一种用于大壁虎运动调控的微电极推进器【推荐论文】

一种用于大壁虎运动调控的微电极推进器【推荐论文】

精品论文一种用于大壁虎运动调控的微电极推进器的研制及初步实验王文波1，刘振东2，王浩1，蔡雷2，戴振东15（1.南京航空航天大学，仿生结构与材料防护研究所，南京；2.南京航空航天大学机电学院，南京）摘要：清醒状态下的脑电刺激和记录研究时，不同的实验动物需要不同的微电极推进器。本文介绍了一种新型的微电极推进器，主要用于大壁虎运动调控的相关研究。这种微电极推进器能够在大面积开颅手术后，牢固的安装在大壁虎颅骨上，并在大壁虎清醒状态下推进微电10极。这套微电极推进器包括一个钛合金箱体和一个螺杆推进机构。钛合金箱体可以屏蔽外界信号并隔离大脑和外界的接触，螺杆机构用于微电极的推进。整个微电极推进器长9.6mm，宽9.8mm，高11.8mm，重量为2.05g。该微电极推进器已经成功用于大壁虎的运动调控实验。安装了这种微电极推进器的大壁虎仍能自由活动。实验中发现，微电极位于不同深度时，电刺激大壁虎能够得到不同的运动行为，并且运动行为可以多次重复。15关键词：大壁虎；电刺激；运动诱导；微电极推进器中图分类号：R318;Q337Aminiaturizedmicrodriveforlocomotioncontrolinfreely-movinglizardsGekkogecko20WangWenbo1,LiuZhendong2,WangHao1,CaiLei2,DaiZhendong1(1.InstituteofBioinspiredStructureandSurfaceEngineering,NanjingUniversityofAeronauticsandAstronautics,NanJing;2.CollegeofMechanicalandElectricalEngineering,NanjingUniversityofAeronauticsandAstronautics,NanJing)25Abstract:Forneuralsimulatingandrecordinginneuroethologicstudies,differentcustomdesignedelectrodemicrodrivesarerequiredfordifferentunrestrainedspecies.HereanovelelectrodemicrodrivewasdesignedandmanufacturedespeciallyforthelocomotioncontrolstudiesoffreemovingLizards,Gekkogecko,inwhichextensiveskullopeningisdemandedforelectrodesimplantationinmidbrain.Themicrodrivesystemmainlyconsistsofanenclosed30titaniumcasetoprotecttheskullopeningandshieldexternalsignalandacrew-and-nutmechanismtodrivetheelectrodeplane.Thewholesystemhasbeenminiaturizedto9.6mm×9.8mm×11.8mminvolumeand2.05ginweight,thatissuitablefortheheadmorphologyandloadingcapabilityoftheanimal.ThissystemhassuccessfullybeenappliedtothelocomotioncontrolstudieswithunrestrainedGekkogecko,inwhichlizardsshoweddiversebehaviors35correspondingtothevariationofelectrodedepthandcouldbeefficientlyguidedtoreorientationlaterally.Keywords:Gekkogecko;electricalstimulation;locomotioncontrol;microdrive0引言40大壁虎在空间中的各个表面（地面，墙面，天花板）上具有超凡自如的运动能力，可以实现在固体表面全空间无障碍运动。为了设计出类壁虎仿生机器人，科学家们对大壁虎的运动机制和粘附机理进行了很多研究[1-3]。