Saito Nae, Itakura Makoto, Sasaoka Toshikuni
Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, Niigata, Japan.
Department of Molecular and Cellular Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
Front Behav Neurosci. 2022 Feb 17;16:751053. doi: 10.3389/fnbeh.2022.751053. eCollection 2022.
Dopaminergic neurotransmission dopamine D1 receptors (D1Rs) is considered to play an important role not only in reward-based learning but also in aversive learning. The contextual and auditory cued fear conditioning tests involve the processing of classical fear conditioning and evaluates aversive learning memory. It is possible to evaluate aversive learning memory in two different types of neural transmission circuits. In addition, when evaluating the role of dopaminergic neurotransmission D1R, to avoid the effects in D1R-mediated neural circuitry alterations during development, it is important to examine using mice who D1R expression in the mature stage is suppressed. Herein, we investigated the role of dopaminergic neurotransmission D1Rs in aversive memory formation in contextual and auditory cued fear conditioning tests using D1R knockdown (KD) mice, in which the expression of D1Rs could be conditionally and reversibly controlled with doxycycline (Dox) treatment. For aversive memory, we examined memory formation using recent memory 1 day after conditioning, and remote memory 4 weeks after conditioning. Furthermore, immunostaining of the brain tissues of D1RKD mice was performed after aversive footshock stimulation to investigate the distribution of activated c-Fos, an immediate-early gene, in the hippocampus (CA1, CA3, dentate gyrus), striatum, amygdala, and prefrontal cortex during aversive memory formation. After aversive footshock stimulation, immunoblotting was performed using hippocampal, striatal, and amygdalar samples from D1RKD mice to investigate the increase in the amount of c-Fos and phosphorylated SNAP-25 at Ser187 residue. When D1R expression was suppressed using Dox, behavioral experiments revealed impaired contextual fear learning in remote aversion memory following footshock stimulation. Furthermore, expression analysis showed a slight increase in the post-stimulation amount of c-Fos in the hippocampus and striatum, and a significant increase in the amount of phosphorylated SNAP-25 in the hippocampus, striatum, and prefrontal cortex before and after stimulation. These findings indicate that deficiency in D1R-mediated dopaminergic neurotransmission is an important factor in impairing contextual fear memory formation for remote memory.
多巴胺能神经传递中的多巴胺 D1 受体（D1Rs）不仅在基于奖赏的学习中，而且在厌恶学习中都被认为起着重要作用。情境性和听觉线索性恐惧条件反射测试涉及经典恐惧条件反射的处理，并评估厌恶学习记忆。可以在两种不同类型的神经传递回路中评估厌恶学习记忆。此外，在评估多巴胺能神经传递 D1R 的作用时，为避免发育过程中 D1R 介导的神经回路改变的影响，使用成熟阶段 D1R 表达被抑制的小鼠进行研究很重要。在此，我们使用 D1R 基因敲低（KD）小鼠，研究了多巴胺能神经传递 D1Rs 在情境性和听觉线索性恐惧条件反射测试中厌恶记忆形成中的作用，其中 D1Rs 的表达可以通过强力霉素（Dox）处理进行条件性和可逆性控制。对于厌恶记忆，我们在条件反射后 1 天使用近期记忆以及在条件反射后 4 周使用远期记忆来检查记忆形成。此外，在厌恶足部电击刺激后，对 D1RKD 小鼠的脑组织进行免疫染色，以研究即刻早期基因激活的 c-Fos 在厌恶记忆形成过程中在海马体（CA1、CA3、齿状回）、纹状体、杏仁核和前额叶皮质中的分布。在厌恶足部电击刺激后，使用 D1RKD 小鼠的海马体、纹状体和杏仁核样本进行免疫印迹，以研究 c-Fos 量的增加以及丝氨酸 187 残基处磷酸化 SNAP-25 的增加。当使用 Dox 抑制 D1R 表达时，行为实验显示在足部电击刺激后的远期厌恶记忆中，情境性恐惧学习受损。此外，表达分析表明，刺激后海马体和纹状体中 c-Fos 的量略有增加，并且刺激前后海马体、纹状体和前额叶皮质中磷酸化 SNAP-25 的量显著增加。这些发现表明，D1R 介导的多巴胺能神经传递不足是损害远期记忆的情境性恐惧记忆形成的一个重要因素。
