Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT_1A receptor-adenylyl cyclase axis

Abstract

Targeting serotonin (5-HT) bioavailability with selective 5-HT reuptake inhibitors (SSRIs) remains the most widely used treatment for mood disorders. However, their limited efficacy, delayed onset of action, and side effects restrict their clinical utility. Endogenous regulator of G-protein signaling (RGS) proteins have been implicated as key inhibitors of 5-HT_1ARs, whose activation is believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS proteins involved remains unknown. We identify RGS6 as the critical negative regulator of 5-HT_1AR-dependent antidepressant actions. RGS6 is enriched in hippocampal and cortical neurons, 5-HT_1AR-expressing cells implicated in mood disorders. RGS6^−/− mice exhibit spontaneous anxiolytic and antidepressant behavior rapidly and completely reversibly by 5-HT_1AR blockade. Effects of the SSRI fluvoxamine and 5-HT_1AR agonist 8-OH-DPAT were also potentiated in RGS6^+/− mice. The phenotype of RGS6^−/− mice was associated with decreased CREB phosphorylation in the hippocampus and cortex, implicating enhanced Gα_i-dependent adenylyl cyclase inhibition as a possible causative factor in the behavior observed in RGS6^−/− animals. Our results demonstrate that by inhibiting serotonergic innervation of the cortical-limbic neuronal circuit, RGS6 exerts powerful anxiogenic and prodepressant actions. These findings indicate that RGS6 inhibition may represent a viable means to treat mood disorders or enhance the efficacy of serotonergic agents.—Stewart, A., Maity, B., Wunsch, A. M., Meng, F., Wu, Q., Wemmie, J. A., Fisher, R. A. Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT_1A receptor-adenylyl cyclase axis.

Deficits in serotonergic neurotransmission have been implicated in a number of mood disorders, including pathological anxiety and depression, which represent large, national socioeconomic health burdens. Anxiety and depression often present as comorbid pathologies, and there is some evidence of a causative link between the two diseases. Current therapeutics target presynaptic reuptake symporters to increase serotonin (5-HT) bioavailability and action at postsynaptic sites primarily located in the hippocampus and prefrontal cortex. Selective 5-HT reuptake inhibitors (SSRIs) remain among the most commonly prescribed drugs in the United States. However, limited efficacy, delayed onset of action, and off-target effects limit their clinical utility, underscoring the need to develop more effectual drugs. Furthermore, because 5-HT activates numerous receptors with divergent and often opposing effects on neuronal signaling, efforts to selectively target receptors associated with the beneficial actions of SSRIs have proven challenging (1), and the exact molecular targets necessary for the antianxiety and antidepressant actions of 5-HT remain unclear.

Nearly all 5-HT receptors belong to the G-protein-coupled receptor (GPCR) class of cell surface receptors and are, thus, subject to regulation by regulator of G-protein signaling (RGS) proteins, GTPase-activating proteins (GAPs) for Gα subunits. By stabilizing the transition state in GTP hydrolysis by Gα, RGS proteins accelerate termination of GPCR signaling and limit the extent of the cellular response to GPCR stimulation (2). Mice expressing a knock-in mutation in Gα_i2 rendering the protein insensitive to RGS protein-mediated regulation exhibit spontaneous anxiolytic and antidepressant behavior due to potentiation of 5-HT receptor 1A (5-HT_1AR) signaling (3). The RGS protein superfamily contains some 30 members, and studies in Gα_i2(G148S)-knock-in mice have failed to identify the specific RGS proteins responsible for anxiogenic and prodepressant blockade of 5-HT_1ARs in vivo.

RGS6 belongs to the R7 subfamily of RGS proteins, characterized by their 3-domain structure. The RGS domain confers functional GAP activity directed toward Gα_i/o, and 2 additional domains, the disheveled, EGL-10, pleckstrin homology (DEP) and Gγ-like (GGL) domains, control protein stability, localization, and protein-protein interactions. The GGL domain facilitates interaction between R7 family members and the atypical Gβ subunit Gβ₅. Complex formation between R7 family members and Gβ₅ is required for the stable expression of both proteins (4). Here we investigate the involvement of RGS6, expressed in hippocampus and cortex, in anxiety and depression behaviors in mice. We found that genetic ablation of RGS6 expression resulted in anxiolytic and antidepressant behavior by enhancing signaling through the 5-HT_1AR-adenylyl cyclase (AC) axis. Our results identify RGS6 as the primary regulator of 5-HT_1AR signaling and a principal modulator of anxiety and depression.

MATERIALS AND METHODS

Materials

WAY-100635, 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT), fluvoxamine, forskolin, and β-actin antibody were obtained from Sigma (St. Louis, MO, USA). SCH-50911 was from R&D Systems (Minneapolis, MN, USA). We developed the RGS6L and pan-RGS6 antibodies in rabbits. Antibodies to Gβ₅ and RGS7 were generously provided to us by Dr. Jason Chen (Virginia Commonwealth University, Richmond, VA, USA). Antibodies for protein kinase B (Akt), phospho-Akt (S473/T308), phospho-mitogen-activated protein kinase (MAPK), glycogen synthase kinase 3β (GSK3β), phospho-GSK3β(S9), phospho-PKA substrate, cyclic AMP (cAMP)-response element-binding protein (CREB), and phospho-CREB(S133) were from Cell Signaling Technology (Boston, MA, USA). 5-HT_1AR antibody and antibody for MAPK were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies targeting microtubule-associated protein 2 (MAP2) and α-tubulin were from EMD Millipore (Billerica, MA, USA).

