Transmitter release assay

Figure 7.3 Dose-response effects of MDMA on the release of preloaded [3H]5-HT (left panel) and [3H]DA (right panel) from synaptosomes in vitro. [3H]Transmitter release is expressed as percent of tritium retained in tissue. Various concentrations of MDMA were incubated with or without the 5-HT uptake blocker fluoxetine (10 n/W) in [3H]5-HT assays, whereas various concentrations of MDMA were incubated with or without the DA uptake blocker GBR12909 (10 n/W) in [3H]DA assays. Data are mean SD for three separate experiments, each performed in triplicate. See Baumann et al.39 for methods.

M. Israel and M. Tomasi, A chemiluminescent catecholamine assay its application for monitoring adrenergic transmitter release, J. Neurosci. Methods., 91, 101-107 (1999). 

Remove 100 pL aliquots from the supernatant (avoid the oil ) for transmitter analysis. (Aliquots may be subdivided permitting measurements of multiple transmitters from a single sample). Treat the samples in the manner appropnate for the type of transmitter ultimately to be measured For amino acids, add TCA to 10% (v/v) for neuropeptides or catecholamines, add 3 vol of methanol 10 Store the samples at-20 C until further analysis (see Note 21). Measurements of diverse transmitters released from hippocampal synaptosomes in a typical release batch assay are shown in Fig. 3. 

We would like to thank G. Scholten for her excellent assistance in the EM-studies. We are thankful to Dr. Verhage for developing the release assays for the different transmitters. We appreciate the cooperation with V. Wiegant and A. Frankhuizen for the neuropeptide RIAs, and Dr, Boomsma for the catecholamine analysis. X N. 0. Zeist is appreciated for packing the HPLC columns for amino acid analysis. 

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