书名：神经生物学实验原理与技术
定价：128.0
ISBN：9787030300324
作者：李云庆，吕国蔚
版次：1
出版时间：2017-02

内容提要：
神经生物学是生命科学的前沿，是应用神经解剖学、神经生理学、神经化学和分子生物学等多学科现代技术，对神经系统进行多层次综合研究的实验性科学。本书作为《生命科学实验指南系列》的重要分册，遵循理性思维与实际操作相结合的原则，全面而系统地介绍了神经生物学实验研究的方法学（第1篇），并将编著者20余年的有关科研成果转化为可操作的实验指导（第2篇），以及提供可供参考的有关实验研究信息（第3篇）。



目录：
目录
序
前言
1 神经生物学实验方法学 1
1.1 科学思维方法学 1
1.1.1 科学技术的历史动力 1
1.1.2 辩证地去求索 3
1.1.3 辩证地去思考 7
1.1.4 辩证地去验证 17
1.1.5 辩证地去训练 24
1.2 实验设计方法学 29
1.2.1 选题 30
1.2.2 专业设计 44
1.2.3 对照设计 54
1.2.4 统计设计 63
1.3 实验分析方法学 79
1.3.1 数据整理 79
1.3.2 统计分析 88
1.3.3 专业分析 100
1.3.4 论文书写 109
1.4 电刺激方法学 117
1.4.1 电刺激的基本原理 117
1.4.2 电刺激的物理特性 119
1.4.3 神经制备的生物特性 123
1.4.4 选择性刺激 127
1.4.5 刺激电流扩散 132
1.5 电记录方法学 136
1.5.1 容积导体内记录 136
1.5.2 诱发电位记录 143
1.5.3 单单位记录 150
1.5.4 计算机辅助的记录 158
1.5.5 细胞内记录 161
1.5.6 膜片钳记录 174
1.5.7 神经纤维速度谱测定 178
1.5.8 轴突分叉点位置测定 182
1.5.9 压脚痛阔测定法 187
1.6 神经化学方法学 190
1.6.1 组织细胞破碎法 190
1.6.2 突触体制备 193
1.6.3 电泳法 198
1.6.4 色谱法 203
1.6.5 高效液相色谱法 213
1.6.6 微透析技术 222
1.7 化学神经解剖学方法学 229
1.7.1 免疫细胞化学技术 229
1.7.2 原位杂交组织化学技术 239
1.7.3 受体定位技术 248
1.7.4 免疫电子显微镜技术 250
1.8 神经形态学方法学 253
1.8.1 辣根过氧化物酶示踪技术 253
1.8.2 荧光素示踪技术 260
1.8.3 放射性核素示踪技术 262
1.8.4 顺行示踪技术 269
1.8.5 激光扫描共焦显微镜技术 271
1.8.6 定量及分析细胞学技术 278
1.9 分子神经生物学方法学 282
1.9.1 核酸分子杂交技术 282
1.9.2 蛋白质印迹法 290
1.9.3 DNA重组技术 296
1.9.4 聚合酶链反应技术 306
1.9.5 DNA序列测定技术 313
1.9.6 mRNA差异显示技术 323
1.9.7 基因芯片技术 329
1.9.8 转基因动物技术 334
1.10 神经行为学实验方法学 338
1.10.1 行为学实验的神经基础及常用动物 339
1.10.2 常用的高级脑功能研究方法 340
1.10.3 常用痛行为研究方法 348
1.11.脑成像 352
1.11.1 计算机辅助体层摄影 352
1.11.2 磁共振成像 356
1.11.3 放射性核素断层成像 362
1.11.4 超声成像 365
2 神经生物学实验与示敬 368
2.1 神经生理学实验 368
2.1.1 家兔外周神经干复合动作电位记录 368
2.1.2 家兔后肢传人神经纤维速度谱 370
2.1.3 扩张肛门对猫慨神经后根放电的影响 372
2.1.4 大鼠脊髓节段性及下行性诱发电位记录 374
2.1.5 脊髓节段性缺血时脊髓诱发电位的变化 375
2.1.6 家兔大脑皮质体感诱发电位记录 377
2.1.7 脑缺血对家兔大脑皮质诱发电位的变化 379
2.1.8 瞻蛤离体脊神经节神经元静息膜电位与动作电位记录 381
2.1.9 大鼠培养脑细胞膜的电学特性 384
2.1.10 大鼠在体脊神经节神经元动作电位的细胞内记录 386
2.1.11 猫脊髓背索突触后神经元的细胞内与细胞外记录 388
2.1.12 猫脊颈束-背索突触后神经元的顺、逆向反应 390
2.1.13 大鼠脊髓背角神经元电活动的细胞内记录 392
2.1.14 大鼠脊孤柬-背索突触后神经元对躯体与内脏传人的反应 394
2.1.15 家兔中缝大核对外周传人刺激的反应 397
2.1.16 家兔丘脑腹后外侧核电活动的细胞外记录 398
2.1.17 躯体内脏传人在脊髓背角的相互作用 400
2.1.18 缺氧预适应鼠脑提取液对ATP敏感性饵电流的作用 401
2.2 神经化学实验 408
2.2.1 缺氧耐受小鼠脑匀浆提取液的抗缺氧作用 408
2.2.2 急性重复缺氧小鼠脑单胺类含量的变化 411
2.2.3 不同强度躯体刺激对家兔脑脊液中CaH、MgH含量的影响 414
2.2.4 不同强度躯体刺激对家兔脑脊液中单胶类含量的影响 417
2.2.5 兔脑内腺昔的微透析法测定 420
2.2.6 缺氧对小鼠大脑应质突触体LDH透出率的影响 421
2.2.7 低氧预适应小鼠脑匀浆提取液对PC12细胞的保护效应 423
2.3 神经组织免疫细胞化学实验 425
2.3.1 延髓背角和中缝太核内的P物质样阳性结构免疫细胞化学或免疫荧光细胞化学染色法 425
2.3.2 大鼠三叉神经节内阿片μ受体与降钙素基因相关肤共存的阳性神经元免疫荧光细胞化学双重标记染色法 428
2.3.3 大鼠延髓背角浅层内P物质样阳性终未与啻钙结合蛋白神经元的联系免疫荧光细胞化学双标染色及激光扫描共焦显微镜观察 430
2.3.4 面口部注射甲醒榕液后大鼠延髓背角内的FOS样阳性神经元观察一一免疫细胞化学染色法 432
2.3.5 大鼠中缝核簇内5 起色胶样阳性神经元表达FOS蛋白一一免疫细胞化学双标染包法 435
2.3.6 大鼠延髓背角内向丘脑投射的FOS 样阳性神经元逆行标记与免疫细胞化学双标染色法 437
2.3.7 大鼠三叉神经节内钙结合素mRNA 阳性神经元的分布放射性核素标记的原位杂交组织化学法 440
2.3.8 大鼠中脑导水管周围灰质内的5-羟色胶样阳性亚微结构免疫电镜法 444
