPase) activity is the essential H(+)-K(+)-ATPase activity under normal conditions, and that HKAalpha2 (colonic H(+)-K(+)-ATPase) is induced and mediates increased proton-secretion under K-depleted conditions. To better understand the role of H(+)-ATPase (potassium-independent) in acid secretion and the relationship between H(+)-ATPase and a specific HKA isoform, we examined H(+)-ATPase activity in the H(+)-K(+)-ATPasealpha1 knockout (KO) mice under normal and K-depleted conditions. Mice were fed a potassium-free diet and studied after 7 days. Segments of the OMCD were perfused in vitro, and intracellular pH (pH(i)) was measured by ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. The isolated OMCD tubules obtained from mice fed a potassium-free diet were examined by fluorescent immunocytochemistry with an antibody to the 31-kDa subunit of H(+)-ATPase (E-11) and were compared with those obtained from a normal diet. In the absence of Na(+) and K(+), the H(+)-ATPase-mediate pH(i) recovery rates were 6.7 +/- 1.1 x 10(-4) units/s (n = 7 ICs) in wild-type (WT) mice and increased to 8.7 +/- 1.8 x 10(-4) (P < 0.05; n = 6) in HKAalpha1 KO mice. K-independent proton transport activity was significantly inhibited by the H(+)-ATPase inhibitor bafilomycin A(1) (BAF, 10 nM) with luminal applied in both WT and KO mice. Comparison of the results indicated upregulation of BAF-sensitive H(+)-ATPase activity in KO mice. To determine the intracellular localization of H(+)-ATPase in the intercalated cells of OMCD, we dissected the OMCD and performed fluorescent immunocytochemistry with the H(+)-ATPase antibody in the WT and KO mice. In the WT mice, on normal diet, H(+)-ATPase staining distributed diffusely throughout the intercalated cells and was slightly polarized to the apical plasma membrane in the KO mice, consistent with increase in the H(+)-ATPase-mediate pH(i) recovery in the KO mice. One week of a potassium-free diet resulted in a significant increase in the degree of H(+)-ATPase polarization at the apical plasma membrane in both WT and KO mice. Hypokalemia stimulates H(+)-ATPase in the intercalated cells of OMCD of both WT and KO mice. The enhanced activity of H(+)-ATPase plays an important role in compensatory proton secretion in the HKAalpha1 KO mice under normal conditions.     

Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis

We have previously demonstrated that interleukin (IL)-10-deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4(+) T cells from IL-10 KO animals and that the cotransfer of CD4(+) T cells from H. hepaticus-infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4(+) cells are contained within the CD45RB(low) fraction and unexpectedly were found in both the CD25(+) and the CD25(-) subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25(+) and CD25(-) CD45RB(low) CD4(+) cells block colitis involves IL-10 and not transforming growth factor (TGF)-beta, as treatment with anti-IL-10R but not anti-TGF-beta monoclonal antibody abrogated their protective effect. In vitro, CD45RB(low) CD4(+) cells from infected WT mice were shown to produce IL-10 and suppress interferon-gamma production by IL-10 KO CD4(+) cells in an H. hepaticus antigen-specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.     

Impaired kisspeptin signaling decreases metabolism and promotes glucose intolerance and obesity

The neuropeptide kisspeptin regulates reproduction by stimulating gonadotropin-releasing hormone (GnRH) neurons via the kisspeptin receptor KISS1R. In addition to GnRH neurons, KISS1R is expressed in other brain areas and peripheral tissues, which suggests that kisspeptin has additional functions beyond reproduction. Here, we studied the energetic and metabolic phenotype in mice lacking kisspeptin signaling (Kiss1r KO mice). Compared with WT littermates, adult Kiss1r KO females displayed dramatically higher BW, leptin levels, and adiposity, along with strikingly impaired glucose tolerance. Conversely, male Kiss1r KO mice had normal BW and glucose regulation. Surprisingly, despite their obesity, Kiss1r KO females ate less than WT females; however, Kiss1r KO females displayed markedly reduced locomotor activity, respiratory rate, and energy expenditure, which were not due to impaired thyroid hormone secretion. The BW and metabolic phenotype in Kiss1r KO females was not solely reflective of absent gonadal estrogen, as chronically ovariectomized Kiss1r KO females developed obesity, hyperleptinemia, reduced metabolism, and glucose intolerance compared with ovariectomized WT females. Our findings demonstrate that in addition to reproduction, kisspeptin signaling influences BW, energy expenditure, and glucose homeostasis in a sexually dimorphic and partially sex steroid–independent manner; therefore, alterations in kisspeptin signaling might contribute, directly or indirectly, to some facets of human obesity, diabetes, or metabolic dysfunction.     

