[1]卢建,金文均,覃涵,等.内嗅皮层-海马投射末梢基于光纤技术的钙活动记录[J].第三军医大学学报,2016,38(11):1229-1234.
 Lu Jian,Jin Wenjun,Qin Han,et al.Optical fiber-based recording of Ca2+ activity dynamics of entorhinal-hippocampal projections in mice[J].J Third Mil Med Univ,2016,38(11):1229-1234.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
38卷
期数:
2016年第11期
页码:
1229-1234
栏目:
基础医学
出版日期:
2016-06-15

文章信息/Info

Title:
Optical fiber-based recording of Ca2+ activity dynamics of entorhinal-hippocampal projections in mice
作者:
卢建金文均覃涵谌小维
第三军医大学基础医学部脑研究中心;华中科技大学武汉光电国家实验室
Author(s):
Lu Jian Jin Wenjun Qin Han Chen Xiaowei

Brain Research Center, College of Basic Medical Sciences, Third Military Medical University, Chongqing, 400038, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430074, China

关键词:
内嗅皮层海马光纤钙活动轴突末梢GCaMP5G
Keywords:
entorhinal cortex hippocampus optical fiber Ca2+ activity axon terminal GCaMP5G
分类号:
R319; R329.25; R338.26
文献标志码:
A
摘要:

目的      运用光纤探测系统建立轴突末梢Ca2+活动的光学记录方法,并初步探究麻醉和自由活动状态下小鼠皮层-海马通路的活动规律。      方法      向8~10周龄雄性C57BL/6小鼠内侧内嗅皮层(medial entorhinal cortex,MEC)浅层(Ⅱ层)注射神经元特异并携带Ca2+荧光指示蛋白基因的腺相关病毒(AAV-Syn-GCaMP5G),继续饲养1个月后将光纤植入到海马齿状回(dentate gyrus, DG)分子层上沿,记录麻醉和自由活动状态下轴突末梢的Ca2+活动;相同方法记录AAV-Syn-增强绿色荧光蛋白(eGFP)小鼠作为对照。向MEC浅层注射河豚毒素(tetrodotoxin, TTX),同步记录轴突末梢Ca2+活动,注射人工脑脊液(artificial cerebrospinal fluid, ACSF)作为对照。      结果      内嗅皮层Ⅱ层神经元投射至海马DG的轴突纤维密集表达GCaMP5G。在麻醉和自由活动的小鼠中均能稳定记录到轴突末梢Ca2+信号,eGFP对照小鼠未记录到类似Ca2+信号。MEC浅层注射TTX后,轴突末梢Ca2+信号100 s内最高幅度降至注射前的12.6%。与ACSF比较,TTX对轴突末梢Ca2+活动有明显阻断作用(P<0.01)。内嗅皮层投射至DG的轴突末梢处Ca2+活动在麻醉状态下以一定节律同步发放,自由活动状态下则表现为无节律非同步发放,且CaCa2+信号幅度有所增强。      结论      通过GCaMP5G特异标记了内侧内嗅皮层Ⅱ层投射至DG的轴突纤维,并运用光纤探测系统建立了长远投射轴突末梢Ca2+活动的光学记录方法。与麻醉状态相比,自由活动状态下内嗅-海马通路呈现更强的Ca2+活动发放。

Abstract:

Objective      To establish a method of recording Ca2+ activity at axonal terminals of neural projection and then study the activity pattern of the pathway from the entorhinal cortex to the hippocampus of mice under anesthetized and freely behaving conditions.       Methods      AAV-Syn-GCaMP5G was injected into the medial entorhinal cortex (MEC) superficial layer of male C57BL/6 mice at age of 8~10 weeks. In 1 month later, optical fiber was implanted onto the stratum moleculare of dentate gyrus (DG) to record Ca2+ activity at the axonal terminals under both anesthetized and freely behaving conditions. The AAV-Syn-eGFP injected mice were recorded as the control group. Ca2+ activity dynamics at the axon terminal sites were compared before and after tetrodotoxin (TTX) was injected into the MEC superficial layers of the anesthetized mice. The mice in the control group were injected with artificial cerebrospinal fluid (ACSF).       Results      The axonal fibers projecting from the entorhinal cortex to DG were densely labeled by GCaMP5G. Ca2+ signals from the axonal terminals can be recorded steadily under both anesthetized and freely behaving conditions by using optical fiber photometry. However, no signals were detected in eGFP-labeled mice. After injecting TTX into the superficial layers of MEC, the maximum amplitude of Ca2+ signals within 100 s from axonal terminals descended to 12.6% of Ca2+ signals recorded before injection. Compared with the ACSF-injected mice, TTX significantly decreased Ca2+ activity at the axon terminals (P<0.01). The Ca2+ activity from the axonal terminals in the hippocampus exhibited unsynchronized pattern and higher amplitude in freely behaving condition as compared with the rhythmic-synchronized pattern under anesthetized condition.       Conclusion       Utilizing specific labeling of neuronal projection with GCaMP5G and optical fiber photometry, Ca2+ signals from the axonal terminal sites of long-range projections can be recorded reliably under both anesthetized and freely behaving conditions. The Ca2+ activity from the axonal terminals of entorhinal-hippocampal projections may exhibit higher level of dynamics in freely behaving mice.

