Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor.

adding immense diversity to the function of the receptor in vivo. Furthermore, recent studies using animal models suggest that RNA editing of the 5-HT 2C receptor is a dynamic process. For example, chronic stress increases the degree of RNA editing of the 5-HT 2C receptor in both mice (Englander et al., 2005) and rats (Iwamoto et al., 2005). Variation in 5-HT 2C receptor RNA editing has also been implicated in a variety of neuropsychiatric disorders, with the most reproducible fi nding being an increase in RNA editing in brains of suicide victims (Niswender et al., 2001;Gurevich et al., 2002;Dracheva et al., 2008a,b). Despite these fi ndings, defi ning the in vivo functional consequences of 5-HT 2C receptor RNA editing has been limited due to technical constraints presented by the immense diversity of receptor RNA isoforms in brain.
In vitro studies in cultured cells transfected with cDNA encoding a single receptor isoform show that differentially edited 5-HT 2C receptor isoforms have unique signaling features, with increased editing generally leading to decreased function. For example, the edited isoform valine-serine-valine (at amino acid positions 157, 159, 161 in humans) has four-fold reduced constitutive activity and four-to fi ve-fold reduced serotonin potency to activate phospholipase C (PLC) relative to the non-edited isoleucineasparagine-isoleucine isoform (INI); the function of the fully edited valine-glycine-valine (VGV) isoform is reduced even further (Fitzgerald et al., 1999;Herrick-Davis et al., 1999;
A distinctive feature of the G-protein coupled 5-HT 2C receptor is its ability to be modifi ed posttranscriptionally by RNA editing (Burns et al., 1997). RNA editing is an enzymatic process that converts adenosine to inosine at fi ve nucleotide positions in the 5th exon of the 5-HT 2C receptor RNA transcript, encoding the second intracellular loop, a region important for G-protein coupling. Selective RNA editing can generate up to 32 mRNA isoforms, predicting 24 protein isoforms of the 5-HT 2C receptor, potentially signifi cantly different from the expected 50% (one sample t-test, t = 3.0949, df = 3, p > 0.05). The fi nding that the frequency of VGV isoform in the heterozygous VGV/X mice is about 50%, combined with subsequent results showing that receptor density in VGV/X mice is about halfway between WT and VGV/Y mice, suggests that there is not signifi cant bias in X-inactivation. We also verifi ed that the increase in the percentage of the VGV isoform in brains of VGV/X mice did not alter the remaining distribution of isoforms ( Figure 1B).

GTP-SENSITIVE HIGH-AFFINITY AGONIST BINDING IN MEMBRANES
Agonists, such as DOI, bind preferentially to 5-HT 2C receptors existing in a high-affi nity state, a receptor conformation that is coupled to G-protein. In vitro, 5-HT 2C-VGV receptors show no basal coupling to G-proteins (Herrick-Davis et al., 1999;Niswender et al., 1999). We tested whether this recapitulates in brain. Mice were briefl y anesthetized using isofl urane and decapitated. Unless otherwise indicated, brains were removed, placed in a slicing mold, and sliced coronally at the level of the optic chiasm. Tissue anterior to the cut, defi ned here as forebrain, was assayed. Tissue was homogenized in 20 ml of ice cold binding buffer (50 mM Tris, 10 mM MgCl 2 , 0.1% ascorbic acid, pH 7.4). Homogenates were centrifuged at 15,000 × g for 20 min. at 4°C. Supernatant was decanted, and the pellet was re-suspended in fresh binding buffer and incubated at 37°C for 15 min to dissociate endogenous ligand from receptors. After a second centrifugation, supernatant was decanted, and the pellet re-suspended in fresh, cold binding buffer. Binding assays were run on the same day as the membrane preparation to avoid a decrease in signal due to freezing.
[ 125 I]-DOI saturation binding was performed on membranes from VGV/Y and WT mice to test potential differences in the proportion of receptors in the agonist high-affi nity conformation. Assays were carried out in triplicate and incubated for 90 min in a 37°C water bath at fi nal volume of 600 µl, consisting of 500 µl of membrane suspension, 1 µM spiperone to mask 5-HT 2A and 5-HT 7 binding sites and increasing concentrations of [ 125 I]-DOI. Non-specifi c binding was determined in the presence of 100 µM methysergide. Data were analyzed using nonlinear regression (GraphPad Prism 5.02, Graphpad Inc., USA). In a separate experiment, GTP sensitivity was evaluated using a single concentration of [ 125 I]-DOI (1 nM) in the absence and presence of 200 µM guanylyl-5′-imidodiphosphate (Gpp(NH)p), a non-hydrolyzable analog of GTP that prevents G-protein coupling. Total density was estimated in each membrane preparation using a single saturating concentration (30 nM) of [ 3 H]-mesulergine. 1999). These studies suggest dramatic alterations will accompany changes in RNA editing of the 5-HT 2C receptor; however, it is widely accepted that functional properties demonstrated in cell culture studies of cloned receptors may not be reproduced in vivo.
Since the majority of 5-HT 2C receptors in brain are edited (Burns et al., 1997), studies of the in vivo functional consequences of 5-HT 2C receptor RNA editing are critically needed. Here we take advantage of mutant mice solely expressing a single isoform, the fully-edited 5-HT 2C-VGV receptor, to characterize for the fi rst time the signal transduction consequences of RNA editing of the 5-HT 2C receptor in vivo. We show that the proximal event in cell signaling, receptor:G-protein coupling, is nearly abolished for the fully edited 5-HT 2C-VGV receptor in brain. We further show that changes in the degree of editing signifi cantly alters the density of receptor binding sites within the brain, suggesting that the pattern of protein isoforms, and hence the inferred overall function of the receptor protein, may not refl ect the pattern of RNA isoforms. This fi nding has signifi cant ramifi cations for basic as well as clinical studies of disease-related alterations in RNA editing of the 5-HT 2C receptor.

