Bidirectional perisomatic inhibitory plasticity of a Fos neuronal network [Ribotag]

Behavioral experiences activate the Fos transcription factor (TF) in sparse populations of neurons that are critical for encoding and recalling specific events. However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos-activated cells. Additionally, it is unknown if Fos is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos-expressing hippocampal CA1 pyramidal neurons by parvalbumin (PV)-interneurons (INs) is enhanced, while perisomatic inhibition by cholecystokinin (CCK)-INs is weakened. This bidirectional modulation of inhibition is specific to Fos-expressing neurons and is abolished when the function of the Fos TF complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling, and chromatin analyses, combined with electrophysiology reveal that Fos activates the transcription of Scg2 (secretogranin II), a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As PV- and CCK-INs mediate distinct features of pyramidal cell activity, the Scg2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo. Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to CA1 theta are significantly altered with loss of Scg2. Together these findings reveal an instructive role for Fos and Scg2 in establishing a network of Fos-activated neurons via the rewiring of local inhibition from an initially broad to a selectively modulated state. The opposing plasticity mechanisms on distinct inhibitory pathways may support the consolidation of memories over time. CaMK2a-Cre; lox-STOP-lox-Rpl22-HA or PV-Cre; lox-STOP-lox-Rpl22-HA mice were intraperitoneally injected with kainic acid (KA) or phosphate buffered saline (PBS). After 6 hours, hippocampal CA1 tissue was rapidly dissected from mice and subsequently used for isolation of ribosome-bound mRNAs. Immunopurification of ribosome-bound mRNAs was performed as previously described (Sanz PNAS 2009) with 10mM Ribonucleoside Vanadyl Complex present in the lysis buffer and using the mouse monoclonal anti-HA antibody (Sigma HA-7, H3663, 12 ug/IP). A small fraction of lysate before the immunoprecipitation (IP) was used as input for each sample. For CaMK2a-Ribotag, 3 bioreplicates were used per condition, with CA1 tissue from 4 mice pooled per bioreplicate. For PV-Ribotag, 4 bioreplicates were used per condition, with CA1 tissue from 9-10 mice pooled per bioreplicate.

行为经验可在稀疏神经元群体中激活Fos转录因子（transcription factor, TF），而这类神经元对于特定事件的编码与提取至关重要。然而，目前学界对行为经验如何驱动神经环路重塑以建立Fos激活神经元网络的机制尚缺乏充分认知。此外，除作为近期神经活动的标志物外，Fos在该过程中是否发挥其他必要功能，以及若存在此类功能，其众多基因靶点中哪些参与调控神经环路重塑，目前仍不明确。

本研究发现，当小鼠对全新环境进行空间探索时，小白蛋白（parvalbumin, PV）阳性中间神经元（interneurons, INs）对表达Fos的海马CA1锥体神经元的胞周抑制作用增强，而胆囊收缩素（cholecystokinin, CCK）阳性中间神经元对其的胞周抑制作用则减弱。这种双向调控的抑制效应仅特异性作用于表达Fos的神经元，且当Fos转录因子复合物的功能被阻断时，该调控作用会消失。

结合单细胞RNA测序、核糖体关联mRNA谱分析、染色质分析与电生理学实验结果，本研究证实Fos可激活分泌粒蛋白II（secretogranin II, Scg2）的转录——该基因编码多种不同的神经肽，以此协调上述抑制作用的变化。由于PV阳性与CCK阳性中间神经元分别介导锥体神经元活动的不同特征，依赖Scg2的抑制性突触输入重塑，有望在体内影响神经环路功能。与该预测相符的是，当Scg2缺失时，海马γ节律以及锥体神经元与CA1θ波的耦合均发生显著改变。

综上，本研究结果揭示了Fos与Scg2的指导性功能：它们可通过将局部抑制作用从初始的广谱状态重塑为选择性调控状态，从而建立Fos激活神经元网络。针对不同抑制通路的双向可塑性机制，或可支持记忆随时间的巩固过程。

将CaMK2a-Cre; lox-STOP-lox-Rpl22-HA或PV-Cre; lox-STOP-lox-Rpl22-HA小鼠经腹腔注射海人酸（kainic acid, KA）或磷酸盐缓冲液（phosphate buffered saline, PBS）。给药6小时后，快速摘取小鼠海马CA1组织，随后用于分离核糖体结合mRNA。核糖体结合mRNA的免疫纯化实验按照此前报道的方法（Sanz等,《美国国家科学院院刊》, 2009）进行：裂解液中添加10mM核糖核苷钒复合物，并使用小鼠单克隆抗HA抗体（Sigma HA-7, 货号H3663, 12 μg/免疫沉淀反应）。免疫沉淀（immunoprecipitation, IP）前的少量裂解液用作每个样本的输入对照。对于CaMK2a-Ribotag实验，每组设置3个生物重复样本，每个生物重复样本混合4只小鼠的CA1组织；对于PV-Ribotag实验，每组设置4个生物重复样本，每个生物重复样本混合9~10只小鼠的CA1组织。