美国国立卫生研究院R01 DK55619，美国国立卫生研究院R21 CA139063

1。动物<ol><li>所有的程序获得批准的机构动物护理和使用委员会新泽西医学院大学和牙科。 </li><li>集团内部雄性C57BL / 6小鼠在12小时light/12小时黑暗的时间表，并允许自由采食获得水和食物。牺牲在4-5个月之久。使用麻醉的手术平面和安乐死的次要形式（ 即通过隔膜穿透切口进入胸腔）进行小鼠安乐死。这是关于安乐死的AVMA准则是一致的。 </li></ol> 2。灌注液，盖玻片，玻璃滴管，与媒体的制备<ol><li>使由2.5mM的氯化钾，7毫摩尔MgCl 2，1.25毫的NaH 2 PO 4，28 mM的的NaHCO 3，0.5mM的氯化钙的1L灌注溶液，7毫摩尔葡萄糖，1mM的抗坏血酸盐，并在蒸馏水DH 2 O 3 mM的丙酮酸调节渗透压〜300毫渗量使用约80g / L蔗糖。含氧化合物通过鼓泡用95％O 2/5％CO 2和pH值调节到7.4。每个实验将需要大约200毫升灌注液。等分试样可被储存在-20℃下长达2个月。 </li><li>使含有的Neurobasal媒体不含葡萄糖，2.5毫米的葡萄糖，和100 U / ml青霉素链霉素250毫升的Neurobasal股票。用蔗糖调整的Neurobasal股票的渗透压摩尔浓度为280毫渗量。让500毫升休眠含Hibernate的一个媒体没有葡萄糖，2.5毫米的葡萄糖，和100 U / ml青霉素链霉素股票。 </li><li>使用Stericup真空过滤单元挑起两只股票好，过滤灭菌。要小心，不要透过玻璃或搅拌棒采用葡萄糖或其他污染物。包装瓶的贴膜，保护媒体的光，并在4℃下保存长达2个月。 </li><li>火焰4蒸压玻璃吸液管（9）的提示，这样的提示是没有卤味NGER尖锐和开口直径大的（〜0.9毫米），中大（约0.7mm）上，介质（〜0.5mm）的，和小（〜0.3mm）上。最大的应该勉强抛光和最小的应该是关于直径的三分之一。 </li><li>在收获的神经元，用70％的乙醇，并通过在本生灯火焰干净425毫米盖玻片的前一天。将每个盖玻片在无菌35毫米培养皿中，加2毫升无菌1％多聚-D-赖氨酸在DH 2 O。能把碗碟放进培养箱（37℃）过夜。第二天，洗用无菌DH 2 O 3倍，并允许盖玻片充分干燥。 </li><li>设为1M的乳酸和GlutaMAX的200微升等分75微升等分;储存于-20°C。使用前充分解冻乳酸和GlutaMAX的等分，以确保均匀性。 </li><li>采伐神经元的日子，让加入30ml新鲜培养基组成的休眠-A含有1mM乳酸传输，0.5mm的GlutaMAX，和2％B27的无胰岛素股票。 VORTEX并用1N的NaOH调节pH值至7.4。 </li><li>放入冰60毫米的玻璃培养皿10毫升培养基，放2毫升15毫升锥形管上的冰，并保持剩余介质在室温下。 </li><li>上收获的神经元的天，使加入25ml新鲜的生长培养基包括含有1mM乳酸，0.5毫米的GlutaMAX，和2％B27无胰岛素的Neurobasal股票。涡旋并用1N的NaOH调节pH值至7.4。让新鲜的生长培养基中，以在培养箱中（37℃，5％CO 2）的平衡至少30分钟。 </li></ol> 3。心脏灌注<ol><li>充分解冻200毫升灌注溶液和含氧化合物用95％O 2/5％CO 2在冰上至少15分钟。 </li><li>用丙酮，70％乙醇和DH 2 O洗vibratome刀片放置在vibratome刀片。 </li><li>冲洗vibratome冷却室和灌注管，带DH 2 O。填补冷却室（4℃）与灌注溶液，并继续充氧。 </li><li>麻醉小鼠用戊巴比妥钠50毫克/毫升，1:1稀释，用无菌水，腹膜内注射。验证鼠标完全被捏的尾巴，观察无反应麻醉。 </li><li>倒入冷灌注液注入灌流水库和管道之前，为了清扫术。除20-30毫升的大脑解剖，并继续氧合冰上。从提升的60毫升注射器通过管道自流和20号针头提供足够的流量。让解决方案来运行，直到气泡被冲了出去。继续充氧。 </li><li>切开背部皮肤鼠标的胸腔以上。提起肋骨，并通过膜片仔细做一个横向切。使两侧切口，通过胸腔向上朝手臂，露出心脏。使用镊子忍住胸腔。 </li><li>做小了2 mm切割在右心房提供一个退出点血被淘汰的vascu的拉尔系统。 </li><li>穿刺左心室灌注针头，就足以插入针开幕。保持针就位，用一只手，开始灌注溶液用另一只手的流动。经过10〜15毫升灌注液，血液应该被淘汰，出水将清除。 </li></ol> 4。脑切片和解剖<ol><li>心脏灌注后，转移充氧冷灌注液在冰上用前一个培养皿。 </li><li>快速斩首，从头骨取出大脑，并将大脑进入灌注液。为了消除大脑：皮肤拉向前，做一个中线切向眼窝头骨，使2向每只眼睛外侧切口，用血管钳拉回来头骨的每一侧，使眼窝之间的冠状切口，并用刮刀轻轻哄大脑伸到灌注液。用剪刀在必要时切断视神经束。不要触摸下丘脑c区和不拉视束，因为这使在正中隆起及相关丘脑组织中的压力太大了。 </li><li>用剃刀刀片和针横向修整脑组织大约3mm后壁和前壁至下丘脑而组织浸入（囟-3.52毫米）。对于前侧切口为电平使大脑将直坐于vibratome夹头它是特别重要的。 </li><li>从溶液中取出剩余的脑组织，用滤纸吸走的解决方案从安装的大脑部分。 </li><li>将强力胶民建联在vibratome夹头和传播胶水到大脑的大小。把脑组织上胶的顶部与前侧朝下，下丘脑面向叶片。威克走多余的胶水，然后将吸盘到vibratome冷却室。谨慎使用，因为它会冒泡周围脑时，放入溶液中不留下太多胶水。 </li><li>用振动磨机的E设置为慢速（2级）和高振幅（9级），使300-500微米的切片，直到到达下丘脑区域。现在做薄100微米片切割从后到前。视觉提示是如下。后路下丘脑第三脑室将是非常小的（前囟-2.70至-2.30毫米）。于前囟-2.30毫米，第三脑室分为背，腹部分的冠状切片上。 </li><li>一旦第三脑室保险丝的部分，使两台500微米的切片，其中将包含VMH的正确的区域（前囟-2.18至-1.22毫米）。前向VMH，第三脑室不再延伸到脑的腹侧地板8（-1.06至-0.82囟）。 </li><li>转移这些切片在含冰培养基的培养皿中。解剖VMH（VMN + ARC）， 如图1。用吸管轻轻转让VMH片在2毫升培养基上冰。 </li></ol> 5。解离和文化<ol><li>从冰中取出VMH并放置在室温下。加入20 U / ml的木瓜蛋白酶〜4毫升培养基。颠倒混合并置于34℃水浴。检查并颠倒混匀每分钟，直到消化媒体不再浑浊。过滤（0.22微米）到无菌烧瓶中，并在组织转移到消化介质。 </li><li>在100转摇在34℃30分钟消化的组织。 </li><li>制备1ml含在消化期间8％牛血清白蛋白（BSA）的介质。