Major depression is hypothesized to involve impaired 5-HT neurotransmission in the brain; this may in part be due to excessive 5-HT1B autoreceptor activity in 5-HTergic axon terminals. These receptors are desensitized by SSRIs, an important class of antidepressant drugs. However, the mechanism underlying this observation is unknown and these receptors are not well understood. I hypothesize that SSRI, by increasing presynaptic 5-HT1B activation by endogenous 5-HT at presynaptic terminals, downregulates 5-HT1B receptor levels. I further propose that 5-HT1B receptor synthesis is downregulated at the mRNA level in dorsal raphe neurons following chronic receptor activation. The goal of this study will be to isolate the regulatory phenomena that apply specifically to 5-HT1B autoreceptors in 5-HTergic neurons (in contrast to the many postsynaptic non-5-HTergic neurons) that also express 5-HT1B receptors. Two approaches will be used: (1) In rat brain, I will investigate whether chronic activation of terminal 5-HT1B autoreceptors reduces 5-HT1B mRNA levels in the dorsal raphe cell lines. This will be achieved by steady infusion of a selective 5-HT1B agonist into discrete forebrain regions innervated by these neurons. I will then compare 5-HT1B mRNA levels between raphe neurons that do or do not project to the site of infusion. I will similarly test the effects of chronic, localized infusion of SSRI on 5-HT1B autoreceptor regulation, (2) The RN46A cell line was recently developed from embryonic rat raphe neurons. It displays 5-HTergic phenotype including 5-HT1B receptor expression. I will characterize the effect of depolarization, hormones, and trophic factors on the levels of 5-HT1B receptor and mRNA in RN46A cells. I will also determine whether SSRI and 5-HT1B agonists downregulate this receptor in these cells as they do in rat brain. Three indices of 5-HT1B levels and activity will be used: mRNA levels in situ hybridization in rat brain, ribonuclease protection assay in cultured neurons), [125I]-cyanopindolol binding to brain sections or RN46A membranes, and 5-HT1B autoreceptor inhibition of 5-HT release (agonist potency determinations using fast cyclic voltammetry in brain slices).