Mice

We generated RGS6^−/− mice as described previously (5). Unless otherwise noted, experiments were performed with age-matched wild-type (WT; RGS6^+/+), heterozygous (RGS6^+/−), and knockout (RGS6^−/−) mice (10–12 wk of age) backcrossed onto a C57BL6 background for 5 generations. Mice were housed on a 12-h light-dark cycle, and experiments were performed during the light cycle. Animals of both sexes were used for behavioral experiments, as we observed no sex-specific differences in mouse performance. Animals naive to each paradigm were used for all behavioral experiments. Drugs were administered 30 min prior to behavioral testing via intraperitoneal (i.p.) injection unless otherwise noted. For all animal experiments, analyses were performed by an observer blinded to mouse genotype and drug treatment. All animal experiments were performed in agreement with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Animal behavioral tests

Anxiety was assessed in mice placed in an automated 40.6- × 40.6- × 36.8-cm open field (San Diego Instruments; San Diego, CA, USA) for 30 min. Center activity was defined as the number of photobeam breaks occurring in the center (15.2×15.2 cm) and expressed as a percentage of the total. Rearing was also monitored. The elevated plus maze apparatus has been described previously (6). After placement of mice in the maze center, behavior was documented for 5 min and scored for total time spent in the open arms of the maze and the number of open arm entries. Novelty-induced hypophagic behavior was assessed in nonstarved animals placed in home or novel cage environments, as outlined previously (7). Latency to drink and total volume consumed were measured. In the marble-burying test, mice were placed in a clear polycarbonate box (18×28×13 cm) containing 5 cm of bedding with 15 marbles arranged in a 5 × 3 grid. The total number of glass marbles buried (covered by 2/3 or more) during a 30-min testing period was assessed. For the forced-swim test, animals first underwent a 15-min preswim in a glass cylinder (18 cm diameter) in ∼10 cm of 25°C water. On a subsequent testing day, behavior was videotaped for 10 min and scored for immobility, climbing, and swimming. Finally, in the tail-suspension test, individual mice were suspended by the tail using adhesive tape from a plastic bar (30-cm elevation) enclosed on all but one side (8). Behavior was videotaped for 5 min, and videos were scored for total immobility time.

Acute measurements of core body temperature

At 5 d before drug injection, mice were individually housed and initially habituated by measurement of core body temperature using a rodent rectal probe (Physitemp Instruments, Clifton, NJ, USA). On the day of testing, core body temperature was measured 30 min after administration of indicated doses of 8-OH-DPAT (3, 9).

Cortical neuron isolation and culture

Cultures of dissociated cortical neurons from 1-d-old mouse pups were isolated as described previously (10). Briefly, the prefrontal cortex was dissected from P1 mouse pups, washed with HBSS, and then digested in 0.05% trypsin-EDTA (Life Technologies, Carlsbad, CA, USA) for 10 min at 37°C. Tissue was washed and resuspended in complete neurobasal medium (Life Technologies) supplemented with 2 mM glutamine and B27, and cells were dissociated by repeated pipetting. Neurons were plated onto tissue culture dishes coated with poly-l-lysine (Sigma) and laminin, and the medium was replaced after 12 h. After 1 d in culture, neurons were treated with forskolin (50 μM, 15 min) followed by 8-OH-DPAT (1 μM, 15 min) and were processed for immunoblotting.

cAMP measurements

Isolated WT and RGS6^−/− cortical neurons were first cultured in DMEM containing 1% bovine serum albumin (BSA) and 500 μM 3-isobutyl-1-methylxanthine (IMBX). After 20 min, cells were treated with and without forskolin (50 μM, 5 min), concurrent with 8-OH-DPAT (1 μM, 6 min) where indicated. Reactions were terminated by liquid nitrogen addition directly to the culture dishes. cAMP measurements were performed with the Direct cAMP ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA), according to the manufacturer's protocol.

Immunoblotting

Whole hippocampi, dorsal striatum, and prefrontal cortex were rapidly dissected from WT and RGS6^−/− mice following drug treatment, where indicated, and flash-frozen in liquid nitrogen. Tissue homogenates and cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Sigma), quantified, and probed as previously described (11). Protein (20 μg/sample) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using standard techniques. Immunoblots were visualized using the Odyssey Imaging System with appropriate fluorescently labeled secondary antibodies (LI-COR Biosciences; Lincoln, NE, USA). Densitometric quantification of Western blots was performed utilizing ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA), and expression of indicated proteins was normalized to actin or α-tubulin-loading controls or total protein levels (phosphorylated proteins) and expressed relative to control conditions.

