This work was supported by a California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Grant (KAW), a grant from Pfizer (WS753098, SH), and an NIH MARC fellowship (#2T34GM008253-21, JAF). The authors would like to thank Michael Sawchuk, Anusha Allawala, Nina Bubalo, Remie Mandawe and Peter Nguyen for their technical support.

适当的国家和机构伦理应该进行动物实验前获得。 1，拆卸EDL肌肉和神经的<ol><li>称量并深深麻醉成年小鼠吸入异氟烷用5％异氟醚蒸发器加上1.5升/分钟的氧气流量或钟形罩，底部异氟醚浸泡的棉花。 </li><li>确保鼠标是深度麻醉，不给脚趾捏回应。快速斩首使用大，锋利的剪刀或断头台。 </li><li>请通过用剪刀肋骨下腹正中切口和除去内脏。 </li><li>通过抓住从颈部区域的皮肤和拉过去脚部皮肤的动物。 </li><li>通过切割上述的臀部除去腿部和放置面皮腿与冷藏（4℃）的培养皿，carbogenated（95％O 2，5％CO 2）低钙，高镁的碳酸氢盐缓冲的盐水溶液周转箱宁以mM：128氯化钠，氯化钾1.9，1.2 KH 2 PO 4，26的NaHCO 3，0.85氯化钙 ，16 6.5 MgSO 4干燥，和10葡萄糖（7.4±0.05的pH）。低钙，高镁溶液在解剖抑制突触传递。 </li><li>将腿背侧的菜和脚的腿和臀部向下用针或昆虫针，使得膝关节和踝关节是在一个90°的角。放置在脚的每一端与一个销上的每个前侧和后侧的大腿的销来固定到位的组织。 </li><li>使用Castroviejo弹簧剪刀，抬起肌的大腿上的顶层，并切割以暴露坐骨神经直接下方的中线。坐骨神经位于正下方的顶端肌肉层和运行从其出口股骨附近的臀部上方，直到其分支成刚膝关节前腓总和胫神经。 </li><li>删除上述坐骨神经肌肉，直到POINT在此深腓神经的分支潜入拇长屈肌（FHL）肌肉可见。 </li><li>使用＃55钳子，解剖周围腓分支的结缔组织，从腓肠肌和比目鱼肌，这是对内侧胫骨释放它。切断腓肠肌和比目鱼肌的肌腱和小心地从神经下将其删除。 </li><li>除去在胫骨的侧面浅表肌肉以暴露三个不同的腱束在踝关节，从内侧到外侧：FHL，EDL，和胫骨前肌（TA），与EDL隐藏在TA下方。 </li><li>切助教肌腱在踝关节，从EDL解除助教起来了，并切断肌肉膝盖附近将其删除。 </li><li>切FHL肌腱在脚踝，将其返回到到达那里的神经进入FHL的区域。切略低于该点并删除FHL肌肉〜2/3。 </li><li>切断坐骨神经尽可能靠近髋关节为POSsible，轻轻去掉所有的神经分支除外腓深神经分支。 </li><li>切筋EDL在两个用大弹簧剪刀踝关节和膝关节。 </li><li>使用锋利的剪刀，从周围组织中取出的EDL，剩余FHL和神经通过在膝关节切削穿过胫骨和中途的大腿。切去其余的胫骨，这样就在EDL中，信望爱和神经的一部分保留下来。 </li></ol> 2，安装EDL肌肉和神经进入组织浴（图1A） <ol><li>在实验开始之前，准备的组织浴中。