Lateralization of responses to vibrissal stimulation: connectivity and information integration in the rat sensory-motor cortex assessed with fMRI

Rats move their whiskers or vibrissae to gain sensory information about the world surrounding them. A single whisker can work as an independent detector but normal whisking involves the use of several vibrissae in a bilateral fashion. Here we used blood oxygen level dependent (BOLD) contrast to acquire functional magnetic resonance images (fMRI) of the rat brain activity during uni- and bilateral whisker stimulations with different timing schemes under Isoflurane anesthesia. Experiments were performed to assess the integration of bilateral information produced by normal whisking behavior. First, we showed that it was possible to obtain BOLD whisker activations using Isoflurane harmless for the animals and thus allowing for future repetitive/longitudinal studies. Second, we obtained different BOLD activation patterns depending on the number of stimulated whiskers and timing of the stimulation scheme. Third, we found lateralization of BOLD activations in the somatosensory-motor cortex. It manifested itself in considerably larger activations in the right hemisphere during equal bilateral whisker stimulation. Fourth, we found Granger Causality Analysis (GCA) to be a useful tool in information integration analysis, as it reproduced the stimulus specific Cross-correlation Analysis results. Both analyses showed that the amount of whiskers stimulated and the timing of stimulation lead to specific dynamic connectivity patterns. Finally, by adding directionality information GCA revealed meaningful lateralization of information processing in the rat whisker system consistent with the observed BOLD activation patterns.     

A role for thymic stromal lymphopoietin in CD4(+) T cell development

Thymic stromal lymphopoietin (TSLP) signals via a receptor comprising the interleukin (IL)-7 receptor alpha chain and a distinctive subunit, TSLP receptor (TSLPR), which is most related to the common cytokine receptor gamma chain, gamma(c). We have generated TSLPR knockout (KO) mice and found that although these mice had normal lymphocyte numbers, gamma(c)/TSLPR double KO mice had a greater lymphoid defect than gamma(c) KO mice. This indicates that TSLP contributes to lymphoid development and accounts for some of the residual lymphoid development in gamma(c) KO mice and presumably in patients with X-linked severe combined immunodeficiency. Injection of TSLP into gamma(c) KO mice induced the expansion of T and B cells. Moreover, sublethally irradiated TSLPR KO mice showed weaker recovery of lymphocyte populations than wild-type (WT) littermates, even when neutralizing anti-IL-7 antibodies were injected. Interestingly, TSLP preferentially stimulated the proliferation and survival of CD4(+) single positive thymocytes and peripheral T cells in vitro. Additionally, CD4(+) T cells from TSLPR KO mice expanded less efficiently than WT CD4(+) T cells in irradiated hosts, and TSLP preferentially expanded CD4(+) T cells both in vitro and in vivo. Thus, as compared with other known cytokines, TSLP is distinctive in exhibiting a lineage preference for the expansion and survival of CD4(+) T cells.     