Immunohistochemistry

Formaldehyde (4%)-perfused frozen brain sections from WT and RGS6^−/− mice were processed to examine protein expression and localization. Briefly, cryosections were washed in phosphate-buffered saline (PBS), blocked with 5% BSA, and incubated overnight at 4°C with and without (control) indicated antibodies. Following washing 4 times in PBS (10 min each), sections were incubated for 1 h at room temperature with Alexa Fluor secondary antibodies (Life Technologies). Sections were visualized using confocal microscopy, as described previously(5).

Statistical analysis

Data were analyzed by Student's t test or 2-way analysis of variance (ANOVA) with the Bonferroni post hoc adjustment as appropriate. Statistical analyses were performed using Prism software (GraphPad Software; La Jolla, CA, USA). Results were considered significantly different at values of P < 0.05. Values are expressed as means ± sem.

RESULTS

Serotonergic deficits in brain regions comprising the cortico-limbic-striatal neuronal circuit have been implicated in anxiety and depression. We detected expression of multiple RGS6 isoforms in hippocampus and cerebral cortex of WT mice that were absent in RGS6^−/− mice. The R7 family member RGS7 also exhibits robust expression in these tissues and likely contributes to the stabilization of Gβ₅ in the absence of RGS6 (Fig. 1A). Immunohistochemical staining of brain sections confirmed our Western blot analysis results, showing specific expression of RGS6 in prefrontal cortex and the dentate gyrus (DG), cornu ammonis 3 (CA3), and CA1 regions of the hippocampus (Fig. 1B). To confirm neuron-specific RGS6 localization, we costained sections with the somatodendritic neuronal marker MAP2. RGS6 and MAP2 exhibit identical expression patterns in hippocampus and cortex, confirming RGS6 localization in hippocampal (CA3/CA1) and cortical pyramidal neurons (Fig. 1C). In the DG, RGS6 expression appears most prominent in the granule cell layer (Fig. 1C), a pattern we also observed in cerebellum (11).

Figure 1.

RGS6 is expressed in the somatodendritic compartment of hippocampal and cortical neurons. A) Immunoblotting confirmed expression of multiple RGS6 isoforms, RGS6-binding partner Gβ₅, and R7 family member RGS7 in hippocampus and cortex. B) Immunohistochemical staining revealed robust expression of RGS6 in the cerebral cortex and the dentate gyrus (DG), cornu ammonis 3 (CA3), and CA1 regions of the hippocampus that was lost in RGS6^−/− tissue. Scale bar = 20 μm. C) RGS6 colocalizes with somatodendritic neuronal marker MAP2 in cortical neurons and hippocampal neurons of the CA1, CA3, and DG. In merged images, nuclei are stained blue using DAPI. Scale bar = 20 μm.

On the basis of these results, we hypothesized that loss of RGS6-dependent Gα_i/o GAP activity might contribute to the anxiolytic and antidepressant phenotype observed in mice harboring the Gα_i2(G148S) mutation. Therefore, we observed the performance of our RGS6^−/− mice in behavior paradigms used to quantitate rodent anxiety and depression-related behaviors. The open-field test is used to assess rodent response to stress from social isolation and novel environment, while simultaneously measuring locomotor activity. RGS6^−/− mice exhibit mild hyperactivity (Fig. 2A), but no difference in anxiety-related behaviors, including time spent in the center of the field (Fig. 2B) or rearing (Fig. 2C). A similar lack of anxiolytic behavior in this test was observed in RGS-insensitive Gα_i2-knock-in mice, and, while they exhibited no increase in acute locomotor activity (3), these mice do display increased activity in the home cage (12). Conversely, RGS6^−/− mice exhibit no hyperactivity in the home cage (unpublished data), indicating that the increased activity in the open field is likely a response to the novel environment.

Figure 2.

RGS6^−/− mice exhibit spontaneous anxiolytic and antidepressant behavior. A–C) RGS6 deficiency promotes mild hyperactivity (significant effect of genotype, F_1,90=83.52, P<0.0001) in the open field (A) but no difference in anxiety-related behaviors, including relative time spent in the center of the field (B) or rearing (C; WT, n=13; RGS6^−/−; n=19). D) RGS6^−/− mice spend more time in the open arms. E) RGS6^−/− mice exhibit a greater number of open-arm entries in the elevated plus maze (WT: n=9; RGS6^−/−: n=14). F, G) RGS6^−/− animals show reduced anxiety in the novelty-induced hypophagia paradigm (WT: n=9; RGS6^−/−: n=13). Data were analyzed by 2-way ANOVA, revealing a significant effect of genotype on latency to drink (F; F_1,40=12.51, P=0.001) and solution consumption (G; F_1,40=260.74, P<0.0001). H) Obsessive-compulsive disorder (OCD) behavior measured by the marble-burying test is reduced in RGS6^−/− mice (WT: n=10; RGS6^−/−: n=13). I) Mice lacking RGS6 show reduced depression-like behavior in the forced-swim test (WT: n=9; RGS6^−/−: n=11). Data are presented as means ± sem. *P < 0.05, **P < 0.001.

