Interrelationships between Cellular Density,Mosaic Patterning,and Dendritic Coverage of VGluT3 Amacrine Cells

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax-mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax-KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population.SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina.     

ONRAB® oral rabies vaccine is shed from,but does not persist in,captive mammals

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.     

鼠巨细胞病毒感染C57BL/6小鼠急性肝炎动物模型的建立与鉴定

目的：建立鼠巨细胞病毒（MCMV）感染C57BL/6小鼠急性肝炎模型并对其感染特点进行分析及鉴定。方法：将24 只C57BL/6小鼠随机分为阴性对照组（n =12）及病毒感染组（n =12），病毒感染组腹腔注射1.0×106 PFU（200 μL）MCMV悬液，阴性对照组注射等体积小鼠胚胎成纤维细胞（MEF）悬液。于感染后第3天和第7天取外周血分离血清检测谷丙转氨酶（ALT）及谷草转氨酶（AST）。同时进行肝组织病毒分离、组织病理学及MCMV IE和M55基因、细胞因子白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子α（TNF-α）的检测。结果：病毒感染组肝组织匀浆病毒分离均为阳性，肝炎发生率为100%。在感染后第3天即发生肝炎病理改变，病毒感染组血清ALT及AST较阴性对照组明显升高（P ＜0.01）；病毒感染组肝脏HE染色第3天可见局灶性炎性细胞浸润及肝脏点灶状坏死，持续至第7天，Ishak评分较阴性对照组明显升高（P ＜0.01）；在感染后第3天病毒感染组肝组织内可检测到MCMV IE及M55基因，且在感染后第7天仍可测得IE基因；感染后第3天及第7天病毒感染组炎性细胞因子IL-6、TNF-α及IL-1β mRNA表达水平明显升高（P ＜0.05）。 结论：成功建立MCMV感染C57BL/6小鼠急性动物肝炎模型，其感染表现主要集中在急性感染前期。     

电子线照射对小鼠胰岛素生长因子相关蛋白表达的影响

目的：探讨电子线对胰岛素生长因子-1家族蛋白表达的影响，为研究辐射对肝脏的早期损伤提供依据。方法：采用电子线照射小鼠，照射剂量为1、2、4 Gy，用Western blot法检测照射后12、48和72 h小鼠肝脏中胰岛素生长因子1受体（IGF-1R）、胰岛素生长因子结合蛋白1（IGFBP1）、胰岛素生长因子结合蛋白4（IGFBP4）和胰岛素生长因子1（IGF-1）蛋白表达水平的改变。结果：照射剂量为1 Gy时，与对照组相比，IGF-1蛋白在照射后48 h时表达增加，IGFBP1、IGFBP4和IGF1R蛋白的表达在照射后12、48和72 h均表达减少。照射剂量为2 Gy时，IGF-1和IGFBP4蛋白在照射后48 h时表达增加，其余时间点表达减少。IGFBP1蛋白在照射后12 h时表达增加，48和72 h时表达减少。IGF1R蛋白在照射后3个时间点均呈现为表达减少。照射剂量4 Gy时，肝脏中IGF-1、IGFBP1和IGFBP4蛋白均表达减少，IGF1R蛋白在照射后12 h时表达增加，48、72 h时均表达减少（均为P < 0.05）。结论：电子线照射后小鼠肝脏中胰岛素生长因子-1家族蛋白多以下调表达为主，与前期基因表达的结果相一致，有可能作为反映放射性工作人员早期肝脏损伤的生物标志物之一。     

不同浓度氯氮平对小鼠空腹血糖和骨骼肌葡萄糖转运蛋白4基因表达的影响

目的探讨氯氮平对雄性C57BL/6小鼠空腹血糖和骨骼肌葡萄糖转运蛋白4(GLUT4)基因表达的影响。方法将63只雄性C57BL/6小鼠随机分为3组,每组21只,分别灌胃给予蒸馏水、氯氮平4mg/kg及氯氮平20mg/kg,于给药后3h、1周、4周以试纸法测定各组空腹血糖,用逆转录-聚合酶链反应测定GLUT4mRNA表达。结果(1)灌药后3h、1周氯氮平4mg/kg组和氯氮平20mg/kg组空腹血糖和GLUT4mRNA的表达与空白对照组相比,差异无统计学意义(P>0.05);(2)灌药后4周氯氮平4mg/kg组和20mg/kg组的空腹血糖值[(5.6±0.5)mmol/L和(5.8±0.5)mmol/L]高于空白对照组[(4.6±0.6)mmol/L],而GLUT4mRNA的表达(0.50±0.14和0.48±0.12)却低于空白对照组(0.85±0.27),差异均有统计学意义(P<0.01)。结论氯氮平可以慢性升高空腹血糖,降低GLUT4mRNA的表达,可能是抗精神病药长期应用后血糖升高的发生机制之一。     

大鼠脑组织单胺类递质及其代谢产物的检测方法研究

目的 :研究大鼠脑组织中单胺类递质及其代谢产物的高效液相反相离子对色谱测定法。方法 :采用LiChrosorbC18,10 μm色谱柱 ,流动相为甲醇 :水 (4 0 :60 ) ,含 0 .0 2 8g LEDTANa2 ,0 .15g LSDS ,0 .2ml LH2 SO4(pH 2 .5～ 3 ) ,荧光检测波长 :λEX=2 85 ,λEM=3 3 3。结果 :对 87只大鼠脑组织中 4种单胺类递质及其代谢产物的含量进行了同时测定 ,高香草酸 (HVA) 2 .5 0～ 40 .0 μg ml、去甲肾上腺素 (NE) 0 .0 1～ 0 .5 0 μg ml、多巴胺 (DA)0 .0 5～ 1.0 0 μg ml、5 羟色胺 (5 HT) 0 .0 2 5～ 0 .5 0 μg ml,峰面积与其含量呈良好的线性关系。 结论 :该法操作简便、快速、准确 ,为组织中单胺类递质及其代谢产物检测的一种理想方法 ,并适用于临床相关研究。     

