Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently-developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1 AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Methods
To facilitate the application of these inhibitory-neuron specific viral vectors to studies of the primate cortex, we have performed a thorough validation and characterization of the laminar expression of reporter proteins mediated by several enhancer/promoter-specific AAVs. Here we report results from the two vector types that have shown the greatest specificity of transgene expression in marmoset primary visual cortex (V1); specifically, we have tested three serotypes of the h56D promoter-AAV that restricts gene expression to GABAergic neurons (Mehta et al., 2019), and one serotype of the S5E2 enhancer-AAV that restricts gene expression to PV cells (Vormstein-Schneider et al., 2020). Using injections of these viral vectors in marmoset V1, combined with immunohistochemical identification of GABA and PV neurons, we find that the laminar distribution of reporter protein expression mediated by the GABA- and PV-enhancer AAVs validated in this study resembles the laminar distribution of GABA-immunoreactive (GABA+) and PV-immunoreactive (PV+) cells, respectively, in marmoset V1

在哺乳动物新皮层中，抑制性活动对于动态平衡兴奋性信号，并塑造神经元与神经环路的响应特性至关重要。学界普遍认为，抑制作用的多样计算功能由不同类型的抑制性神经元介导，而多个物种中均存在丰富的抑制性神经元亚型多样性。目前学界对不同抑制性神经元亚型的功能与连接模式的认知，主要来源于转基因小鼠的相关研究。然而，尚不清楚从小鼠研究中获得的认知是否适用于与人类亲缘关系最近的非人灵长类模型。缺乏能够选择性靶向抑制性神经元亚型的病毒工具，一直是制约灵长类动物中该类神经元功能研究的主要障碍。

在此研究中，我们对多款新近开发的病毒载体进行了全面验证与特征化，这些载体可将转基因表达限定于γ-氨基丁酸能（GABAergic）神经元，或是其小白蛋白（parvalbumin, PV）亚型，并筛选出两款在狨猴初级视觉皮层（V1）中展现出高特异性与高效性的载体。研究表明，在狨猴V1皮层中，腺相关病毒（AAV）-h56D可介导γ-氨基丁酸能神经元的转基因表达，其特异性最高可达91%-94%，转染效率达79%，但该效果依赖于病毒血清型与皮层脑区分层。而AAV-PHP.eB-S5E2则可在全皮层分层的PV神经元中介导转基因表达，其特异性最高可达98%，转染效率为86%-90%，且该效果同样受脑区分层影响。综上，这些病毒载体有望成为研究灵长类皮层中γ-氨基丁酸能神经元与PV神经元功能及连接模式的理想工具。

## 方法
为推动这些抑制性神经元特异性病毒载体在灵长类皮层研究中的应用，我们对多款增强子/启动子特异性腺相关病毒介导的报告蛋白皮层分层表达模式进行了全面验证与特征化分析。本研究报道了两款在狨猴初级视觉皮层（V1）中转基因表达特异性最优的载体的相关结果：具体而言，我们测试了三款可将基因表达限定于γ-氨基丁酸能神经元的h56D启动子型腺相关病毒（Mehta等，2019），以及一款可将基因表达限定于PV神经元的S5E2增强子型腺相关病毒（Vormstein-Schneider等，2020）。通过将这些病毒载体注射至狨猴V1皮层，并结合γ-氨基丁酸与PV神经元的免疫组化鉴定，我们发现本研究验证的γ-氨基丁酸能与PV增强子型腺相关病毒介导的报告蛋白分层表达模式，分别与狨猴V1皮层中γ-氨基丁酸免疫阳性（GABA+）与PV免疫阳性（PV+）神经元的分层分布特征一致。