Hypothalamic transcriptome plasticity in two rodent species reveals divergent differential gene expression but conserved pathways

We have addressed the question of how different rodent species cope with the life-threatening homeostatic challenge of dehydration at the level of transcriptome modulation in the supraoptic nucleus (SON), a specialised hypothalamic neurosecretory apparatus responsible for the production of the antidiuretic peptide hormone arginine vasopressin (AVP). AVP maintains water balance by promoting water conservation at the level of the kidney. Dehydration evokes a massive increase in the regulated release of AVP from SON axon terminals located in the posterior pituitary, and this is accompanied by a plethora of changes in the morphology, electrophysiological properties, biosynthetic and secretory activity of this structure. Microarray analysis was used to generate a definitive catalogue of the genes expressed in the mouse SON, and to describe how the gene expression profile changes in response to dehydration. Comparison of the genes differentially expressed in the mouse SON as a consequence of dehydration with those of the rat has revealed many similarities, pointing to common processes underlying the function-related plasticity in this nucleus. In addition we have identified many genes that are differentially expressed in a species-specific manner. However, in many cases, we have found that the hyperosmotic cue can induce species-specific alterations in the expression of different genes in the same pathway. The same functional end can be served by different means, via differential modulation, in different species, of different molecules in the same pathway. We suggest that pathways, rather than specific genes, should be the focus of integrative physiological studies based on transcriptome data. Animals. Adult male C57BL/6 mice (Harlan Sera-Lab, Loughborough, UK) were group housed (4 per cage) under controlled temperature (21+ 2ºC) and diurnal light conditions (14-h light, 10-h dark, lights on at 05.00). Food and water were available ad libitum until the experiment commenced. Complete fluid deprivation was imposed for 48 hours starting at 11.00 a.m. Control animals maintained free access to drinking water, and both groups had access to standard laboratory rodent chow. Experiments on adult male rats described previously (27). All procedures were conducted in strict accordance with the Animal Scientific Procedures Act (1986), UK, and were approved by the local University of Bristol Ethical Review Process. Tissue collection. Mice were killed using cervical dislocation and the brain was carefully removed from the cranium and snap frozen using powdered dry ice and stored at -80oC for no more than 14 days. Sections of brain (14ÎŒm) were cut using an RNase free cryostat and mounted onto RNase free membrane coated glass slides (P.A.L.M. Membrane slides; P.A.L.M. Microlaser Technologies). Immediately after sectioning, frozen sections were thawed and fixed (30s; in 95% [v/v] EtOH), rehydrated (30s in each of 75% [v/v] and 50% [v/v] EtOH) before being stained (60s 1% [v/v] cresyl violet). Sections were then dehydrated in a graded EtOH series (30s in each of 50% [v/v], 75% [v/v] and 95% [v/v]. then 2x 30s in 100% [v/v]). Laser microdissection was performed using a P.A.L.M. MicrolaserSystem (P.A.L.M. Microlaser Technologies). The SON was identified with reference to Franklin and Paxinos (28) and the tissue from each animal was independently pooled into collection vials containing RNAlater® (Ambion, Huntingdon, UK). A single operative carried out all dissections. Total RNA was isolated without delay (within 24h) according to standard procedures that accompany the Ambion RNAqueous MicroKit (Ambion). Microarray analysis. Separate microarrays (n=4) were probed using independently generated target. For each completely independent replicate, tissue from 1 mouse was used for RNA extraction.本研究探讨了不同啮齿类物种如何在视上核（supraoptic nucleus, SON）的转录组调控层面，应对危及生命的脱水稳态挑战。视上核是特化的下丘脑神经分泌核团，负责合成抗利尿肽激素精氨酸加压素（arginine vasopressin, AVP）。AVP通过促进肾脏的水重吸收以维持机体水平衡。脱水可引发位于神经垂体的SON轴突末梢AVP调控性释放大幅增加，同时伴随该核团在形态学、电生理特性、生物合成与分泌活性上的诸多改变。本研究采用基因芯片分析（microarray analysis）构建了小鼠SON的明确基因表达目录，并解析了脱水刺激下小鼠SON的基因表达谱变化。将脱水诱导的小鼠SON差异表达基因与大鼠的对应基因进行比较后，发现二者存在大量相似性，提示该核团功能相关可塑性的共同分子机制。此外，本研究还鉴定出诸多以物种特异性方式差异表达的基因。不过在多数情况下，高渗刺激可诱导同一通路内不同基因的表达产生物种特异性改变。同一通路的不同分子在不同物种中可通过差异化调控，以不同途径达成相同的功能终点。因此我们提出，基于转录组数据的整合生理学研究应聚焦于通路而非单一基因。 实验动物：选用成年雄性C57BL/6小鼠（购自英国拉夫堡的Harlan Sera-Lab公司），每笼4只群居饲养，饲养环境温度控制为21±2℃，光照周期为14小时光照/10小时黑暗（每日05:00开灯）。实验开始前，小鼠可自由采食饮水。自当日上午11:00起，脱水组小鼠完全剥夺体液48小时；对照组小鼠可自由饮用自来水，两组小鼠均能获取标准实验啮齿类饲料。关于成年雄性大鼠的实验已在既往研究中报道（27）。所有实验操作严格遵循英国1986年《动物科学实验法案》，并获得布里斯托大学当地伦理审查委员会批准。 组织收集：采用颈椎脱臼法处死小鼠，小心取出脑组织，使用干冰粉末快速冷冻后置于-80℃冰箱保存，保存时长不超过14天。使用无核糖核酸酶（RNase-free）冰冻切片机将脑组织切成14μm厚的切片，粘贴于无RNase覆膜玻片（P.A.L.M. 覆膜玻片；P.A.L.M. 激光显微切割技术公司）上。切片完成后立即将冰冻切片解冻固定（于95%[体积分数]乙醇中固定30秒），依次经75%、50%[体积分数]乙醇各浸泡30秒进行复水，随后用1%[体积分数]甲酚紫染色60秒。之后再次通过梯度乙醇系列脱水：依次经50%、75%、95%[体积分数]乙醇各30秒，再用100%[体积分数]乙醇浸泡2次，每次30秒。使用P.A.L.M. 激光显微切割系统（P.A.L.M. 激光显微切割技术公司）进行激光显微切割。参照Franklin与Paxinos的脑图谱（28）定位视上核，将每只小鼠的组织单独收集至含有RNAlater®保存液（Ambion公司，英国亨廷顿）的收集管中，由同一名实验人员完成所有切割操作。按照Ambion RNAqueous MicroKit（Ambion公司）配套的标准操作流程，在24小时内及时提取总RNA。 基因芯片分析：采用独立制备的靶标探针与4张独立的基因芯片（n=4）进行杂交。每个完全独立的生物学重复均使用1只小鼠的组织提取RNA。