This study examines the gene structure and types of regulatory mechanisms that are involved in the expression of a G protein-coupled cannabinoid receptor in brain. The cannabinoid receptor is one of the most abundant receptors found in brain (rat, monkey, human) and is involved in mediating the mind- and mood-altering effects of marijuana. The widespread localization of this receptor suggests that its activity likely influences all brain systems (motor, sensory, cognitive, autonomic and """"""""limbic""""""""). In the forebrains of rats, levels of cannabinoid receptor mRNA are extremely high in certain subpopulations of neurons. Results from the studies described here will help define how this gene expresses differential levels of cannabinoid receptor mRNAs in specific subpopulations of neurons. Preliminary data suggest that alternative forms of mRNA which encode identical proteins are transcribed from a single gene. The alternative forms of the cannabinoid receptor mRNAs will be localized in rat brain using in situ hybridization histochemistry (ISHH). Cell lines needed to study the expression of this gene will be found by identifying those which are positive for cannabinoid receptor mRNAs using Northern blot analysis. Primary neuronal cultures will be established and characterized so that various elements suspected of regulating the levels of cannabinoid receptor mRNAs can be tested. These cultures will be characterized using quantitative ISHH. The structure of this gene in brain tissues will be determined and the level at which this gene is regulated (transcriptional or post transcriptional level) will be examined. Gene sequences will be evaluated in terms of their ability to direct tissue specific expression and/or differential levels of mRNA production by expressing fusion genes comprised of 5'-flanking sequences (leader sequences or promoter elements) and lac Z as a reporter gene. Primary neuronal cultures will be evaluated as potential hosts for testing the expression of these fusion genes. This evaluation will involve the use of a herpes simplex virus (type 1)-derived infection and expression system, applying double-labelled ISHH techniques on cultured neurons, and estimating copy number of fusion genes in infected host cells using quantitative polymerase chain reactions (PCR). The information obtained regarding the expression of the cannabinoid receptor gene in neurons will likely also apply to the expression of the cannabinoid receptor gene in peripheral tissues since it appears that a gene similar to that expressed in brain is also expressed in testes. Information obtained from these studies may aid in determining the significance of the cannabinoid receptor in brain function and development. Since mRNA levels for this receptor in the brains of embryonic rats differ substantially from those found in adult rats, it is highly probable that this gene is influenced by developmental cues. Information obtained from this study will also contribute, in general, to our current understanding of the mechanisms involved in regulating the expression of neural genes and to the use of cultured neurons as an alternative to transgenic animals in evaluating genetic elements that are involved with gene regulation.
| Sinha, D; Bonner, T I; Bhat, N R et al. (1998) Expression of the CB1 cannabinoid receptor in macrophage-like cells from brain tissue: immunochemical characterization by fusion protein antibodies. J Neuroimmunol 82:13-21 |