In the elevated plus maze, which exploits the approach-avoidance conflict in rodents, RGS6^−/− mice exhibit a remarkable reduction in anxiety, spending more time in the open arms (Fig. 2D) and displaying a greater number of open-arm entries (Fig. 2E). Similarly, while no difference was observed in the home cage, mice lacking RGS6 show a reduced hyponeophagic response in a novel cage (Fig. 2F). RGS6^−/− animals also consumed a larger volume of sweetened solution in both cages (Fig. 2G), an effect seen in mice treated chronically with SSRIs (7). Consistent with a reduction in anxiety, RGS6^−/− mice buried fewer marbles in the marble-burying test, an approximation of obsessive compulsive disorder (OCD)-related anxiety (Fig. 2H). Depression-like behavior is assessed in rodents using models of behavioral despair, including the forced-swim and tail-suspension tests. Indeed, RGS6^−/− mice show reduced immobility and climbing behavior with a concomitant increase in swimming in the forced-swim test (Fig. 2I). Interestingly, RGS6^−/− mice completely phenocopy Gα_i2(G148S) mice, leading us to hypothesize that this phenotype results from loss of RGS6-mediated inhibition of G-protein signaling.

Treatment with WAY-100635, a 5-HT_1AR antagonist, rapidly reversed the antidepressant phenotype of RGS6^−/− mice in the tail-suspension test (Fig. 3A), as well as the anxiolytic phenotype of RGS6^−/− mice in the elevated plus maze (Fig. 3B, C). Thus, loss of RGS6 promotes anxiolytic and antidepressant behaviors by enhancing 5-HT_1AR-dependent signaling. In contrast, blockade of GABA_B receptors (GABA_BRs), Gα_i/o-coupled GPCRs implicated in RGS6-dependent motor coordination (11), with antagonist SCH-50911 failed to affect performance of mice in the elevated plus maze, regardless of genotype (Fig, 3B, C). SCH-50911 reverses the ataxic phenotype of RGS6^−/− mice (11). This result demonstrates that the mild ataxia observed in RGS6^−/− mice (11) likely does not contribute to their performance. Furthermore, in the novelty-induced hyponeophagia test, WAY-100635 treatment also restored the aberrant latency to drink (Fig. 3D) and solution consumption in the novel cage (Fig. 3E) observed in RGS6^−/− mice to levels comparable to those observed in WT mice. Thus, the reduced latency to drink and increased solution consumption seen as a consequence of RGS6 deficiency in this test also results from potentiation of 5-HT_1AR signaling. Each of the paradigms described can be used in rodents to screen antidepressant and antianxiety medications for predictive efficacy in human patients. Given our findings, we examined whether loss of RGS6 increases the sensitivity of mice to drugs targeting 5-HT_1ARs. For these experiments WT, RGS6^+/−, and RGS6^−/− mice were challenged with a dose of the SSRI fluvoxamine or direct 5-HT_1AR agonist 8-OH-DPAT insufficient to provoke antidepressant effects in WT mice. Treatment with either fluvoxamine or 8-OH-DPAT failed to provoke antidepressant effects in WT mice and had no additional antidepressant effects in RGS6^−/− mice (Fig. 3F), the latter possibly due to a maximal antidepressant phenotype in these mice. In contrast, RGS6^+/− mice, while showing no phenotypic difference from WT mice, were dramatically sensitized to the actions of both drugs (Fig. 3F). In fact, submaximal doses of both drugs decreased immobility of RGS6^+/− mice to levels observed in RGS6^−/− mice (Fig. 3F). Conversely, the hypothermic response to 8-OH-DPAT, solely mediated by 5-HT_1A autoreceptors in the raphe nucleus (13), occurred with equal potency and efficacy in all mice regardless of genotype (Fig. 3G). Taken together, these results indicate that RGS6 has powerful anxiogenic and prodepressant actions through its ability to terminate postsynaptic 5-HT_1AR signaling.

Figure 3.

RGS6 promotes anxiety and depression by inhibiting 5-HT_1AR signaling. A) 5-HT_1AR blockade using the antagonist WAY-100635 (WAY; 0.1 mg/kg s.c.) rescued the antidepressant phenotype of RGS6^−/− mice in the tail-suspension test (WT: n=8–9; RGS6^−/−: n=8–10). Two-way ANOVA revealed a significant effect of genotype (F_1,31=8.12, P=0.0077) and drug (F_1,31=16.81, P=0.0003). B, C) WAY treatment, but not GABA_BR blockade with SCH-50911 (SCH; 30 mg/kg i.p.), reversed the anxiolytic phenotype of RGS6^−/− mice in the elevated plus maze (WT: n=8–10; RGS6^−/−: n=9–13) as measured by both number of open-arm entries (B) and time spent in the open arms of the maze (C). There was a significant effect of genotype (F_1,54=38.80, P<0.0001) and drug (F_2,54=4.19; P=0.02) on number of open-arm entries and genotype (F_1,54=36.06, P<0.0001) on time spent in the open arms by 2-way ANOVA. D, E) WAY treatment rescued the phenotype of RGS6^−/− mice in the novelty-induced hypophagia paradigm (WT: n=7–8; RGS6^−/−: n=8–9) for both latency to drink (D) and liquid consumption (E) in the novel cage. Both genotype (F_1,28=30.03; P=0.0002) and drug (F_1,28=20.09; P=0.0015) affected the outcome for latency to drink by 2-way ANOVA. There was also a significant effect of genotype (F_1,28=41.22; P<0.0001) and drug (F_1,28=9.15; P=0.028) on volume liquid consumed in the novel cage. F) RGS6^+/− mice are sensitized to the antidepressant actions of the SSRI fluvoxamine (0.5 mg/kg) and 5-HT_1AR agonist 8-OH-DPAT (1 mg/kg) (WT: n=8–10; RGS6^+/−: n=8–10; RGS6^−/−: n=9–10). Two-way ANOVA revealed a significant effect of genotype (F_2,74=37.52; P<0.0001) and drug treatment (F_2,74=6.51; P=0.0002). G) Neither RGS6 haploinsufficiency nor genetic ablation sensitized mice to the hypothermic effects of increasing doses of 8-OH-DPAT (WT: n=9; RGS6^+/−: n=6; RGS6^−/−: n=7). Two-way ANOVA revealed a significant effect of drug dose (F_2,41=83.71; P<0.0001) but no effect of genotype (F_1,41=0.20; P=0.98). No genotype-dependent differences in basal core temperature were detected via telemetry (unpublished results). Data are presented as means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT control; ^#P < 0.01 vs. WAY-treated RGS6^−/− mice; ^†P < 0.05 vs. RGS6^−/− control; ^‡P < 0.01 vs. RGS6^+/− control.