2.3.9 大鼠孤束核内GABA 能纤维终末与F物质受体样阳性神经元的突触联系包埋前与包埋后免疫电镜双标记法 446
2.3.10 大鼠延髓背角内GABA 能神经元与F物质能纤维终末的突触联系包埋前免疫电镜双标记法 449
2.4 神经形态学实验 452
2.4.1 大鼠脊髓灰质向孤束核的投射 452
2.4.2 猫脊髓背角神经元向外侧颈核和背索核的分支投射 454
2.4.3 大鼠脊孤束背索突触后神经元的超（亚）微结构 455
2.4.4 大鼠脊孤束背索突触后神经元对躯体感觉核与内脏感觉核的分支投射 457
2.4.5 大鼠脊髓立体定位磁控过半夹断模型 459
2.4.6 大鼠臂旁核向杏仁中央核的投射HRP逆行追踪方法 461
2.4.7 大鼠中脑导水管周围灰质向伏核的5-羟色胶能投射H即逆行迫踪与免疫细胞化学染色相结舍的双标记法 464
2.4.8 大鼠延髓背角内P物质受体样阳性神经元向丘脑胶状质核技射荧光素逆行追踪与免疫荧光染色相结合的双标记方法 466
2.4.9 大鼠中缝大核向脊髓背角和延髓背角的分支投射荧光素双标记法 469
2.4.10 中脑导水管周围灰质和中缝背核内5-羟色胶能神经元的下行分支投射荧光素双标记与免疫荧光染色相结合的三标记法 472
2.4.11 大鼠三叉神经脊柬核吻侧亚核向三叉神经运动核的投射一一植物凝集素（PHA-L）顺行示踪法 474
2.4.12 大鼠延髓背角浅层向臂旁外侧核及丘脑腹后内侧核的投射一-BDA 顺行示踪法 477
2.4.13 大鼠中脑导水管周围灰质中缝大核三叉神经感觉核簇的间接投射PHA-L顺行示踪与HRP逆行追踪相结合的双标记法的光镜观察 480
2.4.14 大鼠中脑导水管周围灰质-中缝大核-三卫神经脊束核尾侧亚核的间接投射PHA-L顺行示踪与HRP逆行追踪相结合的双标记法的电镜观察 483
2.4.15 大鼠延髓背角向丘脑技射神经元与5-羟色胶阳性终末的突触联系HRP逆行追踪与免疫细胞化学染色双标记法 486
2.4.16 大鼠孤束核-臂旁核-中央杏仁核的同接投射通路——溃变与HRP 逆行追踪相结合的双标记法 489
2.5 分子神经生物学实验 492
2.5.1 用差异显示法分离特异表达的基因片段 492
2.5.2 慢性缺氧培养细胞中缺氧诱导因子-1的提取与检测 497
2.5.3 大鼠三叉神经节总RNA的提取及cDNA的制备 500
2.5.4 5-HT，受体亚型mRNA在大鼠三叉神经节的表达 502
2.5.5 乙酷胆碱转移酶在大鼠纹状体的表达及其DNA 片段的回收 505
2.5.6 乙酷胆碱转移酶DNA片段的亚克隆 507
2.5.7 ChAT-pGEM重组质粒DNA的制备及限制性酶酶切分析 510
2.5.8 ChAT-pGEM重组质粒DNA序列的测定 513
2.5.9 乙酷胆碱转移酶表达蛋白的SDS 聚丙烯酷胶凝胶电泳分析 516
2.5.10 乙酷胆碱转移酶在大鼠纹状体分布的Western印迹检测 520
2.5.11.性激素对周围伤害性刺激诱导脊髓PPDmRNA表达上调的影响 523
2.5.12 坐骨神经部分切断后初级感觉神经元（背根节）的差异表达基因克隆 526
2.6 神经行为学实验 530
2.6.1.一足致炎大鼠双足痛感受性的变化 530
2.6.2 甲醛溶液致炎大鼠疼痛行为的观察 532
2.6.3 神经反射在一足致炎大鼠非致炎足痛阔变化中的作用 533
2.6.4 体液因素在一足致炎大鼠非致炎足痛阑变化中的作用 536
2.6.5 急性缺氧预适应对小鼠缺氧耐受性的影响 537
2.6.6 麻醉与兴奋小鼠缺氧耐受性的变化 539
2.6.7 大鼠脊髓横断及半横断模型的复制 541
2.6.8 慢性束缚应激对大鼠空间学习记忆能力的影响 543
2.6.9 创伤后应激障碍模型大鼠的自发活动和焦虑水平检测 548
3 神经生物学资料 552
3.1.神经生物学常见概念 552
3.1.1.生物电学常见概念 552
3.1.2.生物化学常见词汇 558
3.1.3.细胞培养常见词汇 561
3.1.4.分子生物学常见词汇 565
3.2 常用的实验方法 570
3.2.1.电生理学仪器方法 570
3.2.2 动物实验的实施 577
3.3 实验动物常用数据 589
3.3.1 实验动物常用生理数据 589
3.3.2 实验动物常用麻醉剂与肌肉松弛剂 589
3.4 常用试剂、缓冲液、贮存液与酶的配制 592
3.4.1 组织培养常用试剂 592
3.4.2 电泳缓冲剂 593
3.4.3 常用贮存液
3.4.4 常用酶的配制 597
3.5 常用限制性酶识别序列 598
3.6 常用细胞系、细胞培养基、抗生素 603
3.6.1 细胞系 603
3.6.2 常用培养液成分及配方 605
3.6.3 抗生素 607
3.7 核酸、蛋白质常用数据及相对分子质量标准参照物 607
3.7.1 常用核酸的长度与相对分于质量 607
3.7.2 常用蛋白质分子质量标准参照物 607
3.8 赫尔辛基宣言Ⅱ 608
CONTENTS
FOREWORD
PREFACE
1 Experimental Methhodology in Neurobiology 1
1.1 Methodology of Scientific Thinking 1
1.1.1 Historical impetus of science and technology development 1
1.1.2 Dialectically to seek 3
1.1.3 Dialectically to think 7
1.1.4 Dialectically to verify 17
1.1.5 Dialectically to train 24
1.2 Methodology of Experimental Design 29
1.2.1 Project option 30
1.2.2 Professional design 44
1.2.3 Control design 54
1.2.4 Statistical design 63
1.3 Methodology of Experimental Analysis 79
1.3.1 Data manipulation 79
1.3.2 Statistical analysis 88
1.3.3 Professional analysis 100
1.3.4 Paper writing 109