溶解80毫克BSA的1ml培养基和过滤器（0.22微米）到锥形管中。 </li><li>通过转移到5ml培养基在锥形管，慢慢地倒洗一次组织。 </li><li>加入30微升的DNA酶酶至6 ml培养介质和混合，使研磨介质。吸出冲洗介质和，加入3 mL研磨介质。 </li><li>用玻璃吸管交运集团的以磨碎argest到最小。轻轻磨碎10倍，然后等待4分钟的大块来解决。使用第二个吸管转移前2毫升含一个分离的细胞悬液，以一个新的锥形管。加入2ml研磨介质，磨碎10倍，并等待3分钟。使用第三个移液管顶端2毫升转移到细胞悬浮液。加入1 ml的研磨介质，磨碎5倍，并等待2分钟。使用第四个移液管顶端2毫升转移到细胞悬浮液。 </li><li>层上的8％BSA的顶部分离的细胞悬液，小心不要混用。离心机在1,000 rpm离心5分钟。 </li><li>将蒸压6毫米毫米x 8毫米克隆在每个盖玻片的中央圆柱体。盖玻片必须完全干燥。吸和悬浮颗粒在440微升温暖的生长介质。填克隆缸与神经元悬浮液并置于培养箱中20分钟。 </li><li>取出克隆缸和轻轻洗去杂物。填写每道菜的Wi日2毫升生长介质。让细胞恢复至少1小时的任何实验都进行前。 </li></ol> 6。对于MPD成像准备<ol><li>使由121 mM氯化钠，4.7毫米氯化钾，2毫米氯化钙 ，0.1毫米MgCl 2，1.2毫米硫酸镁4，0.97毫米的K 2 HPO 4，0.23毫米KH 2 PO 4，5毫米碳酸氢钠3，和25 mM的录音解决方案HEPES。将pH调节至7.4。 </li><li>添加葡萄糖，使所需的低血糖测试解决方案（0.1-2.0毫米的葡萄糖）。调匀和预定400毫升低血糖录音解决方案。添加葡萄糖其余600毫升使2.5毫米的葡萄糖录音解决方案调匀。 </li><li>从光尽可能地保护染料。加4毫升室温下缓冲液至蓝MPD小瓶中。调匀，加入每毫升录音解决方案5微升染料。保持均匀的溶液用移液管的解决方案之间的混合。染料含有荧光米olecules，它进入细胞去极化，和猝灭剂分子，从而不能穿过细胞膜。等分部分可被存储在-20℃下和在4天内使用。不冻融一次以上。 </li><li>孵育2毫升2.5毫米葡萄糖录音解决方案，加上染料在34℃，30分钟的细胞。干式加热块都可以使用。避光。 </li><li>制备的注射泵和灌注系统以2.5毫米和0.1毫米记录解决方案，加上染料。从60毫升注射器管应连接到一个歧管。 </li><li>停止所有泵后，等待〜在歧管交换解决方案前10秒，以防止压力积聚在注射器中。压力的变化改变流体输送到含有神经元的封闭腔室，图像的实验和失真中的变化引起在显微镜焦点。 </li><li>歧管输出管应连接到一个嵌入式加热器，然后聚乙烯管，它连接到一个封闭的腔室。<html>气泡应该被清除和灌注速度应设定为0.5ml /分钟。避光。 </li><li>转让25毫米盖玻片贴壁神经元的封闭腔，小心不要让盖玻片干，不要引入气泡。滑移是在底部件的凹槽对准，记录溶液几个滴置于沿着侧面和顶片轻轻地嵌合到保持盖玻片到位。 </li><li>使用滴管以填充该腔室与记录溶液中，然后放置一个18毫米盖玻片上顶。轻轻适合白环入腔以保持较小的盖玻片到位。压下速度非常慢，轻轻防止气泡被引入到密封室。 </li><li>启动注射泵为2.5毫米的葡萄糖录音解决方案，摁倒在白色的环，并连接到封闭室。慢慢地从白环释放压力，并连接到管道通往废物容器。 </li></ol>E“&gt; 7。MPD成像<ol><li>使用低亮场光中心的神经元。尽量减少时间的细胞被暴露于光的量。 </li><li>允许灌注系统运行10分钟，以允许温度时，聚焦的稳定化，以及染料的平衡。 </li><li>而吸，打开的MetaMorph程序，并采取神经元的明场图像（10X）。使用自动对焦功能拍摄3栈的荧光图像（150毫秒，窄的Cy3过滤器，10X），并确定在5微米的焦点。在10分钟灌注期间结束，检查对焦一次。 </li><li>开始40分钟记录荧光图像，每30秒。建立基准为2.5毫米的葡萄糖10分钟，然后降低葡萄糖15分钟，最后返回到2.5毫米的葡萄糖15分钟。解决方案是通过停止注射泵，等待10秒，打开一个端口上的歧管，并开始另一个注射泵改变。变化应该发生提前期望的时间点巴在歧管和细胞之间的滞后sed的。 </li><li>记录所有荧光图像后，将神经元的明场图像。 </li></ol> 8。 MPD影像学分析<ol><li>使用的MetaMorph到在实验结束时拍摄的明场图像创建的每个单元格周围的椭圆形区域。区域应该比每个神经稍大，可制成1个像素的单元格的暗边的外侧。不要选择细胞是不健康的，重叠的，或接近碎片。不健康的细胞往往显得较暗。最后，在没有细胞或碎片为背景测量领域建立5个大型的椭圆形区域。 </li><li>区域传送到所有荧光图像，并检查以确认没有发生移动/班。使用测量功能来记录每个区域的荧光强度在一个Excel文件中的所有图像。中的Metamorph使用期刊的建议。 </li><li>在Excel中，为每个fluoresc平均5个背景区域的耳鼻喉科的形象。这表示在每个时间点的平均背景值。对于每一个图像/时间点，减去所有区域测量背景。在扣除背景的荧光强度将被称为强度以下。 </li><li>对于每一个单元格，在计算分钟的录音2-8的平均强度。这表示在2.5毫摩尔葡萄糖单元的基线。在每次治疗的两端只需两分钟的缓冲，以尽量减少噪音。 </li><li>对于每一个细胞，计算从基线的变化百分比：（强度基线）/基线</li><li>从基线的百分比变化与时间创建的线图。 </li><li>计算从基线时18-23分钟的平均百分比变化。计算从基线时35-40分钟的平均百分比变化。不使用时，图像处理，噪声是通过强度的每个单元区域内的平均化，背景扣除和区域的强度在5分钟ø平均降低ř更长。 </li><li>其中2.5毫摩尔葡萄糖录音解决方案交换与2.5毫米的葡萄糖录音解决方案的对照实验必须执行，以确定噪声阈值。计算从基线时至少6菜肴和3只小鼠18-23分钟的平均百分比变化。确定这些值的平均值和标准偏差。 </li><li>对于正的去极化响应平均值加上2个标准差设定的阈值。平均值加上2个标准差对应的差以p &lt;0.05。我们的控制实验显示，从基线的10％变动为相应的阈值。 </li><li>对于每个小区，评估基于2标准的可逆去极化响应。首先，从基线时18-23分钟的平均百分比变化必须大于10％时，在先前步骤中所确定的阈值。第二，从基线时35-40分钟的平均百分比变化必须小于从平均百分比变化的半在18-23分钟基线。第二准则被选择，因为它被认为是比刺激谷氨酸盐或氯化钾更加严格。不健康的神经元通常以刺激谷氨酸或氯化钾反应，即使他们的反应，降低血糖是不可逆的。 </li><li>对于每道菜，计算神经元去极化的％。这个值用于量化GI神经元的％不同治疗组之间。 </li></ol>