We next sought to determine the molecular basis for the anxiolytic phenotype of RGS6^−/− mice. Like RGS6, 5-HT_1A heteroreceptors are located in the soma and dendrites of hippocampal and cortical neurons (14). No difference in 5-HT_1AR expression levels was observed in RGS6^−/− tissue, indicating that the phenotype of RGS6^−/− mice is not due to receptor up-regulation (Fig. 4A). In brain, 5-HT_1AR stimulation activates an array of downstream signaling, including Gβγ-mediated, phosphorylation-dependent activation of GSK3β, Akt, and MAPK and Gα_i-mediated inhibition of the AC-cAMP-PKA signaling axis (15). Contrary to results obtained in Gα_i2(G148S)-knock-in mice (3), no difference in GSK3β phosphorylation (Fig. 4A) was detected comparing WT and RGS6^−/− tissue lysates from the cortex (Fig. 4B), hippocampus (Fig. 4C), or striatum (Fig. 4D). Similarly, no genotype-dependent changes in the levels of phosphorylated MAPK or Akt were seen (Supplemental Fig. S1). These results show that the anxiolytic and antidepressant phenotype of RGS6^−/− mice is not associated with enhanced Gβγ-mediated signaling by the 5-HT_1AR. In contrast, despite considerable heterogeneity in individual responses, we observed a 5-HT_1AR-dependent reduction in hippocampal and cortical phospho-CREB levels in RGS6^−/− animals that did not occur in the striatum, where 5-HT_1ARs are not expressed (Fig. 4). In the cortex, PKA activity, as measured by immunoreactivity of an antibody recognizing the phosphorylated PKA substrate motif (RRXT/S-P), was also reduced in RGS6^−/− tissue lysates, an effect reversible by 5-HT_1AR blockade (Fig. 4B). These findings suggest that loss of RGS6 promotes 5-HT_1AR/Gα_i-mediated inhibition of the AC-cAMP-PKA axis.

Figure 4.

RGS6^−/− mice exhibit potentiation of 5-HT_1AR/Gα_i-dependent signaling in hippocampus and cortex. A) RGS6, 5-HT_1AR, actin, phospho-total GSK3β, and phospho-CREB, total CREB, and phospho-PKA substrate immunoblots from WT and RGS6^−/− mice treated with and without the 5-HT_1AR antagonist WAY-100635 (WAY; 0.1 mg/kg s.c.). Data are presented in triplicate for all experimental conditions. B–D) Densitometric quantification of phospho-GSK3β, phospho-CREB, and PKA substrate phosphorylation immunoblots was performed in cortex (B), hippocampus (C), and striatum (D) (n=3–6). There was no effect of genotype or drug treatment on phospho-GSK3β levels in hippocampus or cortex. Two-way ANOVA revealed a significant effect of genotype on phospho-CREB levels in cortex (F_1,20=7.78; P=0.0113) and hippocampus (F_1,20=5.12; P=0.035). There was also a significant effect of genotype (F_1,8=10.46; P=0.0120) on phospho-PKA substate immunoreactivity in cortex but not hippocampus. Neither genotype nor drug treatment affected phosphorylated protein levels in striatum. Data are presented as means ± sem. *P < 0.05, **P < 0.01.

