Koyama Shiori, Nakayama Kiyomi, Mochizuki Ayako, Dantsuji Masanori, Nakamura Shiro, Maki Koutaro, Inoue Tomio
Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan; Department of Orthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
J Oral Biosci. 2023 Mar;65(1):126-131. doi: 10.1016/j.job.2023.01.009. Epub 2023 Feb 3.
It is difficult to comprehensively study the activity patterns and distribution of neurons in the brainstem that control the act of swallowing, as they are located deep in the brain. In this study, we aimed to evaluate the usefulness of calcium imaging using GCaMP6f in arterially perfused preparations to study the activity of swallowing-related neurons in the brainstem.
Arterially perfused rat preparations were prepared 3-4 weeks after the injection of a neuron-specific virus expressing GCaMP6f. Fictive swallowing was induced by repetitive electrical stimulation of the superior laryngeal nerve (SLN). Simultaneously, the activity of GCaMP6f-expressing neurons in the dorsal brainstem, between 0.1 and 4.8 mm rostral to the obex, was assessed by changes in the intracellular calcium concentration using confocal laser microscopy.
Neurons responding to stimulation of the SLN included swallowing-related neurons (48%), which showed an increase in fluorescence intensity at the time of swallowing bursts in the cervical vagus nerve, and stimulation-related neurons (52%), which showed an increase in fluorescence intensity through stimulation, regardless of the swallowing bursts. Despite a broad search area, swallowing-related neurons were localized exclusively in and around the solitary nucleus. In contrast, most stimulation-related neurons were located in the brainstem reticular formation, which is more rostral than the solitary nucleus.
Calcium imaging using GCaMP in arterially perfused rat preparations is useful for an efficient search of the activity pattern and distribution of neurons located in a wide area of the brainstem.
由于脑干中控制吞咽动作的神经元位于脑深部，全面研究其活动模式和分布具有一定难度。在本研究中，我们旨在评估在动脉灌注标本中使用GCaMP6f进行钙成像，以研究脑干中吞咽相关神经元活动的有效性。
在注射表达GCaMP6f的神经元特异性病毒3 - 4周后制备动脉灌注大鼠标本。通过反复电刺激喉上神经（SLN）诱导假吞咽。同时，使用共聚焦激光显微镜通过细胞内钙浓度的变化，评估在闩前方0.1至4.8毫米之间的脑干背侧中表达GCaMP6f的神经元的活动。
对SLN刺激有反应的神经元包括吞咽相关神经元（48%），其在颈迷走神经吞咽爆发时荧光强度增加；以及刺激相关神经元（52%），其在刺激时荧光强度增加，与吞咽爆发无关。尽管搜索区域广泛，但吞咽相关神经元仅局限于孤束核及其周围。相比之下，大多数刺激相关神经元位于脑干网状结构，其位置比孤束核更靠前。
在动脉灌注大鼠标本中使用GCaMP进行钙成像，有助于高效搜索脑干广泛区域内神经元的活动模式和分布。