ANIMALS
All experiments involving animals were approved by the Institutional Animal Care and Use Committee of Vanderbilt University. Naive adult mice, 3-6 months of age, were used for all experiments. Mice were housed in groups of 2-5 in a temperature controlled, colony room (ambient temperature 22-23°C, 12:12 light: dark cycle). Food and water were available ad libitum, and all testing occurred between 1200 and 1700 during the light phase. Unless otherwise indicated, male mice were used.
Mutant mice solely expressing 5-HT 2C-VGV receptors were generated by homologous recombination using a replacement-type targeting vector with the fi ve edited adenosine residues of the 5-HT 2C receptor gene mutated to guanosine to mimic the base-pairing properties of inosine (Morabito et al., 2007). Mice were maintained on a mixed JAX ® 129S1/Taconic ® 129S6 background. Four genotypes of mice were used in the experiments examining functional characteristics of the in vivo expressed 5-HT 2C-VGV receptor: wildtype males and females (WT), heterozygous females (VGV/X), and hemizygous (the 5-HT 2C receptor is X-linked) males (VGV/Y), expressing solely the VGV isoform. The genotypes of mice were determined by PCR analysis of genomic DNA from tail samples using the RED Extract-N-Amp tissue PCR kit (Sigma, St. Louis, MO, USA), with the following primers: 5′GGG CAA ATA TTC TGA AAA GAT GTT 3′ (reverse) and 5′AAT ATC AAT AGG TAA TTA TAC C 3′ (forward).
To assess the possible contribution of gene dosing, heterozygous mice expressing a mixture of receptor isoforms enriched with the mutant allele were used; these studies were performed in female mice since the 5-HT 2C receptor is X-linked and it is not possible to generate heterozygote males. Using the method of pyrosequencing (Canal et al., 2009), we verifi ed that heterozygous VGV/X females had VGV as the major isoform,while control mice (N = 4, average of 60 sequences per mouse) had less than 1% VGV isoforms. VGV/X females (N = 4, average of 60 sequences per mouse) had an average of 62% of RNA isoforms that translate to VGV ( Figure 1A); although suggestive of biased X-inactivation, this value was not Samples were incubated for 90 min in a 37°C water bath, and the reactions were terminated by the addition of ice-cold 50 mM Tris buffer. Membranes were collected by vacuum fi ltration (Brandel harvester) using Whatman GF/B glass fi ber fi lters presoaked in 0.3% polyethyleneimine. Filters were rinsed three times with cold Tris buffer, and the bound radioactivity was determined by liquid scintillation spectrometry after soaking fi lters overnight in scintillation cocktail (Aquasol-2, PerkinElmer, Boston, MA, USA).
[ Differences in other proportions (e.g., ABD, VNV) refl ect inclusion of VGV in the calculation of the editing measures (%). Heterozygous VGV/X females expressed the 5-HT 2C-VGV isoform at a quantity (mean = 61.9%, 95% CI 49.7-74.1%) that was signifi cantly higher than the rare expression observed in wild type litter mate females (two sample t-test, t = 15.89, df = 6, p < 0.0001) and signifi cantly lower than the 100% expression seen in VGV homozygous females and hemizygous VGV/Y males (one sample t-test, t = −9.92, df = 3, p = 0.002). (B) Percentage distribution of Htr2c RNA excluding the fully edited sequences (ABECD or VGV) shows that the remaining distribution is unaltered by the presence of a VGV transgenc (Fischer's exact Chi-square test p = 0.37).
non-specifi c binding. The addition of 300 nM SB204741, a 5-HT 2B antagonist, did not alter [ 3 H]-mesulergine binding in WT or VGV/Y brain slices.
Slides were incubated for 120 min at room temperature, then washed 4 × 10 min in ice-cold 50 mM Tris buffer, pH 7.4 and dried with a steady stream of dehumidifi ed air.
After air drying for an additional 90 min, slides were placed in autoradiography cassettes and exposed to Kodak Biomax MR fi lm (Carestream Health, Inc., USA) for 24-48 h for [ 125 I]-DOI or 6-8 weeks for 3 H-mesulergine prior to developing (shorter exposures were required for brain sections from VGV/Y mice to accommodate an increased density of 5-HT 2C receptors). Film was scanned (CanoScan 4400F, Canon, USA) onto a PC, and images were analyzed with Image J software (Abramoff et al., 2004). The average of the minimum gray value (darkest area) from three brain slices containing brain area of interest was converted to µCi/g protein using 14 C standards (ARC, Inc., St. Louis, MO) for statistical comparisons of specifi c [ 125 I]-DOI binding, and 3 H standards for statistical comparisons of specifi c [ 3 H]-mesulergine. Data were compared using 2-tailed unpaired t-tests.