1.4 Methodology of Electrical Stimulation 117
1.4.1 Basic principle of electrical stimulation 117
1.4.2 Physical properties of electrical stimulation 119
1.4.3 Biological properties of neural preparation 123
1.4.4 Selective stimulation 127
1.4.5 Stimulating current spread 132
1.5 Methodology of Electrical Recording 136
1.5.1 Recording in volume conductor 136
1.5.2 Evoked potential recording 143
1.5.3 Single unitary recording 150
1.5.4 Computer associated recording 158
1.5.5 Intracellular recording 161
1.5.6 Patch clamp recording 174
1.5.7 Determination of velocity spectrum of nerve fibers 178
1.5.8 Determination of location ofaxonal burfication 182
1.5.9 Determination of pain threshold by paw pressing 187
1.6 Methodology of N eurochemistry 190
1.6.1 Tissue and cell fragmentation 190
1.6.2 Synaptosome preparation 193
1.6.3 Electrophoresis 198
1.6.4 Chromatography 203
1.6.5 HPLC 213
1.6.6 Microdialysis 222
1.7 Methodology of Chemical Neuroanatomy 229
1.7.1 Immunocytochemical technique 229
1.7.2 In situ hybridization histochemical technique 239
1.7.3 Receptor localization technique 248
1.7.4 Immunochemical electron microscopic technique 250
1.8 Methodology of N euromorphology 253
1.8.1 HRP tracing technique 253
1.8.2 Fluorescence tracing technique 260
1.8.3 Isotope tracing technique 262
1.8.4 Phaseolus vulgaris leucoagglutinin anterograde tracing technique 269
1.8.5 Laser scanning confocal microscopic technique 271
1.8.6 Flow cytometer technique 278
1.9.Methodology of Molecular Neurobiology 282
1.9.1 Nucleic acid hybridization technique 282
1.9.2 Immunoblotting technique 290
1.9.3 Recombinant DNA technique 296
1.9.4 Polymerase chain reaction technique 306
1.9.5 DNA sequencing 313
1.9.6 mRNA differential display technique 323
1.9.7 Gene chip technique 329
1.9.8 Transgenic animal technique 334
1.10.Methodology of Neural behavior observation 338
1.10.1 Neural bases and commonlg used animals for the behavioral tests 339
1.10.2 Commonlg used methods to test the higher brain funcions 340
1.10.3 Commonlg used methods to examine nociceptive behavior 348
1.11.Brain Imaging 352
1.11.1 Computerized tomography 352
1.11.2 Magnetic resonance imaging 356
1.11.3 Radionuclide tomography 362
1.11.4 Ultrasonography 365
2 Experiments in Neurobiology 368
2.1 Experiments of Neurophysiology 368
2.1.1 Compound action potentials of peripheral nerve in the rabbit 368
2.1.2 Velocity spectrum of afferent nerve fibers in hind limb of rabbit 370
2.1.3 Effects of extending anus to discharges of sacral nerve in the cat 372
2.1.4 Recording of segmental and desending spinal field potentials in the rat 374
2.1.5 Effects of segmental ischemia on spinal field potentials in the rat 375
2.1.6 Recording of evoked cortical potentials of the rabbit 377