不少爬壁机器人虽然也能实现三维空间无障碍运动，基金项目：国家重点基础研究发展计划项目(2011CB),自然科学基金面上项目(,),高等学校博士学科点专项科研基金(No.035),南京航空航天大学引进人才科研基金(S0888-GXY)资助课题作者简介：王文波，（1971-），男，助理研究员，主要研究方向：神经信息仿生，动物机器人。E-mail:wwb523@163.com-7-\n精品论文但在运动的协调性、平稳性、环境适应性和能源的利用率方面远远落后于大壁虎[4]。研制出像机器人一样听从指挥的动物机器人，实现动物运动的人工诱导，是未来的发展方向。为实45现对大壁虎运动的人工调控，必须对大壁虎运动相关神经核团的时空编码模式、脑电神经信号与运动行为的相互关系、外界输入电信号或者化学刺激下动物运动行为的变化进行深入研究。为了实现以上目标，必须设计和制造出适用于大壁虎的脑立体定位装置和微电极推进器。王文波等提出了一种大壁虎的脑立体定位方法，首次实现了大壁虎准确的脑立体定位[5]，并运用电刺激和化学刺激方法对麻醉状态下和自由运动时大壁虎的运动行为进行了初步探索50[6]。但在以往的实验中，微电极植入后即被固定。在探索不同深度的脑区电刺激诱发的运动行为时，这种方法效率低下。为了提高效率和实验动物利用率，需要设计一种用于植入微电极的微电极推进器。早在1973年Ranck等就设计了一种用于脑神经信号采集的微电极推进器[7]，此后大量的微电极推进器被用于自由运动动物的脑电记录和脑电刺激研究[8-19]。然而，现有的微电55极推进器主要针对大鼠、猫等动物，无法运用于大壁虎的刺激与记录实验。本文设计了一种用于大壁虎脑电刺激的微电极推进器，它能够把微电极植入到大壁虎脑区不同深度的特定核团。该微电极推进器体积小，重量轻，经济且易于装配。能在大规模开颅手术后牢固的安装在颅骨上，并且不影响大壁虎的自由活动。1材料和方法601.1实验动物实验用大壁虎为体重40-70g的成年大壁虎。大壁虎属爬行纲，有鳞目，蜥蜴亚目，壁虎科。经批准由广西南宁购回并经实验室饲养两个月以上。饲养室中模拟大壁虎的自然生存环境，温度保持在30℃，湿度为60%RH。陈振昆和丁光对大壁虎骨骼系统进行了深入研究[20]。大壁虎额骨与顶骨间具有明显的冠65状骨缝——冠状缝；一对顶骨之间也有比较明显的分界线——矢状缝；两块鼻骨以及鼻骨与额骨之间亦存在明显的分界线（图1）。冠状缝与矢状缝的交点即为囟点（图1b）。（）a囟点矢状缝冠状缝（b）图1大壁虎头部结构示意图.(a)侧面观；（b）背面观Fig.1SchematicdiagramoftheGekkogecko’sskull70-7-\n精品论文-7-精品论文75808590951001.2微电极推进器的设计考虑到结构简单和经济实用，微电极推进器采用螺杆驱动，并设计成分体结构（图2、3）。微电极推进器包括底板（图2a）、垫片（图2b）、螺钉（图2c）、箱体（图2d）、滑块（图2e）和连接器（图2f）。底板用于连接箱体和颅骨，用以把微电极推进器牢固的安装在颅骨上。在大壁虎进食时，顶骨和额骨（图1）可以相对转动，因此微电极推进器不能同时固定在顶骨和额骨上，而只能固定在顶骨上。为了充分利用顶骨的狭小空间，底板的整体轮廓设计成类似于顶骨的形状。并且底板的四周设计成阶梯状，以利于牙科水泥的粘结和固定。底板中央的方形孔作为电极植入区域。底板的上方边缘设有燕尾槽，以便与驱动器箱体相连。底板通过两个半圆孔与颅骨进行相对定位。箱体是微电极推进器中体积最大的部件（图2d）。箱体具有一个U形的滑道，一方面限制了滑块的转动，另一方面用于放置螺钉。旋转螺钉就可以驱动滑块在滑道里上下滑动。在箱体的上方有两个槽，用于放置垫片，下面一块垫片用于对螺钉进行垂直方向的定位，上面一块垫片用于限制螺钉上下运动。在箱体的底部设有燕尾槽，实现与底板连接。一个用PCB改造成的孔阵列板粘附在滑块上用于固定微电极。孔阵列板的孔径为0.25mm，孔距为0.5mm，每个孔均可固定一根微电极。旋转螺钉可以带动滑块上的微电极上下移动，以根据需求将微电极植入不同深度的核团。连接器使用标准的1.27mm排针和排母改造而成（如图2f），用于转接外部电路。图2电极推进器组件.a体和颅骨的底板；b垫片；cM1.5*6的螺钉；d箱体，左右两侧为不同视角；e滑块，左边为已装配好的滑块；f用作引线转接接口的1.27的排针排母Fig.2hecomponentsofthemicrodrivesystem.1.3微电极推进器的组装微电极推进器组装过程中最重要的步骤是螺母和孔阵列板的安装固定。首先将螺母安装在滑块的定位槽中，固定时要保证螺母与上下的圆孔同轴。然后，将孔阵列板固定在滑块上方的阶梯槽上。打磨孔阵列板，使滑块可以在箱体中通畅的滑动。完成滑块的组装后，就可以装配微电极推进器。首先将位于下部的垫片装入箱体中，涂抹少量胶将其固定。