Electroosmotic Push鈥揚ull Perfusion: Description and Application to Qualitative Analysis of the Hydrolysis of Exogenous Galanin in Organotypic Hippocampal Slice Cultures
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- 作者：Amy E. Rupert ; Y. Ou ; M. Sandberg ; S. G. Weber
- 刊名：ACS Chemical Neuroscience
- 出版年：2013
- 出版时间：May 15, 2013
- 年：2013
- 卷：4
- 期：5
- 页码：838-848
- 全文大小：409K
- 年卷期：v.4,no.5(May 15, 2013)
- ISSN：1948-7193
文摘
We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate, a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as 鈥減ush鈥?and 鈥減ull鈥?or 鈥渟ource鈥?and 鈥渃ollection鈥?capillaries have a 味-potential that is negative and greater in magnitude than the tissue鈥檚 味-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured; however, we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all d-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the d-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push鈥損ull perfusion of the neuropeptide galanin (gal1鈥?9) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8鈥?2 渭A and 19鈥?0 渭A) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13鈥?9 to gal20鈥?9) are seen with greater frequency in CA3 than in CA1 but there is no statistically significant difference for longer peptides (gal3鈥?9 to gal12鈥?9).