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	<li>Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-sensing+neurons:+are+they+physiologically+relevant?.">Glucose-sensing neurons: are they physiologically relevant?.</a> Physiol. Behav. 76, 403-413 (2002).</li><li>Canabal, D. D., Potian, J. G., Duran, R. G., McArdle, J. J., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+impairs+glucose+and+insulin+regulation+of+nitric+oxide+production+in+glucose-inhibited+neurons+in+the+ventromedial+hypothalamus.">Hyperglycemia impairs glucose and insulin regulation of nitric oxide production in glucose-inhibited neurons in the ventromedial hypothalamus.</a> Am. J. Physiol. 293, 592-600 (2007).</li><li>Canabal, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose,+insulin,+and+leptin+signaling+pathways+modulate+nitric+oxide+synthesis+in+glucose-inhibited+neurons+in+the+ventromedial+hypothalamus.">Glucose, insulin, and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus.</a> American journal of physiology. Reg. Integr. Comp. Physiol. 292, 1418-1428 (2007).</li><li>Murphy, B. A., Fakira, K. A., Song, Z., Beuve, A., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+and+nitric+oxide+regulate+the+glucose+sensitivity+of+ventromedial+hypothalamic+glucose-inhibited+neurons.">AMP-activated protein kinase and nitric oxide regulate the glucose sensitivity of ventromedial hypothalamic glucose-inhibited neurons.</a> Am. J. Physiol. Cell Physiol. 297, C750-C758 (2009).</li><li>Murphy, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fasting+enhances+the+response+of+arcuate+neuropeptide+Y-glucose-inhibited+neurons+to+decreased+extracellular+glucose.">Fasting enhances the response of arcuate neuropeptide Y-glucose-inhibited neurons to decreased extracellular glucose.</a> Am. J. Physiol. Cell Physiol. 296, C746-C756 (2009).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucokinase+is+a+critical+regulator+of+ventromedial+hypothalamic+neuronal+glucosensing.">Glucokinase is a critical regulator of ventromedial hypothalamic neuronal glucosensing.</a> Diabetes. 55, 412-420 (2006).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+hypoglycemia+enhances+glucose+responsiveness+in+some+ventromedial+hypothalamic+glucosensing+neurons.">Prior hypoglycemia enhances glucose responsiveness in some ventromedial hypothalamic glucosensing neurons.</a> Reg. Integr. Comp. Physiol. 294, R784-R792 (2008).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2004).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1998).</li><li>Song, Z., Levin, B. E., McArdle, J. J., Bakhos, N., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergence+of+pre-+and+postsynaptic+influences+on+glucosensing+neurons+in+the+ventromedial+hypothalamic+nucleus.">Convergence of pre- and postsynaptic influences on glucosensing neurons in the ventromedial hypothalamic nucleus.</a> Diabetes. 50, 2673-2681 (2001).</li><li>Song, Z., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+glucose+and+lactate+on+glucosensing+neurons+in+the+ventromedial+hypothalamic+nucleus.">Differential effects of glucose and lactate on glucosensing neurons in the ventromedial hypothalamic nucleus.</a> Diabetes. 54, 15-22 (2005).</li></ol>

