DataSheet1_Harnessing axonal transport to map reward circuitry: Differing brain-wide projections from medial prefrontal cortical domains.pdf

Neurons project long axons that contact other distant neurons. Neurons in the medial prefrontal cortex project into the limbic system to regulate responses to reward or threat. Diminished neural activity in prefrontal cortex is associated with loss of executive function leading to drug use, yet the specific circuitry that mediate these effects is unknown. Different regions within the medial prefrontal cortex may project to differing limbic system nuclei. Here, we exploited the cell biology of intracellular membrane trafficking, fast axonal transport, to map projections from two adjacent medial prefrontal cortical regions. We used Mn(II), a calcium analog, to trace medial prefrontal cortical projections in the living animal by magnetic resonance imaging (MRI). Mn(II), a contrast agent for MRI, enters neurons through voltage-activated calcium channels and relies on kinesin-1 and amyloid-precursor protein to transport out axons to distal destinations. Aqueous MnCl2 together with fluorescent dextran (3–-5 nL) was stereotactically injected precisely into two adjacent regions of the medial prefrontal cortex: anterior cingulate area (ACA) or infralimbic/prelimbic (IL/PL) region. Projections were traced, first live by manganese-enhanced MRI (MEMRI) at four time points in 3D, and then after fixation by microscopy. Data-driven unbiased voxel-wise statistical maps of aligned normalized MR images after either ACA or IL/PL injections revealed statistically significant progression of Mn(II) over time into deeper brain regions: dorsal striatum, globus pallidus, amygdala, hypothalamus, substantia nigra, dorsal raphe and locus coeruleus. Quantitative comparisons of these distal accumulations at 24 h revealed dramatic differences between ACA and IL/PL injection groups throughout the limbic system, and most particularly in subdomains of the hypothalamus. ACA projections targeted dorsomedial nucleus of the hypothalamus, posterior part of the periventricular region and mammillary body nuclei as well as periaqueductal gray, while IL/PL projections accumulated in anterior hypothalamic areas and lateral hypothalamic nuclei as well as amygdala. As hypothalamic subsegments relay CNS activity to the body, our results suggest new concepts about mind-body relationships and specific roles of distinct yet adjacent medial prefrontal cortical segments. Our MR imaging strategy, when applied to follow other cell biological processes in the living organism, will undoubtedly lead to an expanded perspective on how minute details of cellular processes influence whole body health and wellbeing.

神经元通过延伸长轴突与远端神经元建立突触联系。内侧前额叶皮层（medial prefrontal cortex）的神经元投射至边缘系统，以调节机体对奖赏或威胁的应答。前额叶皮层神经活动减弱可导致执行功能受损，进而引发药物成瘾，但介导此类效应的特异性神经环路尚不清楚。内侧前额叶皮层内的不同区域可能投射至不同的边缘系统核团。本研究借助细胞内膜运输与快速轴突运输的细胞生物学机制，对两个相邻的内侧前额叶皮层区域的神经投射进行图谱绘制。我们采用钙类似物Mn(II)，通过磁共振成像（magnetic resonance imaging, MRI）在活体动物体内追踪内侧前额叶皮层的投射通路。作为磁共振成像对比剂的Mn(II)可通过电压门控钙通道进入神经元，并依赖驱动蛋白-1（kinesin-1）与淀粉样前体蛋白（amyloid-precursor protein）将其经轴突转运至远端靶点。研究人员将含荧光葡聚糖的氯化锰水溶液（3~5纳升）通过立体定位术精准注射至内侧前额叶皮层的两个相邻区域：前扣带回皮层（anterior cingulate area, ACA）与内侧区/前边缘区（infralimbic/prelimbic, IL/PL）。首先通过锰增强磁共振成像（manganese-enhanced MRI, MEMRI）在四个时间点以三维模式对投射通路进行活体追踪，随后在组织固定后借助显微镜完成成像验证。通过对ACA或IL/PL注射后配准并归一化的磁共振图像进行数据驱动的无偏体素级统计分析，结果显示Mn(II)随时间推移可显著向更深脑区扩散：包括背侧纹状体、苍白球、杏仁核、下丘脑、黑质、中缝背核与蓝斑。对24小时时点的远端Mn(II)聚集区域进行定量比较发现，ACA与IL/PL注射组在整个边缘系统中均存在显著差异，尤其在下丘脑亚区中差异最为显著。ACA的投射靶点涵盖下丘脑背内侧核、室周区后部、乳头体核以及中脑导水管周围灰质；而IL/PL的投射则聚集于下丘脑前区、下丘脑外侧核与杏仁核。由于下丘脑亚区负责将中枢神经系统的活动信号传递至外周躯体，本研究结果为理解心身交互关系以及内侧前额叶皮层不同但相邻区域的特异性功能提供了全新的研究视角。本研究采用的磁共振成像策略若应用于追踪活体生物中的其他细胞生物学过程，无疑将推动学界对细胞过程的细微细节如何影响全身健康与生存状态的认知拓展。