Our in vivo data were recapitulated in cortical neurons isolated from WT and RGS6^−/− mice. Interestingly, serum-induced phospho-CREB levels were markedly lower in cortical neurons isolated from RGS6^−/− mice compared to their WT counterparts (Fig. 5A, B), possibly reflecting perpetuation of the in vivo phenotype in culture. Forskolin, a direct AC activator, resulted in robust CREB activation in RGS6^−/− cortical neurons, an effect not observed in WT neurons. This suggests that increased Gα_i activity and consequent reduced AC activity underlies the lower level of CREB phosphorylation in RGS6^−/− neurons. Furthermore, RGS6^−/− neurons were remarkably sensitized to inhibition of CREB phosphorylation induced by 8-OH-DPAT, whereas 8-OH-DPAT had no effect on CREB phosphorylation in WT cells (Fig. 5B). To confirm a direct effect of RGS6 knockout on AC activity, we also measured forskolin-stimulated cAMP accumulation in 8-OH-DPAT-treated WT and RGS6^−/− cortical neurons. Forskolin treatment resulted in cAMP generation in cells of both genotypes (Fig. 5C); however, RGS6^−/− cells exhibited a more robust inhibition of cAMP accumulation in response to 8-OH-DPAT (Fig. 5C). These results illustrate the remarkable sensitivity of RGS6^−/− cells to 5-HT_1AR stimulation. Together, our findings indicate that RGS6 is a critical negative regulator of signaling through Gα_i-coupled 5-HT_1A heteroreceptors in brain and isolated neurons and suggest that potentiation of 5-HT_1AR-mediated AC inhibition might underlie the antidepressant phenotype of mice lacking RGS6.

Figure 5.

RGS6 regulates 5-HT_1AR signaling through Gα_i in isolated cortical neurons. A) RGS6, α-tubulin, phospho-CREB, and total CREB immunoblots from WT and RGS6^−/− cortical neurons (n=3/treatment condition) were treated where indicated with 50 μM forskolin (15 min) followed by 1 μM 8-OH-DPAT (15 min). Data are representative of triplicate experiments. B) Quantification of data from panel A by 2-way ANOVA revealed a significant effect of genotype (F_1,12=20.74; P=0.0007) and drug treatment (F_2,12=14.39; P=0.0006). C) cAMP levels from WT and RGS6^−/− neurons (n=3/treatment condition) treated concurrently with forskolin (50 μM, 5 min) and 8-OH-DPAT (1 μM, 6 min) where indicated. Two-way ANOVA revealed a significant effect of genotype (F_1,12=6.31; P=0.0145) and drug treatment (F_2,12=62.94; P<0.0001). Data are presented as means ± sem. *P < 0.05, **P < 0.01 vs. WT control; ^#P < 0.05 vs. forskolin + 8-OH-DPAT treated RGS6^−/− neurons.

In fact, direct AC activation using forskolin was sufficient to completely reverse the reduced immobility of RGS6^−/− mice in the tail-suspension test (Fig. 6A), indicating that Gα_i-mediated inhibition of AC is exclusively responsible for reducing depression in RGS6^−/− animals. As expected, on the basis of its ability to stimulate cAMP production and subsequent PKA activity, forskolin treatment resulted in elevated levels of phospho-CREB in the hippocampus and cortex (Fig. 6B, C) and PKA substrate phosphorylation in cortex and striatum (Fig. 6C, D). Notably, the reductions in both CREB and PKA substrate phosphorylation seen in RGS6^−/− tissue lysates were normalized to WT levels (Fig. 6B–D), highlighting the potential importance of PKA effectors in the anxiolytic and antidepressant phenotype resulting from RGS6 loss. Thus, we propose a model whereby RGS6 counteracts the anxiolytic and antidepressant actions of 5-HT through blockade of postsynaptic 5-HT_1AR-dependent AC inhibition, leading to increases in cAMP and activation of downstream AC effectors, including PKA and CREB (Fig. 7).

Figure 6.

RGS6 deficiency reduces depression by facilitating AC inhibition. A) AC activation by forskolin (7.5 mg/kg, 45 min prior to behavioral testing) rescued the antidepressant phenotype of RGS6^−/− mice in the tail suspension test (WT: n=7–8; RGS6^−/−: n=10–15). Two-way ANOVA revealed a significant effect of genotype (F_1,36=26.16; P<0.0001) and drug (F_1,36=4.17; P=0.0486). B) RGS6, α-tubulin, phospho-CREB, total CREB, and phospho-PKA substrate immunoblots from WT and RGS6^−/− mice treated with and without forskolin (7.5 mg/kg, 45 min). Data are presented in triplicate for all experimental conditions. C–E) Densitometric quantification (n=3) of phospho-CREB, and PKA substrate phosphorylation immunoblots was performed in cortex (C), hippocampus (D), and striatum (E). In cortex, 2-way ANOVA revealed a significant effect of genotype (F_1,8=12.95, P=0.007; F_1,8=13.38, P=0.0065) and drug treatment (F_1,8=79.50, P<0.0001; F_1,8=106.28, P<0.0001) on phospho-CREB and phospho-PKA substrate levels, respectively. There was also a significant effect of genotype (F_1,8=14.78; P=0.0049) and drug treatment (F_1,8=99.51; P<0.0001) on phospho-CREB in hippocampus, but no difference in PKA-substrate immunoreactivity. Genotype did not affect phosphorylated protein levels in striatum, but there was a significant effect of drug on PKA substrate phosphorylation (F_1,8=73.40; P<0.0001). Data are presented as means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT; ^#P < 0.01 vs. forskolin-treated RGS6^−/− mice.

Figure 7.