然后将螺钉放入垫片的孔中，并将上部的限位垫片插入箱体的开槽中。最后把已装配好的滑块按照装配图装入箱体中，并转动螺钉将滑块升到最上端。-7-\n精品论文-7-精品论文1051101151201251301.4微电极推进器手术固定及微电极的植入麻醉与开颅手术：大壁虎采用0.4%戊巴比妥钠溶液，按30mg/kg体重腹腔注射进行麻醉。将麻醉后的大壁虎固定于大壁虎专用的三维脑立体定位仪[5,6]上，使其头部处于标准定位状态。切开颅顶皮肤后暴露囟点与鼻点。以囟点为定位基准，根据实验需要，采用定位高速颅钻在顶骨适当位置按照底板定位孔间距钻两个定位孔，并拧入定位螺钉。把需要涂覆牙科水泥的顶骨打毛，以增强微电极推进器与顶骨间的结合强度。对大壁虎进行开颅手术，剪去硬脑膜和蛛网膜，暴露实验脑区。底板的固定及微电极推进器的固定：首先在顶骨与底板结合区域堆积少量牙科水泥，形成一个水平平台以保证底板水平放置。将底板放置在平台上，并以定位螺钉定位。然后在底板的前后涂覆少量牙科水泥，将底板固定在顶骨上。大壁虎在进食等状况下，额骨与顶骨之间会绕着冠状缝相对转动。因此在固定底板时，牙科水泥不要越过冠状缝。然后把已装配的箱体装配到底板上。在箱体和底板的四周涂覆牙科水泥。微电极植入：根据囟点位置，在所需要的点植入微电极，并把它们固定在孔阵列板上。固定完所有微电极后，将剩余的孔用脑胶封闭。将连接器粘附在箱体上，用于连接微电极和外部电路。最后缝合大壁虎的头部皮肤，对暴露的皮肤和肌肉组织消毒。待术后恢复进行慢性电刺激实验（图3）。(a)(b)(c)(d)图3手术步骤.(a)固定定位螺钉并对大壁虎实施开颅手术;(b)固定底板;(c)固定组装好的其他部件;(d)植入微电极Fig.3.Surgeryprocedures.1.5慢性电刺激实验在大壁虎恢复四天后，在标记有方格（38mm×38mm）的实验场地（1.2m×1m）中对大壁虎进行术后电刺激实验。刺激电流小于100μA。为确定各个位点所诱发的运动行为的可重复性，每个位点重复刺激三次。同一深度的所有位点实验结束后，逆时针旋转螺钉，使微电极下降到一个新的深度（0.35mm）。对新的电极位置，重复以上过程，直到微电极下降到最低点。每一深度的所有位点电刺激实验结束后，休息30分钟。在接下来的两周中，每经过一周时间在下午五点之后重复一次上述实验。重复实验三次后对大壁虎进行过量麻醉-7-\n精品论文-7-精品论文135140145150155处死、颈总动脉灌流固定、切片、染色，对应大壁虎的脑图谱确定刺激位点，将运动行为与脑内核团相关联。2结果设计制作的微电极推进器长为9.6mm，宽为9.8mm，高为11.8mm，重量仅为2.05g。该微电极推进器易于安装制造，而且与颅骨结合牢固，电刺激实验结果稳定。2.1牢固性和耐久性通过对六只大壁虎的实际验证，安装了微电极推进器的大壁虎，在术后2-4天均可以正常饮食和活动。灌流之前固定于六只大壁虎头部的微电极推进器均与颅骨结合牢固结合牢固无松动和脱落现象。2.2制造安装简易微电极推进器中的螺钉螺母从标准件公司购买，其他大部分部件均可用线切割的方法加工制造。孔阵列板是唯一需要部分手工修配的部件。微电极推进器的组装非常简单，唯一的难点就是组装滑块时要保证螺母和滑块的上下孔同轴。微电极推进器的操作简易，使用者只需顺时针或者逆时针转动螺钉就可以调节微电极的深度位置。2.3电刺激稳定性对术后恢复，清醒状态下的大壁虎所进行的电刺激实验中，可以诱导出大壁虎的左右转、鸣叫、跳跃、横爬等运动，而且重复实验中，这些运动行为仍然能被再次诱发。多次的实验显示，微电极推进器与颅骨之间结合牢固，即使壁虎做出剧烈的运动，微电极的植入位置仍然稳定可靠。实验中对同一植入电极对应的不同深度施加电刺激后，大壁虎表现出左右转、横爬、躲避、鸣叫等一些列差异性运动反应。图4为b1’电极所在位置脑切片与脑图谱的对应图。表1给出了b1’电极不同深度施加电刺激时所诱导的运动行为。其中图5给出了h6深度所诱导出的右转行为录像的截图。图4实验大壁虎组织学切片.图中左侧为慢性电刺激实验后大壁虎脑切片；右侧为标准的脑图谱。微电极不在冠状平面上，因此针道是不完整的。左右两侧的比例尺标尺相同，图中网格最小刻度为：100μm.Fig.4.Histologicalsectionofthelesion.-7-\n精品论文-7-精品论文160165170175180图5.h6位点施加电刺激诱发的右转行为.图片中左下角的数字为截屏图片的帧序号（录像25帧/秒）Fig.5.Rightturningmotioninducedbyelectricalstimulationinfree-movingGekkogecko.3讨论本文中设计的微电极推进器的主要目标是实现植入大壁虎脑区的微电极阵列的在体可调。