Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: Differential roles of glycinergic and GABAergic neural transmission
Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: Differential roles of glycinergic and GABAergic neural transmission
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DOI：
10.1152/jn.1997.77.4.1853
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发表时间：
1997-04-01
影响因子：
2.5
通讯作者：
Feldman, JL
中科院分区：
文献类型：
--
作者：
Shao, XM;Feldman, JL
用于在哺乳动物中产生呼吸节律的神经起搏器和传统网络模型之间的一个关键区别是是否需要吸气和呼气神经元之间的阶段性相互抑制相互作用。在产生呼吸相关节律的新生大鼠的髓质切片中，我们使用全细胞膜片钳在假设的节律产生位点前 Botzinger 复合体（前 BotC）中测量了呼气神经元的相位抑制输入。呼气神经元在呼气期间产生强直脉冲活动，表现出与舌下神经根（XIIn）周期性吸气爆发同步的抑制性突触后电位（IPSP）。甘氨酸受体拮抗剂马钱子碱（STR；5-10μM）的沐浴应用可逆地阻断这些吸气相IPSP，而γ-氨基丁酸-A（GABA（A））受体拮抗剂荷包牡丹碱（BIC；10-100μM）对这些IPSP没有影响。用不含 Cl 的溶液替换对照体外沐浴溶液也消除了这些 IPSP。当吸气相 IPSP 被阻断时，与呼吸相关的节律活动并未消失。当使用 STR、BIC 或不含 Cl 的溶液时，XIIn 节律活动的频率和强度增加，并产生癫痫样活动。在建立全细胞贴片条件（贴片移液管含有 7 mM Cl-）后，吸气相 IPSP 保持稳定。在电压钳下，吸气相抑制性突触后电流（IPSC）的反转电位为-75 mV。当细胞外 Cl- 浓度降低 50% (70 mM) 且反转电位降低至 -60 mV，接近新的 Cl-Nernst 电位时，IPSC 的电流-电压 (I-V) 曲线向右移动。在电压钳（保持电位=-45 mV）下的河鲀毒素（0.5 μM）中，在预BotC上局部应用甘氨酸（1 mM）诱导呼出神经元中的外向电流和膜电导增加。 STR (0.8-1 μM) 的浴应用可阻断该作用。局部应用 GABA(A) 受体激动剂 4,5,6,7-四氢异恶唑并[5,4-c]吡啶-3-醇 (THIP, 1 mM) 可诱导外向电流和膜电导增加，但可被 BIC (10-100 mM) 阻断。在电压钳（保持电位 = -45 mV）下，我们分析了呼气神经元呼气期间的自发 IPSC。 BIC (10 μM) 的浴液应用降低了 IPSC 频率（从 2.2 每秒至 0.3 每秒），而吸气相 IPSC 没有变化。 STR (8-10 μM) 的浴应用消除了两种 IPSC。这些结果表明：1）呼气神经元的相互抑制是甘氨酸能的，由甘氨酸激活的Cl-通道介导，而甘氨酸激活的Cl-通道不是新生大鼠髓质切片中呼吸相关节律产生所必需的； 2)内源性GABA和甘氨酸调节呼吸神经元的兴奋性，影响切片制备中的呼吸模式； 3) BotC前呼气神经元上存在甘氨酸和GABA(A)受体，这些受体分别对STR和BIG敏感； 4) 甘氨酸和GABA(A)抑制机制在呼气神经元中发挥不同的功能作用：甘氨酸和GABA(A)受体均调节神经元兴奋性，而单独的甘氨酸能传递负责相互抑制； 5) 这些新生儿呼气神经元的细胞内 Cl- 浓度与成人相似。
A key distinction between neural pacemaker and conventional network models for the generation of breathing rhythm in mammals is whether phasic reciprocal inhibitory interactions between inspiratory and expiratory neurons are required. In medullary slices from neonatal rats generating respiratory-related rhythm, we measured the phasic inhibitory inputs to expiratory neurons with the use of whole cell patch clamp in the hypothesized rhythm generation site, the pre-Botzinger complex (pre-BotC). Expiratory neurons, which generate tonic impulse activity during the expiratory period, exhibited inhibitory postsynaptic potentials (IPSPs) synchronized to the periodic inspiratory bursts of the hypoglossal nerve root (XIIn). Bath application of the glycine receptor antagonist strychnine (STR; 5-10 mu M) reversibly blocked these inspiratory-phase IPSPs, whereas the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (BIC; 10-100 mu M) had no effect on these IPSPs. Replacing the control in vitro bathing solution with a Cl--free solution also abolished these IPSPs. Respiratory-related rhythmic activity was not abolished when inspiratory-phase IPSPs were blocked. The frequency and strength of XIIn rhythmic activity increased and seizurelike activity was produced when either STR, BIC, or Cl--free solution was applied. Inspiratory-phase IPSPs were stable after establishment of whole cell patch conditions (patch pipettes contained 7 mM Cl-). Under voltage clamp, the reversal potential of inspiratory-phase inhibitory postsynaptic currents (IPSCs) was -75 mV. The current-voltage (I-V) curve for IPSCs shifted to the right when extracellular Cl- concentration was reduced by 50% (70 mM) and the reversal potential was reduced to -60 mV, close to the new Cl- Nernst potential. In tetrodotoxin (0.5 mu M) under voltage clamp (holding potential = -45 mV), local application of glycine (1 mM) over pre-BotC induced an outward current and an increase in membrane conductance in expiratory neurons. The effect was blocked by bath application of STR (0.8-1 mu M). Local application of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol (THIP, 1 mM) induced an outward current and an increase in membrane conductance that was blocked by BIC (10-100 mM). Under voltage clamp (holding potential = -45 mV), we analyzed spontaneous IPSCs during expiration in expiratory neurons. Bath application of BIC (10 mu M) reduced the IPSC frequency (from 2.2 to 0.3 per s), whereas the inspiratory-phase IPSCs did not change. Bath application of STR (8-10 mu M) abolished both IPSCs. These results indicate that 1) reciprocal inhibition of expiratory neurons is glycinergic and mediated by a glycine-activated Cl- channel that is not required for respiratory related rhythm generation in neonatal rat medullary slices; 2) endogenous GABA and glycine modulate the excitability of respiratory neurons and affect respiratory pattern in the slice preparation; 3) both glycine and GABA(A) receptors are found on pre-BotC expiratory neurons, and these receptors are sensitive to STR and BIG, respectively; 4) glycine and GABA(A) inhibitory mechanisms play different functional roles in expiratory neurons: both glycine and GABA(A) receptors modulate neuronal excitability, whereas glycinergic transmission alone is responsible for reciprocal inhibition; and 5) intracellular Cl- concentration in these neonatal expiratory neurons is similar to that in adults.