Schematic outlining the role of RGS6 in regulation of 5-HT_1AR signaling in anxiety and depression. Our results indicated that RGS6 promotes anxiety and depression by inhibiting the 5-HT_1A heteroreceptor-AC signaling axis at postsynaptic sites likely located in neurons of the hippocampus and cortex. By selectively blocking Gα_i-dependent AC inhibition, RGS6 facilitates cAMP accumulation and subsequent activation of PKA and CREB, which contribute to rodent anxiety and depression-related behaviors, and counteracts the actions of antidepressant and anxiolytic medications.

DISCUSSION

Here, we identify RGS6 as a critical regulator of 5-HT_1AR- and AC-dependent anxiolytic and antidepressant actions. RGS6 expression is enriched in hippocampal and cortical neuron populations, 5-HT_1AR-expressing cells implicated in multiple mood disorders (14, 15), and loss of RGS6 potentiates 5-HT_1AR-Gα_i-dependent AC inhibition in vivo and in isolated cortical neurons. RGS6^−/− mice exhibit a spontaneous anxiolytic and antidepressant phenotype in multiple behavioral paradigms, including tests affected only by chronic antidepressant treatment. While the phenotype of RGS6 heterozygotes is indistinguishable from WT mice, partial RGS6 deficiency elicits a remarkable sensitization to the antidepressant actions of the SSRI fluvoxamine and 5-HT_1AR agonist 8-OH-DPAT. These data suggest that partial loss or inhibition of RGS6 is sufficient to potentiate the actions of serotonergic drugs. Indeed, our results indicate that under physiological conditions, a single copy of the RGS6 gene is sufficient to completely prevent 5-HT-mediated signaling through postsynaptic 5-HT_1ARs. Furthermore, RGS6^−/− mice exhibit a level of anxiety comparable to that of WT mice treated chronically with therapeutic doses of SSRIs. Unlike Gα_i2(G148S) mutant mice (12), RGS6 loss has no detrimental effects on mouse fertility, development, or body weight, making RGS6 an attractive novel drug target.

The antidepressant phenotype of RGS6^−/− animals is rapidly and completely reversible by 5-HT_1AR blockade. 5-HT_1AR expression is reduced in critical brain regions of patients with depression, and polymorphisms in the 5-HT_1A R gene are associated with disease susceptibility and serotonergic therapy responsiveness (16). Presynaptic 5-HT_1A autoreceptors located in the raphe nucleus promote anxiety by inhibiting 5-HT release. Conversely, 5-HT_1A heteroreceptors activate diverse signaling in postsynaptic neurons located in both hippocampus and cortex and are believed to mediate the antidepressant and antianxiety actions of 5-HT (14, 17). Our data strongly suggest that RGS6 is primarily responsible for regulation of postsynaptic 5-HT_1A heteroreceptors. First, RGS6 heterozygotes are sensitized to the antidepressant actions of 5-HT_1AR stimulation, but 8-OH-DPAT induced hypothermia, an effect dependent on autoreceptor action (13), occurs with equal potency in RGS6^+/− and WT mice. Second, only 5-HT_1A heteroreceptors couple to Gα_i and induce inhibition of AC (18), and the antidepressant phenotype of RGS6^−/− mice is completely dependent on AC inhibition, as it is completely reversed by forskolin. Finally, the robust reduction in anxiety and depression-related behaviors in mice lacking RGS6 is consistent with potentiation of postsynaptic receptor signaling rather than enhanced inhibition of 5-HT release, which would be expected to increase rather than decrease these behaviors.

Studies in knockout mice have identified 5-HT_1ARs as critical determinants of both baseline anxiety and responsiveness to SSRI therapy, actions due primarily to receptor function during early development (13, 19, 20). However, these observations do not preclude a role for 5-HT_1AR signaling in controlling anxiety and depression in adult animals, especially given extensive historical evidence that hyperactivation of 5-HT_1A heteroreceptors in adult mice produces anxiolytic and antidepressant effects (21–23). Our results demonstrate that singular loss of RGS6-mediated 5-HT_1AR inhibition is sufficient to maximize the antidepressant and anxiolytic capacity of 5-HT_1AR signaling in response to endogenous 5-HT. Thus, RGS6 functions as a critical countermeasure against the antianxiety and antidepressant actions of 5-HT and serotoninergic drugs. While it is possible that RGS6 deletion leads to alterations in development of the circuitry underlying anxiety and depression-related behaviors, two of the observations render this supposition unlikely. First, the antidepressant and anxiolytic phenotype in RGS6^−/− mice is rapidly and completely reversible through 5-HT_1AR blockade. Second, despite the lack of differences in anxiety or depression behaviors in RGS6 heterozygous mice under basal conditions, RGS6^+/− mice are greatly sensitized to subefficacious doses of 5-HT_1AR-targeted drugs.

Previous work in mice expressing RGS-insensitive Gα_i2 demonstrated that endogenous RGS proteins block 5-HT_1AR-mediated GSK3β phosphorylation (3). Given studies identifying GSK3β as an important molecular target of 5-HT (24), it was postulated that increased GSK3β phosphorylation contributed to the anxiolytic phenotype of Gα_i2(G148S) mutants. RGS6^−/− mice show an identical phenotype in the absence of increased phospho-GSK3β, suggesting that GSK3β inactivation does not cause the antidepressant phenotype resulting from loss of RGS protein regulation. Instead, RGS6 appears to selectively regulate a specific 5-HT_1AR-effector pathway, namely the Gα_i-AC-PKA-CREB axis, without affecting other targets (e.g., GSK3β). Indeed, the ability of forskolin to reverse the antidepressant phenotype of RGS6^−/− mice suggests that Gβγ effectors (GSK3β, MAPK, Akt) are dispensable for the ability of 5-HT_1ARs to reduce depression.

