Behavioral Functions of Neuropeptides

我们实验室致力于研究甘丙肽抑制作用的行为伴随。神经肽甘丙肽定位于海马区，与乙酰胆碱共存于大鼠隔-海马通路，与去甲肾上腺素共存于蓝斑，抑制谷氨酸、乙酰胆碱、5-羟色胺和去甲肾上腺素的释放，并抑制诱发的腺苷环化酶信号转导。在阿尔茨海默病患者的基底前脑中，甘丙素过度表达。我们过去的实验表明，中枢微量注射甘丙肽会损害大鼠在几个学习和记忆任务中的表现。我们发现，与对照组相比，甘丙肽过表达转基因小鼠(GAL-TG)在学习和记忆任务上表现出类似的缺陷，包括Morris水迷宫空间学习探测试验、食物偏好社会传递中的嗅觉记忆以及痕迹线索恐惧条件反射。Gal-TG小鼠代表了阿尔茨海默病中甘丙素过度表达的小鼠模型，这可以作为一种翻译工具来检验假设并产生治疗阿尔茨海默病特征的记忆丧失的新疗法。 今年，博士后内森·鲁斯塔试图在我们的小鼠甘丙素过度表达模型中增加神经解剖学的特异性。鲁斯泰尔博士使用腺相关病毒载体(AAV)，该载体含有甘丙氨酸基因，由北卡罗来纳大学的Thomas McCown教授开发，并为我们的实验室做出了善意的贡献。方法遵循麦克考恩博士的方案，该方案成功地展示了他的AAV-Galanin的抗癫痫活性。合作者Markus Heilig博士和他的NIAAA实验室成员在切片、绿色荧光蛋白(GFP)标记物的免疫细胞化学可视化以及Galanin mRNA的原位杂交方面为Rustay博士提供了培训。NIMH的Miles Herkenham博士好心地允许Rustay博士在我们PNRC Pod 1C的冰冻箱上切开小鼠的大脑。最初的实验包括将AAV-Galanin微量注射到内侧隔-斜角带或腹侧海马区。神经解剖学分析证明在这两个部位都存在GFP和甘丙素基因。行为分析的时间跨度为2周至2个月。两项学习和记忆任务，莫里斯水迷宫空间学习和痕迹恐惧条件反射，评估了认知功能。与AAV-GFP对照组相比，AAV-Galanin处理的C57BL/6J小鼠在这些任务中的表现没有显著差异。这些发现表明，需要更多的全球甘丙素过度表达，在多个大脑区域高浓度，以产生记忆损伤。结果与临床发现一致，即广泛的甘丙素过度表达与中晚期阿尔茨海默病在时间上是一致的，当认知功能显著受损时。 今年，博士后凯瑟琳·贝利对一种新的Galanin亚型受体GAL-R2基因敲除小鼠的第一个完整的行为表型特征进行了扩展。突变品系是由西雅图Nura公司的合作者John Hohmann博士产生的。GAL-R2基因敲除小鼠在一般健康、家庭笼子行为、神经反射、感觉能力和运动功能方面都是正常的。贝利博士已经证明，GAL-R2缺失突变体在追踪恐惧条件反射或莫里斯水迷宫的空间导航方面没有表现出缺陷。今年，贝利博士证实了她的初步发现，并扩大了她的初步发现，即在高架的PLUS迷宫上，GAL-R2零突变体中存在类似于焦虑的表型。在压力和焦虑样特征的其他指标上，包括压力诱导的体温过高和明/暗探索在内的其他指标的表型在不同的正式型中是相似的，这表明在升高的PLUS迷宫中，GAL-R2突变具有高度选择性的焦虑样效应。此外，贝利博士用GAL-R2小鼠进行的实验扩展到测试社交互动和社交环境中的类似焦虑行为。在我们的自动三室社交任务中，没有检测到基因差异。贝利博士的发现支持了一个新的证据，即甘丙素是一种抑制性神经调节剂，在特定的冲突情况下具有缓解焦虑的作用。

Our laboratory is engaged in investigating the behavioral concomitants of the inhibitory effects of galanin. The neuropeptide galanin is localized in the hippocampus, coexists with acetylcholine in the rat septohippocampal pathway, coexists with norepinephrine in the locus coeruleus, inhibits the release of glutamate, acetylcholine, serotonin, and norepinephrine, and inhibits evoked adenylate cyclase signal transduction. Galanin is overexpressed in the basal forebrain in Alzheimer's disease. Our past experiments revealed that central microinjection of galanin in rats impairs performance on several learning and memory tasks. We discovered that galanin overexpressing transgenic mice (GAL-tg) display analogous deficits on learning and memory tasks including the Morris water maze spatial learning probe trial, olfactory memory in social transmission of food preference, and trace cued fear conditioning, when compared to WT littermate controls. GAL-tg mice represent a mouse model of galanin overexpression in Alzheimer's disease, which can be used as a translational tool to test hypotheses and generate novel therapeutics for treating the memory loss that characterizes Alzheimer's disease. This year postdoctoral fellow Nathan Rustay sought to increase the neuroanatomical specificity in our modeling of galanin overexpression in mice. Dr. Rustay employed an adeno-associated viral vector (AAV) containing the galanin gene, generated