大壁虎颅骨的特殊结构以及极小的可固定面积给本文中的设计带来了很大的挑战。大壁虎可用于固定微电极推进器的位置为顶骨，面积约为9*10mm。本文根据大壁虎顶骨的形状设计底板的形状，实现了底板的最优化设计，最终实现了微电极推进器牢固固定于顶骨。以往用于脑电记录的微电极推进器均是针对与在颅骨上钻孔或者小范围开颅手术的情况，对于大壁虎这种需要大面积开颅手术的动物并不适合，本文通过箱体的封闭式设计，隔绝了大壁虎大脑与外界环境的直接接触，有利于大壁虎的长期存活，为其他需要大面积开颅手术实验的动物设计微电极推进器提供了新的思路。以往的微电极推进器多是在固定位点植入微电极，在保证微电极推进器体积适当的情况下可植入微电极的数量有限，本文提出一种运用PCB板的方法固定微电极，可以在2*3mm的面积内植入8-10根微电极。每根微电极都可以上下调节。螺钉旋转一圈，微电极进给0.35mm。如果我们对深度方向上进行更加精细的调节，就能够得到大壁虎的运动行为与相关脑区更精细的对应关系，为进一步研究大壁虎的各个脑区的功能提供研究基础。使用这套微电极推进器后，能够明显的提高实验的效率和动物的利用率[21]。此外，采用合适的记录电极，本套微电极推进器也可以用于多通道脑电记录实验。-7-精品论文[参考文献](References)-7-精品论文185190195[1]YuM,JiAH,DaiZD.Effectofmicroscalecontactstateofpolyurethanesurfaceonadhesionandfriction.JBionicEng,2006,3(2):87-91[2]GasparettoA,SeidlT,VidoniR.Amechanicalmodelfortheadhesionofspiderstonominallyflatsurfaces.JBionicEng,2009,6(2):135-142[3]WangZY,WangJT,JiAH,etal.Locomotionbehavioranddynamicsofgeckosfreelymovingontheceiling.ChinSciBull.2010,55(29):3356-3362[4]DickinsonMH,FarleyCT,FullRJ,etal.Howanimalsmove:anintegrativeview.Science,2000,288(7):100-106[5]WangWB,DaiZD,TanH,etal.AstereotaxicmethodandapparatusfortheGekkogecko.ChinSciBull.2008,53(7):1107-1112[6]王文波,戴振东,郭策.电刺激大壁虎(Gekkogecko)中脑诱导转向运动的研究.自然科学进展,2008,18(9):979-986[7]RanckJR.Amoveablemicroelectrodeforrecordingfromsingleneuronsinunrestrainedrats.Brainunitactivityduringbehavior,1973:76-79-7-\n精品论文-7-精品论文200205210215220[8]JeffGK,GeorgeLG.Achronicmulti-electrodemicro-driveforsmallanimals.JNeurosciMeth,2002,117:201-206[9]DistelH.Behaviorandelectricalbrainstimulationinthegreeniguana,IguanaiguanaL.:I.Schematicbrainatlasandstimulationdevice.BrainBehavEvol,1976,13:421-450[10]FeeMS,LeonardoA.Miniaturemotorizedmicrodriveandcommutatorsystemforchronicneuralrecordinginsmallanimals.JNeurosciMeth,2001,112:83-94[11]KeithAS,RichardSO.Multi-electrodemicrodrivearray.UnitedStatesPatent:USB1,2010-08-03[12]CarienSL,MattijsB,WietzeB,etal.Asplitmicrodriveforsimultaneousmulti-electroderecordingsfromtwobrainareasinawakesmallanimals.JNeurosciMeth,2007