2.1.7 Changes of cortical evoked potentials during cerebral ischemia in the rabbit 379
2.1.8 Recording of resting and action potentials of rat DRG neurons 381
2.1.9 Electrical properties of cu1tured cell's membrane in the rat 384
2.1.10 lntracellular recording of action potentials of DRG neurons in the rat 386
2.1.11 Intracel1ular and extracellular recording of electrical activity of dorsa1 column postsynaptic neurcns in the cat 388
2.1.12 Antidromic and orthodromic responses of SCT-DCPS neurons in the rat 390
2.1.13 Intracellular and extracellular recording of electrical activities of dorsal horn neurons in the rat 392
2.1.14 Intracellular and extracellular recording of electrical activities of SST-DCPS neurons in the rat 394
2.1.15 Responses of NRM to peripheral afferent inputs in the rabbit 397
2.1.16 Extracellular recording of nucleus VPL of thalamus in the rabbit 398
2.1.17 Interaction between somatic and visceral afferent input in rat spinal cord dorsal horn neurons 400
2.1.18 Role of ATP-sensitive potassium channel in cerebral hypoxia and its preconditioning 401
2.2.Experiments of N eurochemistry 408
2.2.1 Effects of brain extracts of hypoxia tolerant mice on hypoxic tolerance in mice 408
2.2.2 Changes of monoamnie in brain tissue of mice exposed acutely and epeatedly to hypoxia 411
2.2.3 Effects of diflerent local stimulation on monoamine content in rabbit cerebrospinal fluid 414
2.2.4 Effects of different local stimulation on Ca2+，Mg2+ contents in rabbit cerbrospinal fluid 417
2.2.5 Measurement of adenosine of rabbit brain using microdialysis 420
2.2.6 Effects of brain extract of hypoxia preconditioned mice on ccerbral synaptosome activity 421
2.2.7 Protection of brain tissue extracts of hypoxia preconditioned mice from hypoxic insult of PC12 cells 423
2.3 Immunohistochemical Experiments for Neural Tissues 425
2.3.1.Substance P-like immunoreactive structures in the dorsal hom of medulla oblongata and nucleus raphe magnus in the rat 425
2.3.2 μ-opioid receptor-like and calcitonin gene-related peptide-like immunoreactive posivtive
neurons coexisting in the trigeminal ganglion in the rat 428
2.3.3 Connections of substance P-like immunoreactive positive terminals and calcium-binding proteins-like reactive positive neurons in the superficiallaminae of the dorsal horn of medulla oblongata in the rat 430
2.3.4 Observation of FOS-like positive neurons in the dorsal horn of the medulla oblongata after orofacial injection of formalin in the rat 432
2.3.5 5-hydroxytraptamine-like positive neurons in the raphe nuclei expressing FOS protein 435
2.3.6 Projection from neurons in the dorsal hom of medulla oblongata to the thalamus expressing FOS protein 437