5-HT 2C RECEPTOR AGONIST IMPACT ON LOCOMOTOR ACTIVITY IN VGV/Y AND VGV/X
Dose-response studies were conducted to assess the locomotor effects of MK212, a preferential 5-HT 2C receptor agonist (Fone et al., 1998;Gleason et al., 2001;Stiedl et al., 2007;Fletcher et al., 2009) in WT, hemizygous VGV/Y and heterozygous VGV/X mice. Locomotor activity was measured in Plexiglas activity chambers (11 × 11 × 11 in; LxWxH; Med Associates St. Albans, Vermont), equipped with 16 infrared beams to monitor and record beam breaks in the x-y-z coordinates. VGV/Y and VGV/X mice were tested with saline and MK212 at doses of 0.01, 0.03, 0.1, 0.3 and 1.0 mg/kg. Twenty minutes after s.c. injection, individual mice were placed inside the activity chamber and distance traveled was recorded for 10 min using Activity Monitor software version 5 (Med Associates, St. Albans, Vermont, USA). Dose-response data were analyzed by two-way ANOVA for independent groups.

BIOGENIC AMINE TURNOVER IN VGV/Y MICE
Dopamine (DA), serotonin (5-HT) and metabolites were quantifi ed by high-performance liquid chromatography (HPLC) electrochemical detection methods (Vanderbilt Neurochemistry Core). Animals were anesthetized with isofl urane and rapidly decapitated after cervical dislocation. Brains were removed and placed in a chilled stainless steel mold for dissection -the tissue slice 1.7-3.6 relative to bregma is referred to as frontal cortex; striatum represents slice 1.70 to −0.82 relative to bregma with the cortex removed by freehand dissection. Tissue was frozen rapidly on dry ice and stored at −80°C until assay. Thawed samples were homogenized in 250 µl acetonitrile and centrifuged at 13,000 × g for 30 min. The acetonitrile fraction was transferred to a clean tube, washed twice with 125 µl heptane and then evaporated using a stream of nitrogen. The sample was suspended in 75 µl of the HPLC mobile phase (37.5 mmol H 3 PO 4 , pH 8.5) and 50 µl was injected into the equilibrated HPLC. DA turnover was measured by the ratio DOPAC/DA and HVA/DA; 5-HT turnover was measured by the ratio 5-HIAA/ 5-HT. Each measure was predicted by a mixed ANOVA model that incorporated between-subject fi xed effects for genotype (VGV/Y or WT) and drug (saline or SB206553), a within-subject fi xed effect for brain area (frontal cortex or striatum), and a random effect to account for repeated measures in each mouse.