Although our results demonstrate that AC inhibition is clearly an important and essential consequence of 5-HT_1A heteroreceptor activation in reducing anxiety and depression, the exact molecular determinant of this response remains unclear. We demonstrate a clear decrease in PKA substrate phosphorylation in the cortex, and levels of phosphorylated CREB in both the cortex and hippocampus of RGS6^−/− mice that is normalized following 5-HT_1AR blockade or AC activation. However, numerous signaling moieties are mobilized on AC activation, including cyclic nucleotide-gated ion channels and PKA and its substrates, which may number in the hundreds or even thousands in mammals (25). Because of its ability to promote hippocampal neurogenesis believed to contribute to SSRI efficacy (20), the role of CREB in anxiety and depression has been explored but remains controversial, as CREB appears to have prodepressant or antidepressant actions depending on the neuronal population investigated (26, 27). It is important to note that, although forskolin treatment caused a clear increase in phospho-CREB in the hippocampus and cortex, this ∼2-fold increase was insufficient to alter the behavioral phenotype of WT animals. It seems likely, therefore, that increases in CREB activity may alleviate anxiety and depression only after prolonged stimulation, consistent with the necessity for long-term changes in neuronal architecture. In future studies, RGS6^−/− mice represent a useful model system to further delineate the molecular signaling cascades responsible for the antianxiety and antidepressant actions resulting from 5-HT_1AR-mediated inhibition of AC.

Despite the fact that other R7 RGS protein family members, including RGS7, are also expressed in hippocampus and cortex, our results clearly demonstrate that they fail to compensate for the loss of RGS6-mediated regulation of the 5-HT_1A heteroreceptor-Gα_i-AC signaling axis. Our observations do not, however, completely rule out a role for other members of the R7 RGS protein family, which includes RGS6, RGS7, RGS9, and RGS11, in anxiety, depression, and the actions of serotoninergic drugs. Indeed, polymorphisms in the RGS7 gene are associated with panic disorder subtypes and gender-biased disease susceptibility (28). However, RGS7 is unable to regulate 5-HT_1AR-mediated inhibition of AC in vitro (29), indicating that RGS7 may affect this anxiety-related behavior through regulation of a different receptor signaling cascade. Mice lacking RGS9 exhibit no changes in anxiety, though their behavior has only been evaluated in the open-field test (30). Because mice lacking RGS7 or RGS11 have not been evaluated in the behavioral paradigms described in this work, it is unknown whether these R7 family members have any role in modulating anxiety and depression. Our work clearly demonstrates that RGS6 is a critical regulator of 5-HT-mediated antianxiety and antidepressant actions through its ability to inhibit Gα_i-coupled 5-HT_1ARs.

It is also likely that RGS6 is not the sole regulator of 5-HT_1AR signaling in brain. Indeed, deficiency in the RGS2 gene, implicated in human anxiety disorders (31, 32), promotes anxiety and depression-related behaviors in mice by affecting expression and/or activity of 5-HT_1A autoreceptors (33–35). In vivo, RGS4 overexpression inhibits 5-HT_1A autoreceptors (36), and RGS4 inhibition in cortical neurons potentiates 5-HT_1AR signaling (37). These studies demonstrate nondiscriminate and opposing actions of RGS4 on presynaptic and postsynaptic 5-HT_1ARs. In addition, RGS4 also regulates Gα_q-coupled 5-HT receptors (29), known to promote anxiety. Together, these results make predictions regarding effects of RGS4 inhibition on net behavioral outputs challenging. Recent evidence suggests that while RGS4-knockout mice exhibit no baseline difference in anxiety or depression-related behaviors, RGS4 up-regulation in the nucleus accumbens may be important in the acute and chronic actions of antidepressants (38). Thus, different RGS proteins appear to regulate 5-HT_1AR populations in distinct temporal, contextual and spatial manners. RGS6 is the first RGS protein identified that appears to selectively modulate 5-HT_1A heteroreceptor populations in vivo, although the exact mechanisms contributing to this heteroreceptor specificity remain unknown.

In summary, our results demonstrate that RGS6 deletion selectively enhances the actions of endogenous 5-HT at postsynaptic 5-HT_1A heteroreceptors, believed to underlie the beneficial effects of serotonergic drugs, leading to spontaneous anxiolytic and antidepressant behavior. Although the exact downstream molecular determinant of antidepressant phenotype of RGS6^−/− mice remains unclear, signaling cascades classically activated by the Gβγ subunit of the heterotrimeric G-protein complex appear to be dispensable, and, instead, Gα_i-mediated inhibition of AC is required. In short, RGS6 functions as the gatekeeper for 5-HT-mediated anxiolytic and antidepressant actions due primarily to its ability to completely block 5-HT_1AR-mediated inhibition of AC.