2.3.7 Distribution of Calbindin-D28k mRNA positive neurons in the trigeminal ganglion in the rat 440
2.3.8 Serotonin-like positive structure in the periaqueducal gray 444
2.3.9 Synaptic connection of GABAergic terminals and substance P receptors-like positive neurons in the nucleus of the solitary tract in the rat 446
2.3.10 Synaptic connection of GABAergic neurons and substance P-ergic terminals in the dorsal horn of the medulla oblongata in the rat 449
2.4 Experiments of N euromorphology 452
2.4.1 Spinal cord dorsal horn neurons projecting to STN in the rat 452
2.4.2 Spinal cord dorsal horn neurons projecting to both LCN and DCN in the rat 454
2.4.3 Ultra microstructure of rat SST-DCPS neurons 455
2.4.4 SST-DCPS neurons projectng to both somatic and visceral sensory nuclei in the rat 457
2.4.5 A model of electromagent-controlled pinching semi-transection injury in rat spinal cord 459
2.4.6 Projection from the parabrachial nuc1eus to the central nuc1eus of amygdaloid in the rat 461
2.4.7 Serotoninergic projrctions form the midbrain periaqueductal gray to the nucleus accumbens in the rat 464
2.4.8 Substance P receptor-like immunoreactive neurons in the dorsal horn of medulla oblongata send axons to the gelatinosus thalamic nucleus in the rat 466
2.4.9 Collateral projections of nucleus raphe magnus to dorsal horn of niedulla oblongata and spinal cord in the rat 469
2.4.10 Descending collateral projections of serotoninergic neurons in the periaqueductal gray and dorsal raphe nucleus 472
2.4.11 Proj ection from spinal trigeminal nucleus to trigeminal motor nucleus in the rat 474
2.4.12 PHA-L anterograde tracing 477
2.4.13 Projection from superficiallaminae of dorsal horn of medulla oblongata to parabrachial nucleus and ventroposterior medial thalamic nuclues 480
2.4.14 Indirectly projection from the periaqueductal gray to the trigeminal sensory nuclei via the nucleus raphe magnus 483
2.4.15 Indirectly projection fronithe periaqueductal gray to the spinal trigeminal nucleus via the nucleus raphe magnus 486
2.4.16 Synaptic connection of neurons in the dorsa1.horn of medulla oblongata projecting to thalamus and serotonin-like positive terminals 489
2.5 Experiments in Molecular Nueurobiology 492
2.5.1 Isolation of specifically expressed genes by differential display 492
2.5.2 Separation and detection of hypoxia inducible factor 1 from culture cells after chronic hypoxia 497
2.5.3 Total RNA extraction and cDNA preparation of the rat trigeminal ganglion 500
2.5.4 Expression of 5-HT3 receptor subtype mRNA in the rat trigeminal ganglion 502
2.5.5 Expression of choline acetyltransferase in the rat striatum and the recovery of its DNA fragment 505
2.5.6 Subcloning of choline acetyltransferase DNA fragment 507