我们什么都没有透露。

关键是能够研究神经元的活动从成年小鼠是健康的游离神经元的能力。从成年小鼠的下丘脑神经元的解离更难以在协议几个关键步骤相比，从幼年小鼠的神经元。我们已在许多方面克服了这个问题。制作厚500μm的脑片最大限度地减少机械损伤的神经元相比，用于从年轻小鼠脑组织通常的250-350微米的切片。然而，较厚的切片需要更加重视心脏灌注和脑组织的木瓜蛋白酶消化。如果血液被认为是对脑...

大脑通过神经内分泌和自主神经系统调节能量平衡。腹内侧下丘脑（VMH），由腹内侧核（VMN）和弓状核（ARC）的，是对能量平衡的中枢调节的重要。专门的葡萄糖传感神经元，在VMH内，连接神经元的活动和周边葡萄糖稳 ​​态1。有两种类型的葡萄糖传感神经元;葡萄糖激发（GE）的神经元的增加，而葡萄糖抑制（GI）的神经元活动减少细胞外葡萄糖增加。 VMH葡萄糖感受神经元使用电或钙/膜电位敏感染料成像一般研究。 电生理膜片钳技术被认为是在体外神经元活动的研究的黄金标准。在该技术中，玻璃微电极通过高电阻连接到细胞膜（GΩ）封。膜片钳电极允许在单个神经元动作电位的频率（电流钳）或离子电导（电压钳）实时记录改变。而膜片钳技术提供了关于修改某些离子通道电导的详细信息，一个主要的缺点是只有一个神经元可以在同一时间观测到。这大约需要30-45分钟的记录来验证，一个是记录从葡萄糖传感神经元甚至开始一个具体的实验治疗。此外，GI和GE神经元组成&lt;总VMH神经元群的20％。除了这种问题是缺乏对这些神经元的识别细胞标记的，在许多情况下，。因此，很清楚的是，尽管提供了宝贵的电信息的其它技术不能，膜片钳分析是费力，费时且产量低。 采用分离VMH神经元的荧光成像允许对胡的研究神经元的同时ndreds。钙敏感的染料可以被用于测量细胞内游离钙的变化，从而间接的关联，改变神经细胞的活动。膜电位敏感的染料被用于监控膜电位的变化。测量细胞膜电位是神经元活性相比的变化，细胞内钙水平的更直接的指标。此外，膜电位染料（MPD）成像检测潜在的膜电位变化较小，其中动作电位发放不改变，细胞内钙水平可能不会改变。这两种荧光成像技术已被用于研究VMH葡萄糖传感神经元从幼年小鼠2-7。虽然结果比那些与膜片钳电获得的不太详细，成像实验的实力，他们同时评估一个人口众多的细胞不可避免地包括一个显著的葡萄糖传感神经元的。 MPD成像我S对于研究胃肠神经元这是更均匀地分布在整个VMH本地化特别有用，从而提供足够的人口中分离VMH（〜15％GI）的研究。与此相反，而GE神经元密集地定位于腹外-VMN和ARC和VMN之间细胞贫穷地区，但并不代表一个显著数目的VMH内神经元的（&lt;1％GE）。此外，通过研究分离出神经元，星形胶质细胞和突触前效应被消除。这可能是因为生理连接和过程都将丢失在研究一阶神经元的影响的优点，还有一个缺点。 在这两个膜片钳电生理和MPD /钙染料成像的一个限制因素是需要使用年轻的动物（ 例如 ，小鼠或大鼠&lt;8周龄）。这主要是由于在发生随着年龄减少神经元生存力的组合增加结缔组织。在脑切片电螺柱IES，增加的结缔组织使之更难以以可视化的神经元。增加结缔组织也使得它更难分解大量健康的神经细胞的成像研究。此外，从年轻的动物的神经元或者膜片钳记录或在成像过程中存活时间更长。然而，使用年轻小鼠可以是一个重大的限制。神经元的活动和/或反应性神经递质或循环的营养物质变化与年龄​​。例如，由于能量平衡是紧密联系在一起的生殖状态，下丘脑神经元调节能量平衡可能在前期与postpubescent的动物有不同的反应。此外，许多疾病需要长期治疗或不表现，直到成年。这类疾病的最好的例子是饮食或肥胖2型糖尿病。由于葡萄糖传感神经元被认为在这些疾病中发挥作用，我们开发了一种方法，成功地培养健康成人VMH神经元用于MPD成像EXPeriments。