by Professor Thomas McCown at the University of North Carolina and kindly contributed to our laboratory. Methods followed Dr. McCown's protocol, which had successfully demonstrated anti-seizure activity with his AAV-galanin. Collaborator Dr. Markus Heilig and members of his NIAAA laboratory kindly contributed expertise in training Dr. Rustay on sectioning, immunocytochemical visualization of the green fluorescent protein (GFP) marker, and in situ hybridization for galanin mRNA. Dr. Miles Herkenham, NIMH, kindly permitted Dr. Rustay to section the mouse brains on his cryostat in our PNRC Pod 1C. First experiments included microinjections of the AAV-galanin into the medial septum-diagonal band or into the ventral hippocampus. Neuroanatomical analyses demonstrated the presence of GFP and of the galanin gene at both sites. Behavioral analyses were conducted across a time course of 2 weeks to 2 months. Two learning and memory tasks, Morris water maze spatial learning and trace fear conditioning, evaluated cognitive function. AAV-galanin treated C57BL/6J mice displayed no significant differences in performance on these tasks, as compared to AAV-GFP controls. These findings indicate the need for more global galanin overexpression, at high concentrations in multiple brain regions, to produce memory impairments. Results are consistent with clinical findings that widespread galanin overexpression coincides temporally with mid- to late-stage Alzheimer's disease, when cognitive functions are significantly compromised. This year postdoctoral fellow Kathleen Bailey expanded on the first full behavioral phenotyping characterization of a new galanin subtype receptor GAL-R2 knockout mouse. The mutant line was generated by collaborator Dr. John Hohmann at Nura, Inc. in Seattle. GAL-R2 knockout mice were normal on measures of general health, home cage behaviors, neurological reflexes, sensory abilities, and motor functions. Dr. Bailey had shown that GAL-R2 null mutants did not display deficits on trace fear conditioning or on spatial navigation in the Morris water maze. This year Dr. Bailey confirmed and extended her initial findings of an anxiogenic-like phenotype in GAL-R2 null mutants on the elevated plus maze. Phenotypes on other measures of stress and anxiety-like traits, including stress-induced hyperthermia and light/dark exploration, were similar across gentoypes, indicating a highly selective anxiety-like effect of the GAL-R2 mutation in the elevated plus maze. Further, Dr. Bailey's experiments with GAL-R2 mice were extended to test social interactions and anxiety-like behaviors in a social setting. No genotype differences were detected in our automated 3-chambered social task. Dr. Bailey's findings support the emerging evidence that galanin is an inhibitory neuromodulator with anxiolytic actions in specific conflict situations.