Hermann David, van Amsterdam Christoph
Merck Serono Research Darmstadt, Merck KGaA, 64293 Darmstadt, Germany.
Merck Serono Research Darmstadt, Merck KGaA, 64293 Darmstadt, Germany.
J Pharmacol Toxicol Methods. 2015 Jan-Feb;71:54-60. doi: 10.1016/j.vascn.2014.12.001. Epub 2014 Dec 12.
Excitation of Acetylcholine-mediated (Ach) transmission (especially if irreversible) may pose life-threatening adverse events by increasing neuronal network activity. Unfortunately, adequate detection of this liability during early drug development is hampered, because published ex vivo electrophysiological models are very insensitive to this regard. For example, Eserine, which reversibly inhibits acetylcholinesterase (AchE) in the double digit nM range, affects electrically evoked potentials in hippocampal slices only at ≥10μM. Here, a significantly more sensitive method for detecting Ach-mediated alternations is presented by analyzing spontaneous neuronal network activity in hippocampal slices.
The microelectrode array (MEA) technique with an 8×8 electrode grid was applied to analyze evoked and spontaneous extracellular field recordings in parallel from acute rat hippocampal slices. For evoked potentials, the Schaffer collateral CA3-CA1 pathway was electrically stimulated and the resulting field potential analyzed at the CA1 pyramidal layer. Spontaneous spike activity was detected as negative inflections from the 100Hz high pass filtered signal. Spike frequency was analyzed within the whole CA1 region.
Modification of Ach-mediated neuronal transmission via carbachol, Eserine, or Diisopropylfluorophosphate (DFP) does not induce any effects on evoked field potentials at physiologically relevant concentrations. Similar to previous reports, subtle effects were detectable at very high concentrations. By contrast, spontaneous spike frequency was already increased within the expected concentration range. Eserine-induced effects can also be reversed by atropine and washout. On the contrary, effects by the irreversible AchE-blocker DFP could not be washed out.
Compared to evoked field potentials, spontaneous spike activity in the hippocampal CA1 region appears to be a significantly more sensitive parameter for functional electrophysiological analysis of drug induced Ach-mediated effects. This finding may supplement existing models for detection and prediction of drug-related adverse effects like seizure liability already during early development stages.
乙酰胆碱介导（Ach）的传递被激活（尤其是不可逆激活时）可能会通过增加神经网络活动引发危及生命的不良事件。不幸的是，在药物研发早期，对这种风险的充分检测受到阻碍，因为已发表的体外电生理模型在这方面非常不敏感。例如，毒扁豆碱在纳摩尔双位数范围内可逆地抑制乙酰胆碱酯酶（AchE），但仅在≥10μM时才会影响海马切片中的电诱发电位。在此，通过分析海马切片中的自发神经网络活动，提出了一种显著更敏感的检测Ach介导变化的方法。
采用具有8×8电极网格的微电极阵列（MEA）技术，并行分析急性大鼠海马切片中诱发和自发的细胞外场记录。对于诱发电位，电刺激海马体CA3-CA1区的Schaffer侧支通路，并在CA1锥体层分析由此产生的场电位。自发棘波活动被检测为100Hz高通滤波信号的负向偏转。在整个CA1区域内分析棘波频率。
通过卡巴胆碱、毒扁豆碱或二异丙基氟磷酸酯（DFP）对Ach介导的神经元传递进行修饰，在生理相关浓度下不会对诱发场电位产生任何影响。与之前的报道相似，在非常高的浓度下可检测到细微影响。相比之下，自发棘波频率在预期浓度范围内就已增加。毒扁豆碱诱导的效应也可被阿托品逆转并通过洗脱消除。相反，不可逆的AchE阻滞剂DFP产生的效应无法通过洗脱消除。
与诱发场电位相比，海马CA1区的自发棘波活动似乎是药物诱导的Ach介导效应功能电生理分析中一个显著更敏感的参数。这一发现可能会在药物研发早期阶段补充现有的用于检测和预测与药物相关的不良反应（如癫痫发作风险）的模型。