,162:129-138[13]ManuputyRJD.Microdriveforuseinneurophysiologicalresearchofanimals.EuropeanPatent:.0,2009-12-23[14]DavidKB,GaryMM.Alowcost,highprecisionsubminiaturemicrodriveforextracellularunitrecordinginbehavinganimals.JNeurosciMeth,1999,92:87-90[15]FlorentH,SergejusB,CorneliusS.Aminiaturizedchronicmicroelectrodedriveforawakebehavingheadrestrainedmiceandrats.JNeurosciMeth,2010,187:67-72[16]VladimirFP,GalinaNS,MarkYV,etal.Aremovableperipheraldeviceforintracerebralmicroinjectioninfreelymovingrats.BrainResProtoc,1997,2:31-34[17]VictorAK.Miniaturemicrodrive-headstageassemblyforextracellularrecordingofneuronalactivitywithhigh-impedanceelectrodesinfreelymovingmice.JNeurosciMeth,2006,158:179-185[18]YannickJ,YoonHC.Designofatwintetrodemicrodriveandheadstageforhippocampalsingleunitrecordingsinbehavingmice.JNeurosciMeth,2003,129:129-134[19]StevenJE,WangXQ.Chronicmulti-electrodeneuralrecordinginfree-roamingmonkeys.JNeurosciMeth,2008,172:201-214[20]丁光,陈振昆.大壁虎(Gekkogecko)骨骼系统的解剖.云南农业大学学报.1990,5(1):1-6[21]戴振东,刘振东,王文波,等.微电极推进装置及方法.中国专利:8.2,2010-11-99JWKffwvG#tYM*Jg&6a*CZ7H$dq8KqqfHVZFedswSyXTy#&QA9wkxFyeQ^!djs#XuyUP2kNXpRWXmA&UE9aQ@Gn8xp$R#͑Gx^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu##KN&MuWFA5uxY7JnD6YWRrWwc^vR9CpbK!zn%Mz849Gx^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu##KN&MuWFA5ux^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu##KN&MuWFA5uxY7JnD6YWRrWwc^vR9CpbK!zn%Mz849Gx^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmUE9aQ@Gn8xp$R#͑Gx^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu##KN&MuWFA5uxY7JnD6YWRrWwc^vR9CpbK!zn%Mz849Gx^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu##KN&MuWFA5ux^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu##KN&MuWFA5uxY7JnD6YWRrWwc^vR9CpbK!zn%Mz849Gx^Gjqv^$UE9wEwZ#Qc@UE%&qYp@Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z8vG#tYM*Jg&6a*CZ7H$dq8KqqfHVZFedswSyXTy#&QA9wkxFyeQ^!djs#XuyUP2kNXpRWXmA&UE9aQ@Gn8xp$R#͑Gx^Gjqv^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