特异性敲除Apelin基因对单侧输尿管梗阻小鼠肾间质纤维化的影响

目的观察多肽Apelin基因缺失对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的影响,探讨在梗阻性肾病肾纤维化形成中Apelin的作用和可能机制。方法利用CRISPR/Cas9技术构建Apelin基因敲除小鼠(KO)共20只,同时选取野生型小鼠(WT)20只,均为8周龄雄性C57BL/6J种系,采用随机数字表法分为:KO+Sham组、KO+UUO组、WT+Sham组、WT+UUO组,每组各10只。分别对小鼠行单侧输尿管结扎(UUO)或假手术(Sham)。术后2周将小鼠处死,取血浆和手术侧肾脏进行检测。采用Masson染色观察肾脏纤维化程度,免疫组织化学染色检测肾脏基质成分纤维连接蛋白(FN)、Ⅰ型胶原(Col-I)的分布和表达情况。采用Western blotting法检测肾小管上皮标志物E-钙黏素(E-cadherin)、间充质标志物α-平滑肌肌动蛋白(α-SMA),以及致纤维化细胞因子--转化生长因子-β1(TGF-β1)及其受体TGFβ-R1的蛋白表达量。采用酶联免疫吸附法(ELISA)检测小鼠血浆肌酐(Cr)、尿素氮(BUN)水平。结果KO+UUO组小鼠肾脏可见明显的肾小管萎缩和肾间质纤维化,基质成分FN和Col-I表达量显著升高。WT+UUO组小鼠肾脏的纤维化程度和基质的表达量均低于KO+UUO组,差异具有统计学意义(P<0.05)。KO+Sham组和WT+Sham组肾脏无明显的纤维化改变和基质的沉积。KO+UUO组小鼠,可见显著的肾小管上皮-间充质转分化(EMT)表现,E-cadherin减少,α-SMA增多,TGF-β1和TGFβ-R1受体的表达量也明显升高。与KO+UUO组相比,WT+UUO组肾小管上皮E-cadherin的表达较多,α-SMA、TGF-β1和TGFβ-R1受体的表达均较低,差异具有统计学意义(P<0.05)。而WT+Sham组和KO+Sham组小鼠上述标志物的表达趋于正常水平。KO+UUO组小鼠血浆Cr、BUN的水平最高,WT+UUO组小鼠上述标志物的水平均低于KO+UUO组,差异具有统计学意义(P<0.05)。KO+Sham组小鼠血浆Cr、BUN与WT+Sham组相比,差异无统计学意义(P>0.05)。结论Apelin在正常状态下对肾功能无明显影响,但在输尿管梗阻状态下其基因缺失可能是引起肾间质纤维化加重的因素。     

Presynaptic delta opioid receptors regulate ethanol actions in central amygdala

Endogenous opioid systems are implicated in the reinforcing effects of ethanol consumption. For example, delta opioid receptor (DOR) knockout (KO) mice show greater ethanol consumption than wild-type (WT) mice (Roberts et al., 2001). To explore the neurobiological correlates underlying these behaviors, we examined effects of acute ethanol application in brain slices from DOR KO mice using whole-cell patch recording techniques. We examined the central nucleus of amygdala (CeA) because the CeA is implicated in alcohol reinforcement (Koob et al., 1998). We found that the acute ethanol effects on GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) were greater in DOR KO mice than in WT mice. Ethanol increased the frequency of miniature IPSCs (mIPSCs) significantly more in DOR KO mice than in WT mice. In CeA of WT mice, application of ICI 174864 [[allyl]2-Tyr-alpha-amino-isobutyric acid (Aib)-Aib-Phe-Leu-OH], a DOR inverse agonist, augmented ethanol actions on mIPSC frequency comparable with ethanol effects seen in DOR KO mice. Superfusion of the selective DOR agonist D-Pen(2),D-Pen(5)-enkephalin decreased the mean frequency of mIPSCs; this effect was reversed by the DOR antagonist naltrindole. These findings suggest that endogenous opioids may reduce ethanol actions on IPSCs of CeA neurons in WT mice through DOR-mediated inhibition of GABA release and that the increased ethanol effect on IPSCs in CeA of DOR KO mice could be, at least in part, due to absence of DOR-mediated inhibition of GABA release. This result supports the hypothesis that endogenous opioid peptides modulate the ethanol-induced augmentation of GABA(A) receptor-dependent circuitry in CeA (Roberto et al., 2003).     

GSK3抑制剂氯化锂对FMR1基因敲除小鼠的自主活动的干预作用

目的:探讨GSK3抑制剂氯化锂对FMR1基因敲除小鼠的自主活动的干预作用及机制。方法:通过对90只30日龄FMR1基因敲除小鼠连续腹腔注射不同剂量氯化锂5d,行自主活动行为学实验,观察能否改善KO鼠的过度活动的表型;同时利用免疫印迹技术检测KO及WT鼠的海马和皮层的GSK3β和P-GSK3β的变化。结果:在自主活动行为学实验中,KO鼠的活动次数比WT鼠的活动次数增多而站立次数比WT鼠减少,差异具有统计学意义(P<0.05);使用氯化锂后,KO鼠的活动次数明显减少及站立次数明显增多,差异均有统计学意义(P<0.05);免疫印迹实验结果:KO鼠P-GSK3β表达比WT鼠少,用氯化锂后,KO鼠P-GSK3β的表达增多。KO鼠和WT鼠使用氯化锂后,总的GSK3β无明显改变(P>0.05)。结论:GSK3β的抑制剂氯化锂能改善KO鼠的活动过度的表型,可能与氯化锂导致的P-GSK3β的表达增加有关,对FMR1基因敲除小鼠有治疗作用。