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Neurobasal-A Medium (Custom)</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">0050128DJ</td>
			<td style="width:427px;">custom made glucose free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Hibernate-A Medium (Custom)</td>
			<td style="width:115px;">BrainBits</td>
			<td style="width:180px;"></td>
			<td>custom made glucose free</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Penicillin streptomycin (20,000 U/ml)</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">15140</td>
			<td>other vendors acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Stericup vacuum filter units (0.22 &#956;m)</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:180px;"></td>
			<td>other vendors acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">25 mm Glass coverslips</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">#1 25mm round</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">18 mm Glass coverslips</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">#1 18mm round</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">GlutaMAX</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">35050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">B27 minus insulin (50x)</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">0050129SA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Razor blade</td>
			<td style="width:115px;">VWR</td>
			<td style="width:180px;">55411</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Vibratome &#38; cooling chamber</td>
			<td style="width:115px;">Vibratome</td>
			<td style="width:180px;">Series 1000 Sectioning system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Vibratome blades</td>
			<td style="width:115px;">Polysciences</td>
			<td style="width:180px;">22370</td>
			<td>injector or double edge blades from other vendors acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Papain, suspension</td>
			<td style="width:115px;">Worthington</td>
			<td style="width:180px;">LS003124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">BSA, suitable for cell culture</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:180px;"></td>
			<td>other vendor acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">DNAse, for cell culture</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;"></td>
			<td>other vendor acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">cloning cylinders, 6 mm x 8 mm</td>
			<td style="width:115px;">Bellco Glass</td>
			<td style="width:180px;">2090-00608</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Membrane Potential Dye (blue)</td>
			<td style="width:115px;">Molecular Devices</td>
			<td style="width:180px;">R8042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">In-line heater</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">SF-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Syringe pumps</td>
			<td style="width:115px;">WPI</td>
			<td style="width:180px;">sp100i</td>
			<td>other vendor acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Closed chamber</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">RC-43C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Polyethylene tubing</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">PE-90</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Metamorph</td>
			<td style="width:115px;">Molecular Devices</td>
			<td style="width:180px;"></td>
			<td>alternate image analysis software acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Microscope</td>
			<td style="width:115px;">Olympus</td>
			<td style="width:180px;">BX61 WI</td>
			<td>
			used with 10X&#160;objective
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Camera</td>
			<td style="width:115px;">Photometrics</td>
			<td style="width:180px;">Cool Snap HQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Narrow Cy3 Filter Set</td>
			<td style="width:115px;">Chroma</td>
			<td style="width:180px;">41007a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Illumination System</td>
			<td style="width:115px;">Sutter Instruments</td>
			<td style="width:180px;">Lambda DG-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

membrane potential dye imaging of ventromedial hypothalamus neurons from adult mice to study glucose sensing

往往是由于随着结缔组织相关的技术难题进行神经元活动的研究利用老鼠不到2个月的婴儿神经元和下降随着年龄的增长出现的神经元生存力。在这里，我们描述了一种方法从成年小鼠岁健康丘脑神经元的解离。从成人 - 老年小鼠的神经元学习能力允许使用的疾病模型，体现在以后的年龄和可能更准确发育的某些研究。分离神经元的荧光成像可以用来研究神经元群体的活动，而不是使用电来研究单个神经元。当研究一种异构神经元群中的所需的神经元类型是罕见的，如用于下丘脑葡萄糖感受神经元，这是特别有用的。我们利用成人腹内侧下丘脑神经元的膜电位染料成像技术来研究他们的反应，茶民营企业在胞外葡萄糖。葡萄糖传感神经元被认为在能量平衡的中枢调节中发挥作用。研究葡萄糖传感成年啮齿类动物的能力，是因为涉及到不正常的能量平衡（ 例如 ，肥胖），随着年龄的增长疾病的优势是特别有用的。

在VMH远离其他下丘脑区的精确解剖重要的是获得一致的结果。列入的其他领域可能会削弱VMH神经元群，改变计算神经元去极化的％。此外，葡萄糖传感神经元已经在其他丘脑区域，例如下丘脑外侧，这可能与在功能上和机械地从VMH葡萄糖感受神经元识别， 图1示出了用于适当的解剖正确的解剖学位置。按照上述协议，包含正确的VMH区域脑组织可以分离。进一步解剖精确地分离VMN和ARC，...

Watch this Scientific Journal Video about Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing at JoVE.com

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

从成年鼠龄的单神经元的活性可以通过从特定脑区域的神经元解离，并使用荧光膜电位染料成像进行研究。通过测试响应变化的葡萄糖，这种技术可以用来研究成年腹内侧下丘脑神经元的葡萄糖敏感性。

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

membrane-potential-dye-imaging-ventromedial-hypothalamus-neurons-from

Research

JoVE Journal

Neuroscience

13.6K 次观看.  Rutgers New Jersey Medical School. 从成年鼠龄的单神经元的活性可以通过从特定脑区域的神经元解离，并使用荧光膜电位染料成像进行研究。通过测试响应变化的葡萄糖，这种技术可以用来研究成年腹内侧下丘脑神经元的葡萄糖敏感性。 

视频: Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.