参考文献/References:

[1]O’Keefe J, Dostrovsky J. The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat[J]. Brain Res, 1971, 34(1): 171-175. DOI: 10.1016/0006-8993(71)90358-1
[2]Hafting T, Fyhn M, Molden S, et al. Microstructure of a spatial map in the entorhinal cortex[J]. Nature, 2005, 436(7052): 801-806. DOI: 10.1038/nature03721
[3]Sargolini F, Fyhn M, Hafting T, et al. Conjunctive representation of position, direction, and velocity in entorhinal cortex[J]. Science, 2006, 312(5774): 758-762. DOI: 10.1126/science.1125572
[4]Solstad T, Boccara C N, Kropff E, et al. Representation of geometric borders in the entorhinal cortex[J]. Science, 2008, 322(5909): 1865-1868. DOI: 10.1126/science.1166466
[5]Kropff E, Carmichael J E, Moser M B, et al. Speed cells in the medial entorhinal cortex[J]. Nature, 2015, 523(7561): 419-424. DOI: 10.1038/nature14622
[6]Solstad T, Moser E I, Einevoll G T. From grid cells to place cells: a mathematical model[J]. Hippocampus, 2006, 16(12): 1026-1031. DOI: 10.1002/hipo.20244
[7]Nakai J, Ohkura M, Imoto K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein[J]. Nat Biotechnol, 2001, 19(2): 137-141. DOI: 10.1038/84397
[8]Tallini Y N, Ohkura M, Choi B R, et al. Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2[J]. Proc Natl Acad Sci U S A, 2006, 103(12): 4753-4758. DOI: 10.1073/pnas. 0509378103
[9]Wang Q, Shui B, Kotlikoff M I, et al. Structural basis for calcium sensing by GCaMP2[J]. Structure, 2008, 16(12): 1817-1827. DOI: 10.1016/j.str.2008.10.008
[10]Tian L, Hires S A, Mao T, et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators[J]. Nat Methods, 2009, 6(12): 875-881. DOI: 10.1038/nmeth.1398
[11]Akerboom J, Chen T W, Wardill T J, et al. Optimization of a GCaMP calcium indicator for neural activity imaging[J]. J Neurosci, 2012, 32(40): 13819-13840. DOI: 10.1523/ JNEUROSCI.2601-12.2012
[12]Chen T W, Wardill T J, Sun Y, et al. Ultrasensitive fluorescent proteins for imaging neuronal activity[J]. Nature, 2013, 499(7458): 295-300. DOI: 10.1038/ nature12354
[13]Peron S P, Freeman J, Iyer V, et al. A Cellular Resolution Map of Barrel Cortex Activity during Tactile Behavior[J]. Neuron, 2015, 86(3): 783-799. DOI: 10.1016/j.neuron. 2015.03.027
[14]Wertz A, Trenholm S, Yonehara K, et al. PRESYNAPTIC NETWORKS. Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules[J]. Science, 2015, 349(6243): 70-74. DOI: 10.1126/science.aab1687
[15]Hinckley C A, Alaynick W A, Gallarda B W, et al. Spinal Locomotor Circuits Develop Using Hierarchical Rules Based on Motorneuron Position and Identity[J]. Neuron, 2015, 87(5): 1008-1021. DOI: 10.1016/j.neuron. 2015.08.005
[16]Cui G, Jun S B, Jin X, et al. Deep brain optical measurements of cell type-specific neural activity in behaving mice[J]. Nat Protoc, 2014, 9(6): 1213-1228. DOI: 10.1038/nprot.2014.080
[17]Cui G, Jun S B, Jin X, et al. Concurrent activation of striatal direct and indirect pathways during action initiation[J]. Nature, 2013, 494(7436): 238-242. DOI: 10.1038/ nature11846
[18]Gunaydin L A, Grosenick L, Finkelstein J C, et al. Natural neural projection dynamics underlying social behavior[J]. Cell, 2014, 157(7): 1535-1551. DOI: 10.1016/j.cell. 2014.05.017
[19]Fyhn M, Molden S, Witter M P, et al. Spatial representation in the entorhinal cortex[J]. Science, 2004, 305(5688): 1258-1264. DOI: 10.1126/science.1099901
[20]Suh J, Rivest A J, Nakashiba T, et al. Entorhinal cortex layer Ⅲ input to the hippocampus is crucial for temporal association memory[J]. Science, 2011, 334(6061): 1415-1420. DOI: 10.1126/science.1210125
[21]Zhang S J, Ye J, Miao C, et al. Optogenetic dissection of entorhinal-hippocampal functional connectivity[J]. Science, 2013, 340(6128): 1232627. DOI: 10.1126/science. 1232627
[22]Kitamura T, Pignatelli M, Suh J, et al. Island cells control temporal association memory[J]. Science, 2014, 343(6173): 896-901. DOI: 10.1126/science.1244634
[23]Low R J, Gu Y, Tank D W. Cellular resolution optical access to brain regions in fissures: imaging medial prefrontal cortex and grid cells in entorhinal cortex[J]. Proc Natl Acad Sci U S A, 2014, 111(52): 18739-18744. DOI: 10.1073/pnas.1421753111
[24]Heys J G, Rangarajan K V, Dombeck D A. The functional micro-organization of grid cells revealed by cellular-resolution imaging[J]. Neuron, 2014, 84(5): 1079-1090. DOI: 10.1016/j.neuron.2014.10.048

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更新日期/Last Update: 2016-05-29