O'Donovan M, Sernagor E, Sholomenko G, Ho S, Antal M, Yee W
Section on Developmental Neurobiology, NINDS, NIH, Bethesda, Maryland 20892.
J Exp Zool. 1992 Mar 1;261(3):261-73. doi: 10.1002/jez.1402610306.
We have examined the cellular and synaptic mechanisms underlying the genesis of alternating motor activity in the developing spinal cord of the chick embryo. Experiments were performed on the isolated lumbosacral cord maintained in vitro. Intracellular and whole cell patch clamp recordings obtained from sartorius (primarily a hip flexor) and femorotibialis (a knee extensor) motoneurons showed that both classes of cell are depolarized simultaneously during each cycle of motor activity. Sartorius motoneurons generally fire two bursts/cycle, whereas femorotibialis motoneurons discharge throughout their depolarization, with peak activity between the sartorius bursts. Voltage clamp recordings revealed that inhibitory and excitatory synaptic currents are responsible for the depolarization of sartorius motoneurons, whereas femorotibialis motoneurons are activated principally by excitatory currents. Early in development, the dominant synaptic currents in rhythmically active sartorius motoneurons appear to be inhibitory so that firing is restricted to a single, brief burst at the beginning of each cycle. In E7-E13 embryos, lumbosacral motor activity could be evoked following stimulation in the brainstem, even when the brachial and cervical cord was bathed in a reduced calcium solution to block chemical synaptic transmission. These findings suggest that functional descending connections from the brainstem to the lumbar cord are present by E7, although activation of ascending axons or electrical synapses cannot be eliminated. Ablation, optical, and immunocytochemical experiments were performed to characterize the interneuronal network responsible for the synaptic activation of motoneurons. Ablation experiments were used to show that the essential interneuronal elements required for the rhythmic alternation are in the ventral part of the cord. This observation was supported by real-time Fura-2 imaging of the neuronal calcium transients accompanying motor activity, which revealed that a high proportion of rhythmically active cells are located in the ventrolateral part of the cord and that activity could begin in this region. The fluorescence transients in the majority of neurons, including motoneurons, occurred in phase with ventral root or muscle nerve activity, implying synchronized neuronal action in the rhythm generating network. Immunocytochemical experiments were performed in E14-E16 embryos to localize putative inhibitory interneurons that might be involved in the genesis or patterning of motor activity. The results revealed a pattern similar to that seen in other vertebrates with the dorsal horn containing neurons with gamma-aminobutyric acid (GABA)-like immunoreactivity and the ventral and intermediate regions containing neurons with glycine-like immunoreactivity.
我们研究了鸡胚发育中的脊髓中交替运动活动产生的细胞和突触机制。实验在体外培养的分离腰段脊髓上进行。从缝匠肌（主要是髋屈肌）和股胫肌（膝伸肌）运动神经元获得的细胞内和全细胞膜片钳记录显示，在运动活动的每个周期中，这两类细胞同时去极化。缝匠肌运动神经元通常每个周期发放两串动作电位，而股胫肌运动神经元在整个去极化过程中放电，在缝匠肌动作电位串之间有活动峰值。电压钳记录表明，抑制性和兴奋性突触电流是缝匠肌运动神经元去极化的原因，而股胫肌运动神经元主要由兴奋性电流激活。在发育早期，有节律活动的缝匠肌运动神经元中的主要突触电流似乎是抑制性的，因此放电被限制在每个周期开始时的单个短暂动作电位串。在E7 - E13胚胎中，即使臂部和颈部脊髓浸泡在低钙溶液中以阻断化学突触传递，在脑干刺激后仍可诱发腰段运动活动。这些发现表明，到E7时，从脑干到腰段脊髓的功能性下行连接已经存在，尽管不能排除上行轴突或电突触的激活。进行了损毁、光学和免疫细胞化学实验，以表征负责运动神经元突触激活的中间神经元网络。损毁实验用于表明节律性交替所需的基本中间神经元元件位于脊髓腹侧部分。伴随运动活动的神经元钙瞬变的实时Fura - 2成像支持了这一观察结果，该成像显示，高比例的有节律活动细胞位于脊髓腹外侧部分，并且活动可以从该区域开始。大多数神经元（包括运动神经元）中的荧光瞬变与腹根或肌肉神经活动同步发生，这意味着节律产生网络中的神经元动作是同步的。在E14 - E16胚胎中进行了免疫细胞化学实验，以定位可能参与运动活动产生或模式形成的假定抑制性中间神经元。结果揭示了一种与其他脊椎动物相似的模式，背角含有具有γ-氨基丁酸（GABA）样免疫反应性的神经元，腹侧和中间区域含有具有甘氨酸样免疫反应性的神经元。