不断灌注同浴含氧（100％O 2）合成的组织间液（SIF）含（单位mm）123氯化钠，氯化钾3.5，0.7 硫酸镁 ，1.7 磷酸二氢钠，2.0 氯化钙 ，9.5 NAC 6 H 11 O（葡糖酸钠），5.5葡萄糖，7.5蔗糖和10 N -2 - hydroxyethylpiperazine- N&#39;-2 -等hanesulfonic磺酸（HEPES）; pH值7.4±0.05 17。 15-30毫升/分钟的流速被推荐。显示的是25毫升容量与固定在浴缸的底部，有一个组织安装后和安装的力和长度控制器（约计浴缸尺寸2刺激电极市售洗澡8.5厘米×3厘米×1厘米;适合的产品。参照表的材料/设备）。 </li><li>用剩余的FHL组织处理隔离肌肉神经，并将其放入组织浴。放置一小片SYLGARD对培养皿的底部，并用驱虫针通过剩余FHL组织稳定的肌肉。 </li><li>用6-0丝线缝合扎两个筋并加盖一端到组织后，其他的力和长度控制器的控制杆臂（见表材料/设备规范）。使用最小的缝线长度是可行的。取出昆虫针和SYLGARD你绑筋后。注：为便于连接到所述杠杆臂的小片金属丝可以弯曲成“J”形钩和固定到该杆臂与环氧树脂。缝合线可以被连接到导线，而不是拧入于该杆臂的小孔。 </li><li>请从SA的16玻璃吸电极先用玻璃微拉马（热火= 286，拉= 0，速度= 150，时间= 200）;突破尖端背部和手动磨它放在磨刀石，直到有大约3毫米的锥形。使用microforge尖端融化到10之间所需的尖端内径- 100微米取决于神经的一个希望从取样的区域（ 见图1B，1C的电极原理图和表格材料/设备产品信息）。 </li><li>填写预制玻璃吸电极的内银线与SIF。 </li><li>抽吸神经的切断端插入电极和连接到差分放大器的正端口。包裹在电极与氯化的银导线连接到探头的负极端口。从浴缸到探头的接地端口上运行的第二氯化的银线接地的SIF洗澡。另外地灌注管法拉第笼在多个点，以减轻通过灌注泵推出的电噪声。 </li><li>刺激通过安装在该组织浴中的肌肉的任一侧以诱导肌收缩，​​电极的肌肉。可选地放置在神经的切断端部的刺激电极。增加刺激电压，直到峰值收缩力观察，然后增加电压通过一个额外的15％，达到超强的电压（0.5毫秒脉冲宽度）。继续抽搐收缩在超最大电压，用10秒的休息在中间，但改变肌肉的长度，直到峰值收缩力达到找到肌肉的最佳长度（L O）。所有的长坡道和震动将开始与肌肉在这个冷日。 </li><li>允许肌肉的神经制剂保持在该浴中的至少1小时，随后的数据采集之前，以允许所述组织，以达到熔池温度和正常的突触传递，以恢复以下清扫低钙溶液。 </li><li>为了收集数据，在比室温等的温度，将温度探头到附近的肌肉组织浴。慢慢地由穿过组织浴底板泵送加热的水使浴加热到所需温度。环绕SIF水库粘土微波炉加热垫，以帮助保持稳定的温度。 </li></ol> 3，数据收集<ol><li>要确定一个传入的梭传入，在一个0.5毫秒超强电压刺激产生重复发表抽搐收缩每秒一次记录的神经元活动。注：肌梭传入应抽搐收缩18,19（ 图2）期间暂停。 </li><li>使用DATA采集软件适用长度的变化以不同的速度和不同的长度。使用自定义脚本来自动完成这个任务（见补充信息的脚本和方向如何自定义给出的延伸截图）。应用4秒的斜坡和保持伸展为2.5％，5％，7.5％L O伸缩长度为20，40，或60％L O / 15秒的拉伸速度。注：请参阅具体的力量和长度控制器所需的电压 - 毫米换算系数使用手册。 </li><li>在实验结束时，确定肌肉健康在24℃下使用最大等长强直收缩（500毫秒列车，120赫兹串频率，0.5毫秒的脉冲宽度，超最大电压）。峰值收缩力比较先前报道的值（〜24牛顿/厘米2 9,20）。 </li></ol>

