Goldbach R, Allgaier C, Heimrich B, Jackisch R
Institut für Pharmakologie und Toxikologie der Universität Freiburg, Neuropharmakologisches Labor, Germany.
Brain Res Dev Brain Res. 1998 Jun 15;108(1-2):23-30. doi: 10.1016/s0165-3806(98)00026-1.
We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the rat hippocampus. To this end, hippocampal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the cholinergic ingrowth. In hippocampal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.11 (P3) to 0.81% of tissue 3H (P16), the latter value being still much lower than that of hippocampal slices from adult rats (2.89% of tissue 3H). Already at P3 the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release. The muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in hippocampal slices with increasing effectivity from P4 to P16; no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine and the muscarinic antagonist atropine (1 microM, each) exhibited significant inhibitory and facilitatory effects, respectively, only at P15-16. The specific activities of both hippocampal HACU (pmoles/mg protein/min) and ChAT (nmoles/mg protein/min) continuously increased from P3 to P16. It is concluded (1) that cholinergic nerve terminals arriving at the hippocampal formation during postnatal ingrowth are already endowed with the apparatus for action potential-induced, Ca(2+)-sensitive (exocytotic) ACh release; (2) that, in contrast, the expression of presynaptic muscarinic autoreceptors on these cholinergic axon terminals is delayed; and (3) that autoinhibition due to endogenous ACh develops even later, probably when the density of presynaptic terminals in the hippocampus and hence, the concentration of released ACh has reached a suprathreshold value.
我们研究了大鼠海马中乙酰胆碱（ACh）释放以及突触前释放抑制性毒蕈碱自身受体的产后发育情况。为此，将不同出生后年龄（出生后第3天[P3]至P16）大鼠的海马切片（350微米厚）与[3H]胆碱预孵育，并在含有半胱氨酸-3（10微摩尔）的生理缓冲液灌流期间进行两次刺激（S1、S2：360个脉冲，2毫秒，3赫兹，60毫安）。同时，测定半胱氨酸敏感的高亲和力胆碱摄取（HACU，在突触体中）和胆碱乙酰转移酶（ChAT，在粗匀浆中）的活性，作为胆碱能神经纤维长入的标志物。在用[3H]胆碱预孵育的海马切片中，S1时电诱发的3H溢出量从0.11（P3）增加到组织3H的0.81%（P16），后者的值仍远低于成年大鼠海马切片的值（组织3H的2.89%）。在P3时，诱发的3H溢出就已经是Ca(2+)依赖性的，并且对河豚毒素敏感，表明ACh释放是由动作电位诱发的胞吐机制。毒蕈碱激动剂氧化震颤素（1微摩尔）显著抑制海马切片中诱发的ACh释放，从P4到P16抑制作用逐渐增强；在P3时未检测到显著影响。ACh酯酶抑制剂毒扁豆碱和毒蕈碱拮抗剂阿托品（各1微摩尔）仅在P15 - 16时分别表现出显著的抑制和促进作用。海马HACU（皮摩尔/毫克蛋白质/分钟）和ChAT（纳摩尔/毫克蛋白质/分钟）的比活性从P3到P16持续增加。得出的结论是：（1）出生后神经纤维长入期间到达海马结构的胆碱能神经末梢已经具备了由动作电位诱导的、Ca(2+)敏感（胞吐）ACh释放的机制；（2）相比之下，这些胆碱能轴突末梢上突触前毒蕈碱自身受体的表达延迟；（3）内源性ACh引起的自身抑制发育得更晚，可能是当海马中突触前末梢的密度以及因此释放的ACh浓度达到阈上值时。