[ 3 H]-MESULERGINE SATURATION BINDING IN MEMBRANES
The procedures for [ 3 H]-mesulergine saturation binding were essentially identical to [ 125 I]-DOI binding in membranes. Membrane homogenates, binding buffer, spiperone at a fi nal concentration of 1 µM, and increasing concentrations of [ 3 H]-mesulergine up to 36 nM were added. Non-specifi c binding was determined in the presence of 100 µM methysergide.

5-HT 2C-VGV RECEPTORS IN BRAIN EXHIBIT REDUCED HIGH-AFFINITY AGONIST BINDING: MEMBRANE BINDING
In saturation binding assays of membranes from WT littermates, the binding of the radiolabeled agonist [ 125 I]-DOI was best fi t by a two-site equation, as illustrated in the Scatchard plot (Figure 2A). Approximately 10% of the binding sites were in a high affi nity state (K d = 0.51 nM).The low-affi nity site had a K d (20 nM) and density of 137 fmol/mg protein. In contrast, [ 125 I]-DOI binding in membranes from VGV/Y mice was best fi t by a one-site equation, as illustrated in the Scatchard plot ( Figure 2B) with a K d (29 nM) that was consistent with the K d of the low affi nity site in WT mice. The extremely high density of binding sites in VGV/Y mice was confi rmed in [ 3 H]-mesulergine binding experiments (see below).
To corroborate the fi nding of decreased high-affi nity binding sites in brains of VGV/Y mice and to estimate the percentage of G-protein coupled receptors, the binding of a single concentration of [ 125 I]-DOI was determined in the absence and presence of Gpp(NH)p to uncouple the receptor:G-protein complex (Figure 3). Fifty-seven percent of the [ 125 I]-DOI binding in membranes from WT mice was eliminated by addition of Gpp(NH)p; in contrast [ 125 I]-DOI binding in membranes from VGV/Y mice was Gpp(NH)p-insensitive (Figure 3).

VGV/Y MICE HAVE INCREASED SENSITIVITY TO 5-HT 2C RECEPTOR AGONIST: LOCOMOTOR ACTIVITY
Injection of the 5-HT 2C agonist, MK212, dose-dependently decreased total distance traveled in all genotypes, yet the potency of MK212 was greater in mutant mice relative to their respective WT littermates. For WT, female VGV/X and VGV/Y mice (Figure 5), a 2-way ANOVA revealed main effects of dose (F 5,135 = 31.77,   www.frontiersin.org p < 0.0001) and genotype (F 2,135 = 42.46, p < 0.0001). The potency of MK212 was 10-fold higher in VGV/Y and VGV/X mice relative to WT's ( Figure 5; N = 10). For example, 0.1 mg/kg MK212 essentially eliminated locomotor activity in VGV/Y mice (post-hoc Fisher least signifi cant difference (LSD) test, saline vs. 0.1 mg/kg, p < 0.001), whereas this dose did not reduce locomotor activity in WT mice (post-hoc Fisher LSD test, saline vs. 0.1 mg/kg, p = 0.58). There were no gender differences in the motor effects of MK212 in WT mice (data not shown).

VGV/Y MICE HAVE INCREASED SENSITIVITY TO 5-HT 2C RECEPTOR INVERSE AGONIST: DA TURNOVER
Given that the 5-HT 2C receptor has been reported to exert an inhibitory infl uence on DA release, we asked if the DA turnover is decreased in VGV/Y mice and further if SB206553 differentially altered DA turnover. Twenty minutes after treatment with saline or 3 mg/kg of the inverse agonist SB206553, biogenic amine levels were determined in striatum and cortex of VGV/Y and WT male mice ( Table 1). DA turnover was measured by the ratio DOPAC/DA and the ratio HVA/DA, and 5-HT turnover was measured by the ratio 5-HIAA/5-HT. Each measure was predicted by a mixed ANOVA model that incorporated between-subject fi xed effects for genotype (VGV/Y or WT) and drug (saline or SB206553), a within-subject fi xed effect for brain area (cortex or striatum), and a random effect to account for repeated measures in each mouse.
Although basal DA turnover (saline treated) was reduced in cortex of VGV/Y mice (p = 0.039 for DOPAC/DA, p = 0.063 for HVA/DA, least-squared difference post-hoc tests, Figures 6A,B), this effect was small and not reproduced in the striatum. A more striking dopamine phenotype occurred in both cortex and striatum following SB206553 challenge. Signifi cant genotype by drug interactions were observed for the ratios DOPAC/DA (Figure 6A), HVA/DA (Figure 6B), and for 5-HIAA/5-HT ( Figure 6C). Similar interaction profi les of the marginal means were observed for each turnover measure (Figure 6), where VGV/Y mice showed signifi cantly increased turnover after SB206553 challenge compared to saline, but WT mice revealed a relatively fl atter profi le across saline and SB206553 conditions.

