Caldwell Kevin K, Solomon Elizabeth R, Smoake Jane J W, Djatche de Kamgaing Chrys D, Allan Andrea M
Department of Neurosciences, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Department of Neurosciences, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
Neurobiol Learn Mem. 2018 Dec;156:1-16. doi: 10.1016/j.nlm.2018.10.002. Epub 2018 Oct 12.
Studies in clinical populations and preclinical models have shown that prenatal alcohol exposure (PAE) is associated with impairments in the acquisition, consolidation and recall of information, with deficits in hippocampal formation-dependent learning and memory being a common finding. The glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and extracellular signal-regulated kinase 2 (ERK2) are key regulators of hippocampal formation development, structure and functioning and, thus, are potential mediators of PAE's effects on this brain region. In the present studies, we employed a well-characterized mouse model of PAE to identify biochemical mechanisms that may underlie activity-dependent learning and memory deficits associated with PAE.
Mouse dams consumed either 10% (w/v) ethanol in 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-h) access, drinking-in-the-dark paradigm. Male and female offspring (∼180-days of age) were trained using a delay conditioning procedure and contextual fear responses (freezing behavior) were measured 24 h later. Hippocampal formation tissue and blood were collected from three behavioral groups of animals: 20 min following conditioning (conditioning only group), 20 min following the re-exposure to the context (conditioning plus re-exposure group), and behaviorally naïve (naïve group) mice. Plasma corticosterone levels were measured by enzyme immunoassay. Immunoblotting techniques were used to measure protein levels of the GR, MR, ERK1 and ERK2 in nuclear and membrane fractions prepared from the hippocampal formation.
Adult SAC control male and female mice displayed similar levels of contextual fear. However, significant sex differences were observed in freezing exhibited during the conditioning session. Compared to same-sex SAC controls, male and female PAE mice demonstrated context fear deficits While plasma corticosterone concentrations were elevated in PAE males and females relative to their respective SAC naïve controls, plasma corticosterone concentrations in the conditioning only and conditioning plus re-exposure groups were similar in SAC and PAE animals. Relative to the respective naïve group, nuclear GR protein levels were increased in SAC, but not PAE, male hippocampal formation in the conditioning only group. In contrast, no difference was observed between nuclear GR levels in the naïve and conditioning plus re-exposure groups. In females, nuclear GR levels were significantly reduced by PAE but there was no effect of behavioral group or interaction between prenatal treatment and behavioral group. In males, nuclear MR levels were significantly elevated in the SAC conditioning plus re-exposure group compared to SAC naïve mice. In PAE females, nuclear MR levels were elevated in both the conditioning only and conditioning plus re-exposure groups relative to the naïve group. Levels of activated ERK2 (phospho-ERK2 expressed relative to total ERK2) protein were elevated in SAC, but not PAE, males following context re-exposure, and a significant interaction between prenatal exposure group and behavioral group was found. No main effects or interactions of behavioral group and prenatal treatment on nuclear ERK2 were found in female mice. These findings suggest a sex difference in which molecular pathways are activated during fear conditioning in mice.
In PAE males, the deficits in contextual fear were associated with the loss of responsiveness of hippocampal formation nuclear GR, MR and ERK2 to signals generated by fear conditioning and context re-exposure. In contrast, the contextual fear deficit in PAE female mice does not appear to be associated with activity-dependent changes in GR and MR levels or ERK2 activation during training or memory recall, although an overall reduction in nuclear GR levels may play a role. These studies add to a growing body of literature demonstrating that, at least partially, different mechanisms underlie learning, memory formation and memory recall in males and females and that these pathways are differentially affected by PAE.
临床人群研究和临床前模型研究表明，产前酒精暴露（PAE）与信息获取、巩固和回忆受损有关，海马结构依赖性学习和记忆缺陷是常见的发现。糖皮质激素受体（GR）、盐皮质激素受体（MR）和细胞外信号调节激酶2（ERK2）是海马结构发育、结构和功能的关键调节因子，因此是PAE对该脑区影响的潜在介质。在本研究中，我们采用了一种特征明确的PAE小鼠模型，以确定可能是PAE相关的活动依赖性学习和记忆缺陷基础的生化机制。
母鼠使用限时（4小时）暗饮范式，饮用含10%（w/v）乙醇的0.066%（w/v）糖精（SAC）或仅饮用0.066%（w/v）SAC。雄性和雌性后代（约180日龄）采用延迟条件化程序进行训练，并在24小时后测量情境恐惧反应（僵住行为）。从三组行为学实验动物中收集海马结构组织和血液：条件化后20分钟（仅条件化组）、再次暴露于情境后20分钟（条件化加再次暴露组）和未经行为训练的（未处理组）小鼠。通过酶免疫测定法测量血浆皮质酮水平。采用免疫印迹技术测量从海马结构制备的核和膜组分中GR、MR、ERK1和ERK2的蛋白质水平。
成年SAC对照雄性和雌性小鼠表现出相似水平的情境恐惧。然而，在条件化过程中观察到僵住行为存在显著的性别差异。与同性SAC对照相比，雄性和雌性PAE小鼠表现出情境恐惧缺陷。虽然PAE雄性和雌性的血浆皮质酮浓度相对于各自的SAC未处理对照有所升高，但仅条件化组和条件化加再次暴露组的血浆皮质酮浓度在SAC和PAE动物中相似。相对于各自的未处理组，仅条件化组中SAC雄性海马结构的核GR蛋白水平升高，但PAE雄性未升高。相比之下，未处理组和条件化加再次暴露组的核GR水平没有差异。在雌性中，PAE显著降低了核GR水平，但行为组或产前处理与行为组之间没有相互作用。在雄性中，与SAC未处理小鼠相比；SAC条件化加再次暴露组的核MR水平显著升高。在PAE雌性中，仅条件化组和条件化加再次暴露组的核MR水平相对于未处理组均升高。再次暴露于情境后，SAC雄性而非PAE雄性中活化的ERK2（磷酸化ERK2相对于总ERK）蛋白水平升高，并且发现产前暴露组与行为组之间存在显著的相互作用。在雌性小鼠中未发现行为组和产前处理对核ERK2有主要影响或相互作用。这些发现表明在小鼠恐惧条件化过程中激活的分子途径存在性别差异。
在PAE雄性中，情境恐惧缺陷与海马结构核GR、MR和ERK2对恐惧条件化和情境再次暴露产生的信号反应性丧失有关。相比之下，PAE雌性小鼠的情境恐惧缺陷似乎与训练或记忆回忆期间GR和MR水平或ERK2激活的活动依赖性变化无关，尽管核GR水平的总体降低可能起作用。这些研究增加了越来越多的文献表明，至少部分地，雄性和雌性在学习、记忆形成和记忆回忆方面存在不同的机制，并且这些途径受到PAE的不同影响。