DA→LEW大鼠肝脏急性排斥反应模型的技术改进与评价

目的:建立大鼠肝脏移植急性排斥反应模型并改进其手术技巧,对所建模型进行评价。方法:采用近交系雄性DA大鼠60只,Lewis大鼠120只,分为两组,其中A组30例:Lewis大鼠为供体(n=30),Lewis大鼠为受体;B组60例:DA大鼠为供体(n=60),Lewis大鼠为受体。采用改良“二袖套”法建立肝脏移植模型,观察手术成功率、平均存活时间、术后第1、3、5、7和10 d各时相点血清丙氨酸氨基转移酶(ALT)、总胆红素(TBIL)水平以及移植肝脏的病理学变化。结果:手术成功率为93.3%(84/90);总耗时(90.0±15.0)min;无肝期时间(14.2±2.6)min;平均存活时间A组超过100d,B组(16.2±1.4)d;术后第5 d B组血清ALT和TBIL升高,与A组比较差异非常显著(P<0.01);B组移植肝脏病理检查有明显的排斥反应,而A组没有。结论:DA→LEW(Lewis)大鼠的品系组合是稳定的肝脏移植急性排斥反应模型;通过改进手术技巧提高了手术成功率,为进一步研究提供技术保障。     

Neuron transplantation into mice hippocampus alters sensitivity to barbital narcosis

The role of several hippocampal innervations in the sensitivity to barbital-induced narcosis was studied in selected mice strains. The outbred and inbred mouse strains HS/Ibg, SABRA/HUC, C57BL, CBA/LAC, and BALB/c were tested for barbital-induced sleep (315 mg/kg). The relatively short sleeping HS/Ibg (HS) and the longest sleeping BALE/c (BALE) were chosen for further investigation. Cholinergic (ACh), serotonergic (5-HT), and noradrenergic (NE) innervations were studied in HS strain; whereas BALE, which possesses both an unusually high sensitivity to barbital and unique NE innervations in the cortex and hippocampus, was employed in a detailed study of the NE innervations. Transplantation of embryonic NE cells from the mouse embryo into the hippocampus of adult HS mice increased barbital narcosis by 65% (p < 0.05), whereas transplantation of 5-HT cells decreased barbital narcosis by 54% (p < 0.001). Transplantation of ACh cells had no significant effect on barbital-induced narcosis. BALB mice were subjected to NE cell transplantation into the hippocampus and cortex. Similarly to HS, BALB receiving NE transplants into their hippocampus slept 34% longer than control after barbital challenge (p < 0.025). Noradrenergic cell transplantation into frontal cortex had no effect on barbital sleep. The results suggest that (a) enhancement by neural grafting of the NE innervation to the hippocampus accentuates and enhancement of the 5-HT innervations attenuates the sensitivity to barbital narcosis, whereas ACh innervations have no effect on the sensitivity to barbital narcosis, and (b) the unusually high sensitivity of BALB mice to barbital may not be related to its unique NE innervations.     

Nucleus A10 dopaminergic neurons in inbred mouse strains: Firing rate and autoreceptor sensitivity are independent of the number of cells in the nucleus

Inbred mouse strains have different numbers of midbrain dopaminergic neurons; for example, BALB/cJ mice have 20–25% more neurons than CBA/J mice. As the number of cells decrease, for example in Parkinson's disease and in animals with midbrain dopaminergic cell lesions, the activity of their remaining cells increases. The purpose of the present experiment was to determine whether the functional properties of dopaminergic neurons in the ventral tegmental area (nucleus A10) differ in inbred mouse strains which possess different numbers of cells. The firing rate and autoreceptor sensitivity of A10 dopaminergic cells were examined in the in vitro slice preparation in BALB/cJ, C3H/HeJ, CBA/J, and DBA/2J mouse strains. It was observed that the autoreceptors on mouse dopaminergic neurons exhibit pharmacological properties of dopamine autoreceptors; activation of the autoreceptor produced a marked inhibition (50–70%) in cell firing rate by quinpirole (10⁻⁸ M), LY-141865 (10⁻⁷ M), (+)-3-(3-hydroxyphenyl)-N-n-propyl-piperidine (10⁻⁶ M), propyl-norapomorphine (10⁻⁵ M) and dopamine (10⁻⁴ M), and this inhibition was blocked or reversed by specific dopamine D2 receptor antagonists [(−) sulphide and spiroperidol, 10⁻⁶ M]. The baseline firing rates of the A10 cells did not differ among the four inbred strains [range 2.5 ± 0.2 (C3H/HeJ)−3.4 ± 0.3 (CBA/J) spikes/s ±SEM], and there was no significant difference in autoreceptor sensitivity among the mouse strains as assessed either by superfused dopamine (inhibitory dose 50% ≈150 μM), or by superfused quinpirole (inhibitory dose 50% ≈10 nM). These data indicate that differences in A10 cell numbers of 30% do not significantly influence the baseline firing rate or autoreceptor sensitivity of the cells.