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>

<ol>
	<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><tbody><tr><td>Neurobasal-A Medium (Custom)</td><td>Invitrogen</td><td>0050128DJ</td><td>custom made glucose free</td></tr><tr><td>Hibernate-A Medium (Custom)</td><td>BrainBits</td><td></td><td>custom made glucose free</td></tr><tr><td>Penicillin streptomycin (20,000 U/ml)</td><td>Invitrogen</td><td>15140</td><td>other vendors acceptable</td></tr><tr><td>Stericup vacuum filter units (0.22 &amp;mu;m)</td><td>Millipore</td><td></td><td>other vendors acceptable</td></tr><tr><td>25 mm Glass coverslips</td><td>Warner</td><td>#1 25mm round</td><td></td></tr><tr><td>18 mm Glass coverslips</td><td>Warner</td><td>#1 18mm round</td><td></td></tr><tr><td>GlutaMAX</td><td>Invitrogen</td><td>35050</td><td></td></tr><tr><td>B27 minus insulin (50x)</td><td>Invitrogen</td><td>0050129SA</td><td></td></tr><tr><td>Razor blade</td><td>VWR</td><td>55411</td><td></td></tr><tr><td>Vibratome &amp;amp; cooling chamber</td><td>Vibratome</td><td>Series 1000 Sectioning system</td><td></td></tr><tr><td>Vibratome blades</td><td>Polysciences</td><td>22370</td><td>injector or double edge blades from other vendors acceptable</td></tr><tr><td>Papain, suspension</td><td>Worthington</td><td>LS003124</td><td></td></tr><tr><td>BSA, suitable for cell culture</td><td>Sigma</td><td></td><td>other vendor acceptable</td></tr><tr><td>DNAse, for cell culture</td><td>Invitrogen</td><td></td><td>other vendor acceptable</td></tr><tr><td>cloning cylinders, 6 mm x 8 mm</td><td>Bellco Glass</td><td>2090-00608</td><td></td></tr><tr><td>Membrane Potential Dye (blue)</td><td>Molecular Devices</td><td>R8042</td><td></td></tr><tr><td>In-line heater</td><td>Warner</td><td>SF-28</td><td></td></tr><tr><td>Syringe pumps</td><td>WPI</td><td>sp100i</td><td>other vendor acceptable</td></tr><tr><td>Closed chamber</td><td>Warner</td><td>RC-43C</td><td></td></tr><tr><td>Polyethylene tubing</td><td>Warner</td><td>PE-90</td><td></td></tr><tr><td>Metamorph</td><td>Molecular Devices</td><td></td><td>alternate image analysis software acceptable</td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX61 WI</td><td> &lt;p&gt;used with 10X&amp;nbsp;&lt;span style=&quot;font-size: 13px; line-height: 1.6em;&quot;&gt;objective&lt;/span&gt;&lt;/p&gt; </td></tr><tr><td>Camera</td><td>Photometrics</td><td>Cool Snap HQ</td><td></td></tr><tr><td>Narrow Cy3 Filter Set</td><td>Chroma</td><td>41007a</td><td></td></tr><tr><td>Illumination System</td><td>Sutter Instruments</td><td>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 ...

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13.7K 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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

科研

神经科学

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

医学

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

<meta charset="utf8"></head><body>通过小鼠经颅钙成像研究肠脑相互作用

Real-time Analysis of Gut-brain Neural Communication: Cortex wide Calcium Dynamics in Response to Intestinal Glucose Stimulation

Communication between the gastrointestinal tract and the brain after nutrient absorption plays an essential role in food preference, metabolism, and feeding behaviors. Particularly concerning specific nutrients, many studies have elucidated that the assimilation of glucose within gut epithelial cells instigates the activation of many signaling molecules. Hormones such as glucagon-like peptide-1 are renowned as quintessential signaling mediators. Since hormones predominantly influence the brain through circulatory pathways, they slowly modulate brain activity.
However, recent studies have shown two expeditious gut-brain pathways facilitated by the autonomic nervous system. One operates via the spinal afferent neural pathway, while the vagus nerve mediates the other. Consequently, brain responses following glucose assimilation in the gastrointestinal tract are complicated. Moreover, as intestinal stimulation finally induces diverse cortical activities, including sensory, nociceptive, reward, and motor responses, it is necessary to employ methodologies that facilitate the visualization of localized brain circuits and pan-cortical activities to comprehend gut-brain neural transmission fully. Some studies have indicated precipitous alterations in calcium ion (Ca2+) concentrations within the hypothalamus and ventral tegmental area independently through different pathways after intestinal stimulation. However, whether there are changes in cerebral cortex activity has not been known.
To observe cerebral cortex activity after intragastric glucose injection, we developed an imaging technique for real-time visualization of cortex wide Ca2+ dynamics through a fully intact skull, using transgenic mice expressing genetically encoded Ca2+ indicators. This study presents a comprehensive protocol for a technique designed to monitor intestinal stimulation-induced transcranial cortex wide Ca2+ imaging following intragastric glucose injection via an implanted catheter. The preliminary data suggest that administering glucose solution into the gut activates the frontal cortex, which remains unresponsive to water administration.

<meta charset="utf8"></head><body>肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

Gut-brain communication, facilitated by the vagus nerve, is crucial for communication between the gastrointestinal endocrine system and the brain. However, whether intragastric glucose injection can change cortical activity is still not understood. Here, we offer a comprehensive protocol to observe changes in cortical activity after glucose injection into the duodenum.

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

Communication between the gastrointestinal tract and the brain after nutrient absorption plays an essential role in food preference, metabolism, and ...

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

使用基因编码的电压指示器测量 体内 线粒体膜电位

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

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

摘要

探索更多视频

此视频中的章节

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

遗传操纵鼠标的发展下丘脑通过

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

使用基因编码的电压指示器测量体内线粒体膜电位

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

摘要

探索更多视频

此视频中的章节

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

遗传操纵鼠标的发展下丘脑通过

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

肠脑神经通讯实时分析:响应肠道葡萄糖刺激的皮层宽钙动力学

使用基因编码的电压指示器测量 体内 线粒体膜电位

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

使用基因编码的电压指示器测量体内线粒体膜电位