Nilsson O G, Kalén P, Rosengren E, Björklund A
Department of Medical Cell Research, Section of Neurobiology, Lund, Sweden.
Neuroscience. 1990;36(2):325-38. doi: 10.1016/0306-4522(90)90429-8.
Changes in extracellular levels of acetylcholine and choline in the hippocampal formation were measured using intracerebral microdialysis coupled to high performance liquid chromatography with post-column enzyme reaction and electrochemical detection. Various pharmacological and physiological manipulations were applied to awake unrestrained normal rats and rats subjected to a cholinergic denervation of the hippocampus by a complete fimbria-fornix lesion (1-2 weeks previously). Low baseline levels of acetylcholine (about 0.3 pmol/15 min sample) could be detected in the absence of acetylcholinesterase inhibition in all animals. However, in order to obtain stable and more readily detectable levels, the acetylcholinesterase inhibitor neostigmine was added to the perfusion medium at a concentration of 5 or 10 microM and was used during all subsequent manipulations. Addition of neostigmine increased acetylcholine levels approximately 10-fold (to 3.7 pmol 15 min) in the normal rats, which was about 4-fold higher than the levels recovered from the denervated hippocampi. Depolarization by adding KCl (100 mM) to the perfusion fluid produced a 3-fold increase in the extracellular acetylcholine levels, and the muscarinic antagonist atropine (3 microM) resulted in a 4-fold increase in the normal rats, whereas these drugs induced only small responses in the denervated rats. Neuronal impulse blockade by tetrodotoxin (1 microM) resulted, in normal rats, in a 70% reduction in extracellular acetylcholine levels. Sensory stimulation by handling increased acetylcholine levels by 94% in the normal rats, whereas this response was almost totally abolished in the denervated hippocampi. Behavioural activation by electrical stimulation of the lateral habenula resulted in a 4-fold increase in acetylcholine release in normal animals, and this response was totally blocked by a transection of the lateral habenular efferents running in the fasciculus retroflexus. The levels obtained by lateral habenula stimulation were reduced by about 95% in the rats with fimbria-fornix lesions. Following an acute knife transection of the fimbria-fornix performed during ongoing dialysis, acetylcholine levels dropped instantaneously by 70%, indicating that the extracellular acetylcholine levels in the hippocampus are maintained by a tonic impulse flow in the septohippocampal pathway. The extracellular levels of choline were reduced by about 30% after the addition of neostigmine in the normal rats, and increased by about 50% in both normal and denervated rats after addition of KCl to the perfusion fluid. No changes could be detected after atropine, handling, lateral habenula stimulation, or acute fimbria-fornix or fasciculus retroflexus transection.(ABSTRACT TRUNCATED AT 400 WORDS)
采用脑内微透析技术，结合柱后酶反应和电化学检测的高效液相色谱法，测定海马结构中乙酰胆碱和胆碱的细胞外水平。对清醒自由活动的正常大鼠以及海马胆碱能失神经支配的大鼠（1 - 2周前进行完全穹窿 - 海马伞损伤）进行了各种药理学和生理学操作。在所有动物中，若不抑制乙酰胆碱酯酶，可检测到较低的乙酰胆碱基线水平（约0.3 pmol/15分钟样本）。然而，为了获得稳定且更容易检测到的水平，将乙酰胆碱酯酶抑制剂新斯的明以5或10 microM的浓度添加到灌注培养基中，并在所有后续操作中使用。添加新斯的明后，正常大鼠的乙酰胆碱水平增加了约10倍（达到3.7 pmol/15分钟），这比从失神经海马中回收的水平高约4倍。向灌注液中添加氯化钾（100 mM）使细胞外乙酰胆碱水平增加了3倍，毒蕈碱拮抗剂阿托品（3 microM）使正常大鼠的乙酰胆碱水平增加了4倍，而这些药物在失神经大鼠中仅引起微小反应。河豚毒素（1 microM）阻断神经元冲动，在正常大鼠中导致细胞外乙酰胆碱水平降低70%。触摸进行感觉刺激使正常大鼠的乙酰胆碱水平增加94%，而在失神经海马中这种反应几乎完全消失。电刺激外侧缰核引起行为激活，使正常动物的乙酰胆碱释放增加4倍，并且该反应被在回返束中运行的外侧缰核传出纤维横断完全阻断。在穹窿 - 海马伞损伤的大鼠中，外侧缰核刺激所获得的水平降低了约95%。在透析过程中对穹窿 - 海马伞进行急性手术刀横断后，乙酰胆碱水平立即下降70%，这表明海马中的细胞外乙酰胆碱水平由隔海马通路中的紧张性冲动流维持。在正常大鼠中添加新斯的明后，胆碱的细胞外水平降低了约30%，向灌注液中添加氯化钾后，正常大鼠和失神经大鼠的胆碱水平均增加了约50%。在使用阿托品、触摸、外侧缰核刺激或急性穹窿 - 海马伞或回返束横断后，未检测到变化。（摘要截断于400字）
