While primary targets of many antidepressant drugs may be proteins mediating monoamine uptake or catabolism, there may also be postsynaptic actions, including increased cAMP production and a cascade of events resulting from sustained increase in cAMP or the activation of Gsα [1]. Recent results suggest that Gsα/adenylyl cyclase coupling may be one of the targets of antidepressant action, and that chronic treatment is required to observe this effect via altered membrane localization [10]. Previous studies demonstrated that chronic antidepressant treatment-induced increases in Gsα movement from a TX-100 insoluble lipid raft rich domain to a TX-100 soluble membrane domain with a concurrent increase in coupling to adenylyl cyclase [7]. These results are consistent with a study revealing that a number of antidepressant drugs concentrate in detergent insoluble membrane domains, like lipid rafts, subsequent to chronic treatment [11]. Preclinical, platelet, and postmortem brain data indicate that, in depression, the G-protein Gsα is more likely to reside in detergent insoluble lipid rafts [5, 8, 9]. There is evidence that other proteins potentially involved in a mechanism of depression also show altered lipid raft localization. SERT clustering in lymphocyte lipid rafts is altered in a rat model of depression, relative to controls. [26].

The model system used in these studies, C6 glioma cells, has been used for antidepressant studies by this and other labs [4, 7, 11, 12, 23, 25, 37]. Direct comparisons have demonstrated similar effects of prolonged drug treatment as seen in rats, except that 3 weeks in rats were identical to 3 days in cell culture [7]. While the presumption is that neurons are the dominant factor in both depression and antidepressant response, a role for glia (alone or in combination with neurons) is both likely and unresolved.

Chronic antidepressant treatment (in rats and cultured cells) moves Gsα out of TX-100 insoluble lipid raft rich domains and into a domain more amenable to an association with adenylyl cyclase [1, 4], this action may explain the sustained increases in cAMP signaling that accompany antidepressant treatment [2]. A previous study also provided evidence that Gsα signaling was inhibited in TX-100 insoluble lipid raft rich domains [24]. These data are consistent with the hypothesis that liberation of Gsα from these detergent insoluble inhibitory membrane domains by chronic treatment with antidepressants leads to increased coupling between signaling molecules in the detergent soluble non-raft membrane regions.

Lithium has been used as a treatment for bipolar disorder for over 50 years and has been effective in treating both acute manic and depressive symptoms, in addition to reducing their occurrence [27, 28]. A number of studies have demonstrated that lithium inhibits adenylyl cyclase activity [29–32] and more specifically type V adenylyl cyclase [13]. Additionally, one potential mechanism of the antidepressant action of lithium is via inhibition of GSK-3 [14, 33–35]. Recent evidence suggests that lithium has a membrane delimited dual function in inhibiting the activation of G-protein activated K+ channels [36]. This was suggested to be due to decreased affinity of the Gα subunit for the Gβγ subunit and diminished GDP-GTP exchange on the Gα subunit. What is unknown is whether chronic lithium treatment has any effect on the TX-100 insoluble lipid raft membrane localization of Gsα. In this vein, we set out to test whether lithium has the ability to alter Gsα localization in TX-100 insoluble lipid raft rich domains in an antidepressant manner. In addition, we used a structurally disparate mood stabilizer, valproic acid, to see if the effects were similar to lithium. In light of mood stabilizers’ effect on Gsα membrane disposition as determined by membrane fractionation and immunoblotting (Figure 1), these data are not sufficient to suggest that Gsα movement into lipid raft fractions and the decreased cellular production of cAMP associated with lithium or valproic acid treatment [13] are due to a decreased association of Gsα with adenylyl cyclase. Rather, it likely reflects a G protein-independent pathway of adenylyl cyclase regulation.

Curiously, while the above membrane fractionation data demonstrates movement of Gsα into lipid rafts upon treatment with lithium (Figure 1), we did not observe changes in membrane localization of Giα (Figure 2), nor FRAP recovery half-time of either GFP-Gsα or GFP-Giα (Figures 3, 4, 5). One interpretation of this is that the physical coupling between GFP-Gsα or GFP-Giα and adenylyl cyclase is not altered by lithium treatment, and that regulation of cellular cAMP levels by lithium is not effected by direct G protein regulation of adenylyl cyclase. The lack of effect by lithium on escitalopram-mediated Gsα FRAP (Figure 4) further suggests a different locus of action. These effects on Gsα raft domain localization may partially explain why lithium is not effective as a stand alone treatment for chronic depression.

The decreased mobility of GFP-Gsα subsequent to antidepressant treatment was initially surprising to us, as we had hypothesized the “liberation” of Gsα from lipid raft domains would produce the opposite effect, increased mobility of GFP-Gsα and faster recovery of fluorescence after photobleaching (i.e., decreased half-time to recovery). Instead, we found, consistently, that the movement of Gsα out of lipid rafts upon antidepressant treatment was associated with a slowing of GFP-Gsα mobility and an increase in half-time to recovery. We have proposed that this phenomenon is due to the increased association of Gsα, a smaller, peripheral membrane protein, with the larger, multi-transmembrane spanning adenylyl cyclase, which displays extremely slow lateral mobility and has been shown to increase physical association with Gsα subsequent to antidepressant treatment [37]. This “molecular signature” is typical of the many antidepressants tested by our lab (all major groups and several atypical compounds) [4, 7, 12, 23]. Other psychotropic molecules (e.g. antipsychotics and anxiolytics) did not alter mobility of Gsα [12].