The abusive use of many drugs is known to cause the depletion of serotonin (5-HT) and possible damage to 5-HT neurons. In many cases the depletion of 5-HT can be replenished, but degeneration of the 5-HT neurons is permanent. Current methods used to distinguish between the two conditions have produced only inconclusive results. Standard methods capable of revealing 5-HT neurons, such as 5-HT immunocytochemistry, are subject to variations due to changes in 5-HT levels. Uptake ligand-binding methods are confounded by those drugs which block the uptake sites, and are incapable of providing the detailed morphology essential for characterizing degeneration. It has recently become apparent that, in addition to biochemical analysis, anatomical evidence is critical for the verification of degeneration. To date, however, two aspects of neuroanatomical analysis methods remain inconclusive: 5-HT neurons specificity and/or sensitivity to alteration n 5-HT levels caused by the drugs being used in testing. The recent publication of a new protein sequence has made feasible the development of a new probe to circumvent the current problems. The 5-HT transporter (5-HTT) protein serves as a good candidate for this purpose. Experiments using paroxetine ligand binding indicate that 5-HTT is specifically and abundantly located on/in the 5-HT neurons. A specific antibody to this protein will reveal in detail the degeneration characteristics down to a single neuron level, and yet will be free from the complications of 5-HT depletion and uptake blockage. The overall aims of this proposed study are: (a) to create this new probe for the evaluation of 5-HT degeneration, during the process of which antibodies will be produced against synthetic 5-HTT peptides; (b) to test the probe in a degeneration process caused by a known 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT); and (c) to perform a drug test using an amphetamine / MDMA which are known to deplete 5-HT in the brain. The effects of drug abuse on neuronal degeneration versus depletion of 5-HT fibers will be evaluated. We have experience in making polyclonal antibodies against 5-HT in rabbits and mice. In addition, we have brought in Dr. Renee Lin as a consultant. Dr. Lin is very knowledgeable in the making of such antibodies against peptides, and has been collaborating with us on a similar project. We are aware of the analysis of antigenic actors and the major concerns which impact the selection of the proper peptides for preparing antibodies. We are also familiar with the process of degeneration and the plasticity of the 5-HT system. Preliminary data are resented to show the antiserum against one of the synthetic peptide.