VGV/Y MICE SHOW LARGE INCREASES IN 5-HT 2C RECEPTOR DENSITY: [ 3 H]-MESULERGINE SATURATION BINDING
[ 3 H]-Mesulergine saturation binding was performed to determine the maximum density of 5-HT 2C receptor binding sites in brains of WT, VGV/Y and VGV/X mice (Figure 7). All plots were best fi t by a single site equation. The density of 5-HT 2C receptors in the forebrain was increased by 25-fold in VGV/Y mice relative to WT mice (B max = 163.3 ± 5.9 fmol/mg protein in WT vs. 4266 ± 260 in VGV/Y; N = 4). Heterozygous VGV/X mice displayed an approximately 10-fold increase in the density of [ 3 H]-mesulergine binding sites (B max = 1754 ± 149 fmol/mg protein; N = 8), which was intermediate to that observed for WT and VGV/Y mice. The K d values for [ 3 H]-mesulergine were identical in all three genotypes. Furthermore, in competition binding experiments, the Ki values for MK212, SB206553 and spiperone were not altered in membranes from VGV/Y vs WT mice (data not shown).

Frontiers in Neuroscience | Neuropharmacology
widespread increase in 5-HT 2C receptor binding in thalamus, amygdala, posterior cingulate cortex, piriform cortex (Figure 8) appears to reproduce the distribution in WT mice, suggesting that overexpression in the mutant mice does not refl ect an aberrant distribution of the 5-HT 2C receptor.

DISCUSSION
5-HT 2C receptor RNA transcripts undergo fi ve adenosine to inosine RNA editing events, resulting in the generation of distinct amino acids in the second intracellular loop of the protein, a region critical for G-protein coupling (Moro et al., 1993;Ballesteros et al., 1998;Gaborik et al., 2003). Combinations of the fi ve edited nucleotides predict 24 different protein isoforms (Burns et al., 1997); many isoforms display altered signaling properties in transfection experiments in cell lines. The fully-edited VGV isoform, where all fi ve sites are edited, shows the most dramatic decrease in agonist potency, as well as silencing of receptor constitutive activity and elimination of high-affi nity, Gpp(NH)p sensitive agonist binding . The current set of experiments takes advantage of genetically modifi ed mice solely expressing the VGV isoform to show, for the fi rst time in brain, that RNA editing of the 5-HT 2C receptor alters the proximal event in cell signaling, receptor: G-protein coupling. Several techniques were used to highlight the in vivo properties of the 5-HT 2C-VGV receptor including [ 3 H]-mesulergine saturation binding, [ 3 H]-mesulergine autoradiography, [ 125 I]-DOI saturation binding, and [ 125 I]-DOI GTP-sensitive high affi nity binding in membranes and in autoradiography. Nonlinear regression analysis of [ 125 I]-DOI saturation binding revealed two saturable binding sites in membranes from brains of WT mice, with the high affi nity site representing nearly 10% of the total binding. In contrast, nonlinear regression analysis of [ 125 I]-DOI saturation binding to membranes from mice expressing the VGV isoform reveals only a single, low-affi nity site, even though receptor density is increased dramatically. Based on the extended ternary complex model of receptor:G-protein coupling (Samama et al., 1993), it is likely that the loss of high affi nity binding refl ects reduced G-protein coupling. B max (fmol/mg protein) ± SEM: VGV/ Y, 4266 ± 259.9 **(p < 0.001 compared to WT and VGV/ X); VGV/X, I754 ± 149 *(p < 0.001 compared to WT); WT, 163.3 ± 5.9. K d (nM) ± SEM: VGV/Y. 3.18 ± 0.23; VGV/X, 2.49 ± 0.30; WT, 2.13 ± 0.26. Differences between means were determined by a one-way ANOVA followed by a Tukey test.

