Instituto de Neurociencias CSIC-UMH, San Juan de Alicante, Spain.
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
Neuroscience. 2018 Jun 15;381:115-123. doi: 10.1016/j.neuroscience.2018.04.011. Epub 2018 Apr 19.
Focal administration of pharmacological agents during in vivo recordings is a useful technique to study the functional properties of neural microcircuits. However, the lack of visual control makes this task difficult and inaccurate, especially when targeting small and deep regions where spillover to neighboring regions is likely to occur. An additional problem with recording stability arises when combining focal drug administration with in vivo intracellular recordings, which are highly sensitive to mechanical vibrations. To address these technical issues, we designed a micro-holder that enables accurate local application of pharmacological agents during in vivo whole-cell recordings. The holder couples the recording and drug delivery pipettes with adjustable distance between the respective tips adapted to the experimental needs. To test the efficacy of the micro-holder we first performed whole-cell recordings in mouse primary somatosensory cortex (S1) with simultaneous extracellular recordings in S1 and motor cortex (M1), before and after local application of bicuculline methiodide (BMI 200 µM). The blockade of synaptic inhibition resulted in increased amplitudes and rising slopes of "Up states", and shortening of their duration. We then checked the usability of the micro-holder in a deeper brain structure, the striatum. We applied tetrodotoxin (TTX 10 µM) during whole-cell recordings in the striatum, while simultaneously obtaining extracellular recordings in S1 and M1. The focal application of TTX in the striatum blocked Up states in the recorded striatal neurons, without affecting the cortical activity. We also describe two different approaches for precisely releasing the drugs without unwanted leakage along the pipette approach trajectory.
在体内记录期间对药理制剂进行局部给药是研究神经微电路功能特性的一种有用技术。然而，由于缺乏视觉控制，使得这项任务变得困难且不准确，尤其是在针对可能发生溢出到邻近区域的小而深的区域时。当将局部药物给药与体内细胞内记录结合使用时，会出现另一个与记录稳定性相关的问题，因为后者对机械振动非常敏感。为了解决这些技术问题，我们设计了一种微夹具，可在体内全细胞膜片钳记录期间实现药理制剂的精确定位给药。该夹具通过可调距离将记录和药物输送管偶联，适用于实验需求的各个尖端之间的距离。为了测试微夹具的功效，我们首先在小鼠初级体感皮层（S1）中进行了全细胞膜片钳记录，同时在 S1 和运动皮层（M1）中进行了细胞外记录，然后在局部给予荷包牡丹碱甲碘化物（BMI 200µM）前后进行了记录。抑制性突触的阻断导致“Up 状态”的幅度和上升斜率增加，持续时间缩短。然后，我们在更深的脑结构纹状体中检查了微夹具的可用性。我们在纹状体的全细胞膜片钳记录期间施加了河豚毒素（TTX 10µM），同时在 S1 和 M1 中获得细胞外记录。TTX 在纹状体中的局部给药阻断了记录纹状体神经元中的 Up 状态，而不影响皮质活动。我们还描述了两种不同的方法，可精确释放药物，而不会沿着管腔进样轨迹发生不必要的泄漏。
