ROLE OF GAP-43 IN OLFACTORY DEVELOPMENT AND LEARNING

神经生长相关蛋白GAP-43参与了 神经可塑性在发育和学习过程中的调节。我们 GAP-43在嗅觉中表达的检测与调控 在早期学习中的球茎。首先，我们将研究GAP-的表达- 43在嗅球在出生后发育过程中的RNA印迹，核糖核酸酶 保护定量、原位杂交和免疫细胞化学。 因为蛋白激酶C对GAP-43的磷酸化是至关重要的 这种蛋白质的功能，因为最近的数据表明，蛋白质 激酶C可能稳定GAP-43的mRNA，我们还将分析其表达 鳞茎发育过程中的蛋白激酶C同工酶。这些是相同的 分析将对来自具有 接受嗅觉偏好训练。细胞外多巴胺已经被 在早期嗅觉中，新生儿嗅球的数量增加 偏好习得。来自我们实验室的数据表明，释放 多巴胺是由GAP-43调节的。因此，我们假设两者之间存在联系 GAP-43在嗅球中的表达和多巴胺水平。建议 测试这种关系的基因干预实验包括 单纯疱疹病毒1型GAP-43重组体的体内注射 嗅球转化及转基因小鼠的构建 一个GAP-43突变体。我们将使用表达GAP-43反义RNA的HSV-1载体 减少多巴胺能肾小球细胞生长相关蛋白-43的表达 在嗅觉偏好学习过程中的嗅球，并检测 大鼠注射2-脱氧葡萄糖、体内透析和行为学 更倾向于确定这种基因干预的后果。我们 也将使用HSV-1载体在嗅球中过表达GAP-43 在嗅觉偏好学习的敏感期过后，确定 这种操作是否允许表达任何神经或 早期气味偏好习得的行为相关性。最后，我们 将构建转基因小鼠，在基因控制下表达 酪氨酸羟基酶启动子，GAP-43的突变体，其中丝氨酸-41 通常由蛋白激酶C磷酸化的蛋白被一种 甘氨酸残留物。其目的是导致脑内多巴胺释放不足。 表达该突变体的多巴胺能神经元。我们将研究其影响 这种突变对转基因小鼠早期嗅觉学习的影响。

The neuronal growth associated protein GAP-43 has been implicated in the modulation of neural plasticity during development and learning. We propose to examine and manipulate the expression of GAP-43 in the olfactory bulb during early learning. First, we will examine the expression of GAP- 43 in the olfactory bulb during postnatal development by RNA blots, RNAse protection quantification, in situ hybridization, and immunocytochemistry. Because phosphorylation of GAP-43 by protein kinase C is critical for the function of this protein, and because recent data suggest that protein kinase C may stabilize GAP-43 mRNA, we will also analyze the expression of the protein kinase C isozymes in the bulb during development. These same analyses will be performed on the olfactory bulbs from animals that have undergone olfactory preference training. Extracellular dopamine has been shown to increase in the neonatal olfactory bulb during early olfactory preference acquisition. Data from our laboratory suggests that the release of dopamine is modulated by GAP-43. Hence, we postulate a link between GAP-43 expression and dopamine levels in the olfactory bulb. Proposed genetic intervention experiments to test for this relationship include injection of Herpes Simplex Virus One (HSV-1) GAP-43 recombinants in vivo into the olfactory bulb and the construction of transgenic mice expressing a GAP-43 mutant. We will use HSV-1 vectors expressing GAP-43 antisense RNA to decrease the expression of GAP-43 in dopaminergic glomerular-layer cells in the olfactory bulb during olfactory preference learning, and assay the injected rats by 2-deoxyglucose uptake, in vivo dialysis, and behavioral preference to determine the consequences of such genetic intervention. We will also use HSV-1 vectors to overexpress GAP-43 in the olfactory bulb after the sensitive period for olfactory preference learning, to determine whether this manipulation will allow expression of any of the neural or behavioral correlates of early odor preference acquisition. Finally, we will construct transgenic mice expressing, under the control of the tyrosine hydroxylase promoter, a mutant of GAP-43 in which the serine-41 that is normally phosphorylated by protein kinase C is replaced by a glycine residue. The aim is to cause deficiency in dopamine release in dopaminergic neurons expressing this mutant. We will examine the effects of such a mutation on early olfactory learning in transgenic mice.