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total 3 H-mesulergine binding is increased, the proportion of GTP-sensitive high affi nity [ 125 I]-DOI binding is reduced nearly eight-fold in choroid plexus of VGV/Y mice. These experiments confi rm that the blunted G-protein coupling of the VGV isoform described in cell lines is reproduced in a native setting. We conclude therefore that it is valid to interpret in vivo increases in the degree of RNA editing of the 5-HT 2C receptor as a loss of function at the cellular level. An attempt to evaluate the classical effector pathway, Gq mediated-PLC activation, was unsuccessful; 5-HT failed to elicit a PLC signal in cortical tissue from wild-type or VGV/Y mice. Previous studies of 5-HT 2C receptor-mediated PLC activation in rat brain were performed in choroid plexus (Conn and Sanders-Bush, 1986), but this was not feasible in mice.
Our studies also suggest that changes in the proportion of edited isoforms alter the density of 5-HT 2C receptor binding sites within the brain. VGV/Y mice, solely expressing the VGV isoform, have a 25-fold increase in 5-HT 2C receptor density relative to WT mice, similar to that found by Morabito et al. (2007). In heterozygous VGV/X mice, the percentage of RNA isoform encoding VGV and the density of 5-HT 2C receptor binding sites is approximately halfway between WT and VGV/Y mice. This gene-dose dependent increase in binding site density supports the notion that the increases are not the result of erroneous genetic manipulation. In addition, receptor autoradiography suggests that the increased expression of 5-HT 2C To test this further, [ 125 I]-DOI binding was carried out in the presence and absence of a GTP analog. The addition of Gpp(NH)p reduces the binding of [ 125 I]-DOI in brain membranes from WT mice, but has essentially no effect on [ 125 I]-DOI binding in membranes from VGV/Y mice, suggesting that the G-protein coupling capacity is compromised in brains of mice solely expressing the 5-HT 2C-VGV receptors. An unexpected fi nding was the marked increase in total [ 125 I]-DOI binding in brains of VGV/Y mice relative to WT, with a binding site density comparable to that found in the choroid plexus (Conn and Sanders-Bush, 1986). We considered the possibility that Gq/11 protein may become rate-limiting in the case of the large increase in receptor density in brains of VGV/Y mice. However, quantitative studies of Milligan (1993) show that the level of Gq protein in rat frontal cortex is 17 pmol/mg protein and combined Gq/G11, 25 pmol/mg protein, which is fi ve-fold higher than receptor density in VGV/Y mice. Furthermore, we have previously shown that the levels of Gq and G11 protein are not altered in VGV/Y mice (Canal et al., 2009). These fi ndings suggest that Gq/11protein is not rate limiting in the brains of VGV/Y and that the reduction in GTP-sensitivity refl ects reduced coupling capacity of the VGV isoform.
Consistent with a reduction in G-protein coupling capacity found in membranes prepared from brains of VGV/Y mice, receptor autoradiography in intact brain slices reveals that, although  Navailles et al., 2004;Alex and Pehek, 2007), we examined DA turnover as another index of in vivo 5-HT 2C receptor function. 5-HT 2C receptor regulation of DA release is complex, involving direct and indirect pathways; microdialysis studies suggest that the mechanism of regulation differs in the nigrostriatal versus mesocorticolimbic DA systems (Di Matteo et al., 1999;Di Giovanni et al., 2000;see Di Matteo et al., 2008 for review). This may explain the regional difference seen in saline-treated VGV/Y mice, where DA turnover in the mutant mice is decreased in frontal cortex but not in striatum. Microdialysis experiments combined with direct microinjection would be valuable in pursuing this question. The fi nding that SB206553 increases DA turnover in frontal cortex and striatum of VGV/Y mice is consistent with the evidence that 5-HT 2C receptors inhibit DA release in both areas. The ability of SB206553 to increase DA release has been proposed to refl ect constitutive activity of the 5-HT 2C receptor ; however, such an explanation is less plausible in mice solely expressing the VGV isoform, since in vitro data in transfected cells shows that this isoform is essentially devoid of constitutive activity Niswender et al., 1999;Wang et al., 2000;Berg et al., 2008) and the present studies in brain are consistent with that interpretation. An alternative explanation is suggested by our fi nding of increased 5-HT turnover in VGV/Y mice given SB206553; enhanced release of 5-HT onto the up-regulated 5-HT 2C receptors may play a role in the SB206553 modulation of DA release in VGV/Y mice.
In summary, in vitro studies in transfected cell lines have shown that RNA editing of 5-HT 2C receptor RNA transcripts profoundly alters the signaling properties of the 5-HT 2C receptor protein. The current studies utilizing genetically modifi ed mice solely expressing the most extensively edited isoform of the 5-HT 2C receptor show, for the fi rst time, that the G-protein coupling capacity in the brain is altered, i.e., expression of fully edited 5-HT 2C-VGV receptor isoform within the brain blunts receptor:G-protein coupling. We further show that changes in the degree of editing signifi cantly alters the density of receptor binding sites within the brain, suggesting that the pattern of protein isoforms, and hence the inferred overall function of the receptor protein, may not refl ect the pattern of RNA isoforms. This conclusion points to a critical need for a method that differentiates 5-HT 2C receptor protein isoforms in brain tissue. Until such a method becomes available, clinical and laboratory investigations must continue to rely on RNA isoform distribution to infer function. Our studies emphasize the need for caution in these interpretations. receptor in VGV/Y mice does not refl ect aberrant distribution of the receptor. It is possible that the increased density is a compensatory response to reduced cellular function of the edited receptor. In support of this conclusion, transgenic mice solely expressing the non-edited, highly functional, 5-HT 2C-INI receptor have reduced receptor density (unpublished results).