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The open access charges for this manuscript have been paid by Aurora Scientific, Inc., the company that makes the force and length controller and in vitro bath plate used in this manuscript.

这篇文章的目的是描述了一种从肌梭传入在一个孤立的小鼠肌肉，神经的准备记录。我们已经发现，小鼠梭传入响应类似于伸展那些从大鼠，猫和人15，和其它的实验室已经使用小鼠作为模式生物研究中均肌肉和体外的皮肤感觉神经元（例如3,13） 。 肌肉感觉传入可以在两个24°C和34°C的记录至少6-8小时。以最小化处理的EDL和神经，使用FHL的剩余部分，​​以处理组织只要有可能。以下的夹层，等待至少1小时，以启动数据收集，以使组织平衡到浴温度和正常的突触传递，以进行恢复。先前，肌肉健康是在此制备中所用的肌肉的子集，通过确定最大强直禁忌验证由肌肉产生的ctile力是类似的报告由他人在实验15的开始和结束。 肌梭传入进行功能识别在该制备通过在烧制响应于抽搐收缩（ 图2）寻找一个特性停顿以及预期的瞬时频率增加响应长度的变化（ 图3）。根据我们的经验，大多数传入那团火在控制条件是肌梭传入。保留在该制剂（〜7毫米最大）的神经的长度不够长以使用传入传导速度的差异，以确定肌肉传入神经 ​​亚型13。此外，不像猫和人类的动态灵敏度措施未能明确区分组IA和II传入小鼠（进一步讨论见威尔金森等人。15）。传入的其他亚型（ 即，，第III族和第IV）添加物质，如辣椒辣素，ATP，或缓激肽，降低染液的pH，暴露肌肉局部缺血等 3,13,21-23在此制备使用附加的功能测试可以被识别，例如使用抽吸电极可以从多个感觉神经元的活动被记录一次，这增加了数据，这些数据可以从一个单一的肌肉被收集量。该制剂可用于衡量对感觉神经元群体的反应或识别传入的响应扰动的整体效果。如果尖峰形状是独特的是，高达4的感觉神经元可以通过软件进行鉴别（两者的Spike2（剑桥电子设计）和LabChart临（AD仪表）也同样进行）。在神经细胞不能够被辨别的情况下，改变在电极放置或尖端直径可以很容易地实现。 总之，鼠标肌肉神经体外 preparATION是一个简单的实验方法可用于研究的肌肉感觉传入的响应特性，以各种物理化学的干扰，损伤和疾病模型。此外，该制剂特别适合于利用在小鼠中可用的功能强大的遗传工具，包括转基因动物和光遗传学工具的优点。