2.5.7 Preparation of ChAT-pGEM recombinant plasmid DNA and analysis by restriction enzyme digestion 510
2.5.8 Sequencing of ChAT-pGEM recombinant plasmind DNA 513
2.5.9 SDS-polyacrylamide gel electrophoresis analysis of choline acetyItrandferase-expressed protem 516
2.5.10 Western blot analysis of choline acetyltransferase in the rat striatwn 520
2.5.11 Effects of sex hormone to the upregulation of PPD mRNA in the spinal cord induced by peripheral noxious stimulation 523
2.5.12 Cloning of diffeential-expression gene in primary sensory neurons（dorsal root ganglion） after partial transection of sciatic nerve by DD-PCR method 526
2.6 Experiments of Neural Behavior 530
2.6.1 Bilateral changes of nociceptive sensitivity in rats with one paw inflamed 530
2.6.2 Score of nociceptive responses in inflammated rat by formalin injection 532
2.6.3 Role of neural reflex factor in changes of nociceptive sensitivity of non-inflamed side in rats with one paw inflamed 533
2.6.4 Role of humoral factor in changes of nociceptive sensitivity of non-inflamed side in rats with one paw inflamed 536
2.6.5 Effects of acute repeated hypoxia on hypoxic tolerance of mice 537
2.6.6 Changes of hypoxic tolerance in anesthetized and excited mice 539
2.6.7 Reproduction of spinal cord transection and hemitransection model in the rat 541
2.6.8 Effects of the chronic restrain stress on the spatial learn and memory ability in the rat 543
2.6.9 Examinations on the spontaneous activity and anxiety level in the post-trauma stress disorder model of rat 548
3 Information in Nenrobiology 552
3.1.Common Concepts in Neurobiology 552
3.1.1 Common concept of biological electricity 552
3.1.2 Common vocabulary of biochemistry 558
3.1.3 Common vocabulary of cell culture 561
3.1.4 Common vocabulary of molecular biology 565
3 2 Ordinary Experimental Method 570
3 2.1 Customary instrument and its usage in electrophysiology 570
3.2.2 Animal m.anagenient in common use 577
3.3 Regular Data of Experimental Animals 589
3.3.1 Natural data of physiology 589
3.3.2 Nonnal anesthetics and musc1e relaxants 589
3.4 Preparation of Regular Reagents，Buffers，Stocks Solution and Enzymes 592
3.4.1 Reagents of tissue culture 592
3.4.2 Electrophoretic buffers 593
3.4.3 Regular stock solution 595
3.4.4 Preparation of regular enzymes 597
3.5 Recognition Sequences of Ordinary Restriction Enzymes 598
3.6 Cell Line，Cell Culture Medium and Antibiotics 603
3.6.1 Cellline 603
3.6.2 Composition and prescription of regular cell culture medium 605
3.6.3 Antibiotics 607
3.7 Data of Nucleic Acid and Protein in Common Use 607
3.7.1 Length and molecular weight of nuc1eic acid 607
3.7.2 Molecular weight marker of protein 607
3.8 The Declaration of Helsinski Ⅱ 608

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