Cannabis is a widely used illegal drug in western society, and because of its widespread use, it is important to understand its mode of action in as great a detail as possible. The main cannabinoid receptor, CB-1, is primarily found in the dendrite of GABAergic neurons, and especially strong labeling by CB-1 antibodies is found in neurons in the hippocampus. Study of the effect of CB-1 expressing neurons is complicated by the tremendous morphological, neurochemical and electrophysiological diversity of hippocampal neurons. Heavy and regular use of cannabinoids leads to tolerance. Whatever molecular changes that occur with induction of tolerance are likely to be focused near the regions of high CB-1 density, which is the synapse of CB-1 expressing neurons. We have recently developed a protein fingerprinting technology that is six orders of magnitude more sensitive than current protein analysis technologies, and we have used the technology to fingerprint protein expression in single somatic cells. In this CEBRA grant application, we propose the use of this technology to fingerprint changes in protein expression in the axon of single CB-1 expressing neurons grown in culture, to determine the distribution in protein expression between neurons, and to determine the change in protein fingerprint following chronic cannabinoid treatment. To the best of our knowledge, there have been no studies of the changes in protein fingerprint in single axons to probe changes associated with cannabis tolerance. This study will provide the foundation to identify those proteins whose expression changes with tolerance and to map the interaction between those proteins at the level of a single axon.
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| Hu, Shen; Le, Zhang; Krylov, Sergey et al. (2003) Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis. Anal Chem 75:3495-501 |
| Hu, Shen; Le, Zhang; Newitt, Richard et al. (2003) Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins. Anal Chem 75:3502-5 |
