Kuwana Shun-ichi, Tsunekawa Naoko, Yanagawa Yuchio, Okada Yasumasa, Kuribayashi Junya, Obata Kunihiko
Department of Physiology, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo 173-8605, Japan.
Eur J Neurosci. 2006 Feb;23(3):667-74. doi: 10.1111/j.1460-9568.2006.04591.x.
The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre-Bötzinger complex. We used 67-kDa isoform of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) (Delta neo) knock-in (GAD67(GFP/+)) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre-Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre-Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP-positive neurons in the pre-Bötzinger complex were recorded in a perforated whole-cell patch-clamp mode. Six of 32 GFP-positive neurons were respiratory and the remaining 26 neurons were non-respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post-synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP-negative neurons were recorded in the pre-Bötzinger complex. GFP-positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP-positive inspiratory neurons had bipolar, triangular or crescent-shaped somata and GFP-negative inspiratory neurons had multipolar-shaped somata. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre-Bötzinger complex.
由于迄今为止在活体组织中难以识别GABA能神经元，参与呼吸控制的GABA能神经元的特征尚未完全明确。在本项体外研究中，我们成功分析了前包钦格复合体中GABA能神经元的电生理和形态学特征。我们使用了谷氨酸脱羧酶-绿色荧光蛋白（GAD67-GFP）（Delta neo）敲入（GAD67(GFP/+)）小鼠的67-kDa亚型，这使我们能够在活体组织中识别GABA能神经元。我们从这些新生小鼠制备了包含前包钦格复合体的延髓横切片。在前包钦格复合体区域的荧光强度在延髓腹侧区域中相对较高。在前包钦格复合体中以穿孔全细胞膜片钳模式记录GFP阳性神经元的活动。32个GFP阳性神经元中有6个是呼吸性的，其余26个神经元是非呼吸性的；呼吸性神经元均为吸气性的，在吸气期接受兴奋性突触后电位。此外，在前包钦格复合体中记录到30个GFP阴性神经元中的6个吸气性神经元和1个呼气性神经元。GFP阳性吸气性神经元表现出高膜电阻以及对去极化电流脉冲的尖峰频率轻度适应。GFP阳性吸气性神经元具有双极、三角形或新月形的胞体，而GFP阴性吸气性神经元具有多极形的胞体。GFP阳性吸气性神经元的胞体比GFP阴性吸气性神经元的胞体小。我们认为，参与前包钦格复合体呼吸神经元网络的GABA能抑制作用不是由呼气性神经元介导，而是由具有特定电生理和形态学特性的吸气性神经元介导。