骨骼肌是由感觉神经元提供对中枢神经系统具有对肌肉环境的重要信息支配。肌梭是高度专业化的梭内肌纤维分布在梭外肌纤维平行的由封装结构。主轴是由集团IA和II传入编码改变肌肉的长度和梭内纤维音通过动态伽玛支配运动神经元1规定支配。 Ib族传入位于肌纤维和它们的肌腱插入之间，并且在肌肉2收缩的变化，肌肉力量，例如敏感。第Ia，Ib和II传入提供有助于适当的电机控制本体的反馈。位于整个肌肉肌肉传入的附加种群（组III和IV），用于发信号的代谢物的积累，伤害性刺激的存在下，和肌肉温度<sup&gt; 3。此肌肉感觉信息是维持体内平衡，防止肌肉损伤和调节运动的关键。肌肉的传入可以根据以往的经验4改变他们的射击模式。伤害感受器的活化可导致慢性疼痛状态5和从肌肉本体感受器异常信号的诱导可导致问题的平衡或运动6。我们使用的分离的肌肉神经的体外制备，研究肌肉感觉神经元受体末梢从两种性别的成年小鼠的响应（示出的是从2-4个月大的C57BL / 6小鼠的响应）。鼠标是模型的遗传物质在哺乳动物的研究。这种准备需要坐骨神经腓深支和趾长伸肌（EDL）的肌肉，小腿外侧部发现腓骨组的快肌的隔离。该EDL经常用于研究肌肉收缩特性7-9，具有德ndons，很容易分离和足够小，以允许足够的扩散供氧在休息和合理的收缩工作周期10。其他的肌肉在这个区域（例如比目鱼肌和胫骨前肌）具有类似尺寸的和该制剂可以很容易地修改从这些肌肉传入记录。一吸入电极被放置在神经记录肌肉感觉传入神经烧成的切断端。单个神经元可被识别和分析的基础上使用穗排序软件的尖峰形状。刺激在洗澡或对神经的电极可用于唤起肌肉收缩。肌肉长度和力可以被控制，并使用市售的系统测得的。类似体外制剂已被用于在啮齿动物研究III族和第IV肌肉传入在大鼠EDL 11，Ia族和II梭传入在大鼠第四蚓状趾肌12和III族和第IV肌肉传入的叔他鼠标跖肌13。 一种体外系统具有药理访问和直接控制如温度和pH值灌洗液和物理变量的优点。 体外方法消除体内的潜在混淆的麻醉和肌肉的灌注状态。而肌肉的神经制剂允许的扰动对传入的直接反应的研究中，研究主轴敏感度等综合应对伽玛传出调制的能力是可以在体内14或离体3准备的牺牲有这个准备没有神经元胞体。 这种准备是以前用来表征鼠标肌梭传入到斜坡的电池反应并保持伸展和震动，它被确定鼠标梭传入responsES类似于所报告的其他物种例如大鼠，猫和人类。鼠标梭传入的反应被认为是在两个24°C和34℃的水浴温度相似，虽然在34℃的绝对发射率和更快的传入更能够更快地响应长度变化15。我们在下面说明如何编写可用于确定和研究的肌梭传入。此外，该制剂可以很容易地进行修改，以研究其他肌肉传入神经 ​​亚型13的响应，以响应于药物或疾病状态或分类的其他变量（ 例如年龄，性别，基因敲除）比较感觉传入的属性。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Force and Length Controller &#38; Tissue Bath</td>
			<td>Aurora Scientific, Inc.</td>
			<td>300C-LR &#38; 809B-IV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive Perfusion Pump &#38; Easy Load II Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07523-80 &#38; EW-77200-60</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Channel Microelectrode AC Differential Amplifier &#38; Headstage Amplifier</td>
			<td>A-M Systems</td>
			<td>Model 1800; 700000 &#38; 700500</td>
			<td></td>
		</tr>
		<tr>
			<td>Simulator</td>
			<td>Grass 
			OR 
			Aurora Scientific, Inc.</td>
			<td>S88 
			OR 
			701 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Swift</td>
			<td>892070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Light Source</td>
			<td>Fiberlite</td>
			<td>Model 190</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog to Digital Board</td>
			<td>AD Instruments</td>
			<td>PL3508/P (PowerLab 8/35)</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition &#38; Analysis Software</td>
			<td>AD Instruments</td>
			<td>LabChart Pro 7</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Holder</td>
			<td>A-M Systems</td>
			<td>672445</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Glass</td>
			<td>Dagan</td>
			<td>SA16</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-80/PC</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflourane</td>
			<td>MWI Veterinary Supply</td>
			<td>18627</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Surgical Tools</td>
		</tr>
		<tr>
			<td>Large Dissecting Scissors</td>
			<td>Fine Science Tools</td>
			<td>14014-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15006-09</td>
			<td></td>
		</tr>
		<tr>
			<td>#5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>#55 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp Scissors</td>
			<td>Fine Science Tools</td>
			<td>14059-11</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk Suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Chemicals for Low Calcium- High Magnesium aCSF and SIF 
			(Low Calcium solution is equilibriated with 95% 02/5% C02 gas and pH of 7.4 &#177; 0.05; SIF is equilibriated with 100% 02 gas and pH of 7.4 &#177; 0.05)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S9888-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>Sigma</td>
			<td>230391-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma</td>
			<td>223506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Gluconate</td>
			<td>Sigma</td>
			<td>S2054-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S8282-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5767-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S5016-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P3911-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S6014-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro adult mouse muscle-nerve preparation for studying the firing properties of muscle afferents

肌肉感觉神经支配肌梭和高尔基腱器官编码的长度和力量的变化必需的本体。额外传入纤维监视其他特征肌肉环境，包括代谢物的积累，温度，和感受伤害的刺激。总体而言，感觉神经元的异常激活可能导致运动障碍或慢性疼痛综合症。我们描述趾长伸肌（EDL）的肌肉和神经的，在成年小鼠拉伸诱发传入的反应在体外研究中的隔离。感官的活动记录从神经与抽吸电极和个人传入可以用秒杀排序软件进行分析。 体外准备允许在感觉传入了很好的控制研究没有麻醉的潜在的困惑或改变肌肉的灌注。在这里，我们描述了一个协议来识别和测试肌梭传入响应舒展。重要的是，这种准备还支持肌肉的传入，响应特性下列药物的应用和强大的遗传方式和疾病模型小鼠中掺入其他亚型的研究。

