The anatomical organization of the rodent claustrum remains obscure due to lack of clear borders that distinguish it from neighboring forebrain structures. Defining what constitutes the claustrum is imperative for elucidating its functions. Methods based on gene/protein expression or transgenic mice have been used to spatially outline the claustrum but often report incomplete labeling and/or lack of specificity during certain neurodevelopmental timepoints. To reliably identify claustrum projection cells in mice, we propose a simple immunolabelling method that juxtaposes the expression pattern of claustrum-enriched and cortical-enriched markers. We determined that claustrum cells immunoreactive for the claustrum-enriched markers Nurr1 and Nr2f2 are devoid of the cortical marker Tle4, which allowed us to differentiate the claustrum from adjoining cortical cells. Using retrograde tracing, we verified that nearly all claustrum projection neurons lack Tle4 but expressed Nurr1/Nr2f2 markers to different degrees. At neonatal stages between 7 and 21 days, claustrum projection neurons were identified by their Nurr1-postive/Tle4-negative expression profile, a time-period when other immunolabelling techniques used to localize the claustrum in adult mice are ineffective. Finally, exposure to environmental novelty enhanced the expression of the neuronal activation marker c-Fos in the claustrum region. Notably, c-Fos labeling was mainly restricted to Nurr1-positive cells and nearly absent from Tle4-positive cells, thus corroborating previous work reporting novelty-induced claustrum activation. Taken together, this method will aid in studying the claustrum during postnatal development and may improve histological and functional studies where other approaches are not amenable.

中文翻译：

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一种简单可靠的小鼠跨年龄屏状核定位方法

由于缺乏将其与邻近前脑结构区分开来的清晰边界，啮齿动物屏状体的解剖结构仍然模糊。定义屏状核的构成对于阐明其功能至关重要。基于基因/蛋白质表达或转基因小鼠的方法已用于在空间上勾勒屏状体，但经常报告在某些神经发育时间点期间标记不完整和/或缺乏特异性。为了可靠地识别小鼠中的屏状体投射细胞，我们提出了一种简单的免疫标记方法，该方法将屏状体富集和皮质富集标记的表达模式并列。我们确定，对富含屏状核的标记物 Nurr1 和 Nr2f2 具有免疫反应性的屏状核细胞缺乏皮质标记物 Tle4，这使我们能够将屏状核与相邻的皮质细胞区分开来。通过逆行追踪，我们证实几乎所有屏状核投射神经元都缺乏Tle4，但不同程度地表达Nurr1/Nr2f2标记。在 7 至 21 天的新生儿阶段，屏状核投射神经元通过其 Nurr1 阳性/Tle4 阴性表达谱进行鉴定，在这个时期，用于定位成年小鼠屏状核的其他免疫标记技术无效。最后，接触新奇的环境增强了屏状核区域神经元激活标记物 c-Fos 的表达。值得注意的是，c-Fos 标记主要局限于 Nurr1 阳性细胞，而 Tle4 阳性细胞几乎不存在，从而证实了先前报告的新奇诱导的屏状核激活的工作。总而言之，这种方法将有助于研究出生后发育期间的屏状体，并可能改善其他方法不适合的组织学和功能研究。