ACKNOWLEDGMENTS
The molecular mechanism of the substantial density alteration in mutant mice is unknown. In the initial characterization of the VGV/Y mouse phenotype, Morabito et al. (2007) found no change in the steady state levels of 5-HT 2C receptor RNA transcripts was detected. Other possible mechanisms include differences in translation effi ciency or stability of different protein isoforms. The latter is suggested by studies in cultured cells showing that the VGV isoform has diminished ability to bind to beta-arrestin and translocate into intracellular compartments (Marion et al., 2004). It is important to point out that the RNA isoform distribution, even in heterozygous mice where the VGV isoform accounts for approximately 50%, is markedly different from the native situation where the VGV isoform represents less than 5%. It is possible that some aberrant regulatory mechanism, which is not engaged under normal circumstances, is responsible for the marked increase in 5-HT 2C-VGV receptor density in the mutant mice. This question can only be defi nitively answered when a method becomes available for isolation and quantifi cation of individual protein isoforms.
Although our pharmacological studies in brains of mice expressing the VGV isoform document reduced G-protein coupling capacity, the marked increase in 5-HT 2C receptor site density may mitigate this diminished cellular signal in the intact animal. To investigate the functional consequences in vivo of these opposing effects, we employed a behavioral assay of 5-HT 2C receptor activation. 5-HT 2C receptor agonists, such as MK212, have long been known to induce hypolocomotion (Fone et al., 1998;Gleason and Shannon, 1998;Gleason et al., 2001;Stiedl et al., 2007), although there is debate about their specifi city. Recent studies in 5-HT 2C receptor knockout mice (Fletcher et al., 2009), showing that MK212-induced hypolocomotion is eliminated, suggest that, in mice, this behavior is a valid measure of in vivo 5-HT 2C receptor activation. We found that MK212 elicits a dose-dependent decrease in locomotor activity in both WT and VGV/Y mice, but the sensitivity to MK212 is dramatically increased in VGV/Y mice. This observation confi rms the work of Kawahara et al. (2008), and importantly shows that the marked behavioral sensitization is reproduced on another background strain. These data suggest that the large increase in receptor density overcomes the reduced G-protein coupling of the VGV receptor. The fi nding that 5-HT 2C antagonists increase locomotor activity in VGV/Y mice (Kawahara et al., 2008; data not shown) agrees with this interpretation. Given emerging evidence suggesting that the 5-HT 2C receptor attenuates DA release and 5-HT 2C receptor antagonists augment the action of dopamine releasing agents such as cocaine (Filip and Cunningham, 2003;De Deurwaerdere et al.,