肌肉传入的反应可以下面的各种扰动被记录，这取决于其传入亚型正在研究上。肌梭传入肌肉收缩和坡道和保持弹力代表答复如下图所示。 图2来识别传入的梭传入，抽搐收缩给出每秒一次（0.5毫秒脉冲宽度），看看是否有在收缩18,19烧成一个暂停。显示的神经元活动和肌肉紧张时代表跟踪这些肌收缩。抽搐时不收缩的神经元活动时观察到，如预期的梭传入。如果所记录的传入是Ib族高尔基腱器官传入，增加了收缩时的燃烧速率将期望。 在控制条件下最传入一个普通的放电模式（〜12脉冲/秒，在24℃〜〜32脉冲/秒，在34℃下）是肌梭传入。梭传入的一个子集，只会火拉伸过程中（在我们的手中〜梭传入的11％）15。 图3A显示了两种肌梭传入具有代表性的原始跟踪响应斜坡和保持受力和长度控制器产生的弹力。 LabChart的峰值直方图功能是用来识别和分析两个单个神经元的瞬时发射频率分开（ 图3B-3C）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51948/51948fig1highres.jpg" width="500" /> 图1：隔离肌肉制备A）趾长伸肌（EDL）和支配坐骨神经被安装在一个孤立的组织浴灌注含氧合成间质液（SIF）。细胞外放大器连接到sucti对电极记录神经活动。双力和长度控制器，用适当的软件控制和措施的肌肉力量和长度。组织浴电极提供刺激来产生抽动或强直收缩。通过内部浴盘频道抽热水控制组织浴温度。 二）拉玻璃吸电极的理想针尖形状，因为这是用microforge产生的吸力电极〜3毫米锥形尖端，C）的放大写照。 <a href="https://www.jove.com/files/ftp_upload/51948/51948fig1highres.jpg" target="_blank">请点击这里查看本图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51948/51948fig2highres.jpg" width="500" /> 图2主轴传入响应抽搐收缩。神经activitY（上图）和30抽搐的肌肉收缩张力（底部的迹线）叠加在黑色的顶部重叠的痕迹。在收缩引起的张力增加，这是肌梭传入的特征响应传入活动暂停。收缩的过程中支架和箭头上面的顶部曲线表示的时候活动已暂停。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51948/51948fig3highres.jpg" width="600" /> 图3主轴传入响应斜坡期间斜坡和保持舒展）两个传入（上部曲线）原始神经活动和保持应用到EDL（在底部的迹线显示肌肉的长度）。 二）瞬时发射频率（研究所舒展。大一，对参展单位从A L O活动的每秒脉冲）（以蓝色显示）。 三）Instantane较小的单位项发射频率，在拉伸唯一火灾（图中橙色）。这两个单位的拉伸是肌梭传入1特征中表现出的秒杀频率调整。

Watch this Scientific Journal Video about An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents at JoVE.com

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

肌肉感觉神经元参与本体感受器的信号，并在代谢状态和伤害相关的事件报告。我们描述了成年小鼠的体外肌肉神经准备在拉伸激活肌肉传入的研究。

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

an-vitro-adult-mouse-muscle-nerve-preparation-for-studying-firing

Research

JoVE Journal

Neuroscience

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

16.8K 次观看.  San José State University. 肌肉感觉神经元参与本体感受器的信号，并在代谢状态和伤害相关的事件报告。我们描述了成年小鼠的体外肌肉神经准备在拉伸激活肌肉传入的研究。 

视频: An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

科研

神经科学

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

一个<em在体外</em制备与生理变动，为激发和记录进给电机程序<em&gt;海兔夜蛾</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

体外大鼠结肠传入纤维的电生理特性

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...