Synergistic Roles for G-protein γ₃ and γ₇ Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

Background: Specificity of G-protein function may be determined by a specific αβγ composition.

Results: Combinatorial disruption of γ₃ and γ₇ produces a severe seizure phenotype not observed with either gene alone.

Conclusion: This reflects distinct roles for γ₃ and γ₇ in G_i/o- and G_olf-signaling pathways that modulate seizure susceptibility.

Significance: The γ subunits direct the assembly of distinct G-protein αβγ heterotrimers that specify diverse receptor actions.

Abstract

The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ₃ and γ₇ subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3^−/−Gng7^−/− double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ₃ subunit is a component of a G_i/o protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ₇ subunit is a component of a G_olf protein that is responsible for A_2A adenosine or D₁ dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Introduction

Proper functioning of the central nervous system requires the coordination of several hundred receptors whose actions may be mediated by a similarly large number of distinct G-protein αβγ heterotrimers. Identifying the specific G-protein αβγ subunit combinations functioning in particular signaling pathways has been a challenge. Although specificity of G-protein function was originally ascribed to the various α subtypes, there is a growing recognition that diverse βγ dimers may impart an additional level of selectivity (1–3). Compared with the five β subtypes, the 12 γ subtypes are more structurally diverse, suggesting the in vivo specificity observed among different βγ dimers is most likely due to the γ component (2, 4, 5). Providing a rigorous test of this hypothesis, we produced Gng3^−/− and Gng7^−/− mice, which lack the closely related γ₃ and γ₇ subunits. Subsequent characterization of these animals revealed distinct neurological phenotypes, reflecting their roles in different receptor signaling pathways (6–8). Offering a mechanistic basis for their diverse roles, biochemical analyses of these animals identified a critical role for the γ subunit in directing the assembly of distinct G_i/o and G_olf heterotrimers (6–8). Taken together, these results support the notion that even closely related γ subtypes have distinct signaling roles and biological functions in the context of the animal.

In this study, we sought to extend these findings by exploring a novel interaction between signaling pathways requiring γ₃ and γ₇ subunits in brain. Suggesting this possibility, Gng7^−/− mice exhibit a nearly 40% up-regulation of the γ₃ protein in the striatum (8). The increased γ₃ abundance could reflect a compensatory mechanism aimed at replacing the lost γ₇ protein that is required for the G_olf-dependent signaling pathway. Alternatively, this change could reflect an adaptive mechanism arising from interaction between G_i/o- and G_olf-dependent signaling pathways that converge on a common neurological process. To distinguish between these possibilities, we produced Gng3^−/−Gng7^−/− double knock-out mice and characterized them at the behavioral, neurological, electrophysiological, cellular, and biochemical levels. Collectively, these results showed that double knock-out mice exhibit a progressive seizure disorder and premature death that is not observed for either single knock-out strain alone on the same genetic background. We speculate the severity of the phenotype results from simultaneous disruption of G_i/o- and G_olf-dependent signaling pathways in different neuronal populations that normally operate together to limit seizure initiation, seizure propagation, or seizure-induced damage.

EXPERIMENTAL PROCEDURES

Mice and Husbandry

Animal use was approved by the Geisinger Institutional Animal Care and Use Committee. Every effort was made throughout the study to minimize usage, pain, and discomfort of the animals. The generation of Gng3^−/− and Gng7^−/− single knock-out mice was described previously (6, 7). On a mixed genetic background (129, FVB, and B6), the Gng3^−/− mice experienced more handling-induced seizures (24%) compared with their littermate controls (8%) (7). However, after backcrossing onto the C57BL/6J (B6) background (The Jackson Laboratory, Bar Harbor, ME) for five or more generations, the Gng3^−/− mice showed no signs of seizure activity or premature death compared with their littermate controls (7). On either a mixed (129, BALB/c, and B6), or a B6 background, the Gng7^−/− mice did not display any evidence of seizure activity (6). Accordingly, to minimize any effect of the genetic background, the Gng3^−/− and Gng7^−/− mice were maintained on a B6 background (≥N7 backcross) in this study. For experimental purposes, Gng3^+/− or Gng7^+/− mice were intercrossed to produce single knock-out and control groups, whereas Gng3^+/−Gng7^−/− were intercrossed to generate double knock-out and control groups. Immediately after weaning, mice were genotyped and assigned to experimental groups that were similarly matched for age and sex.

Survival and Video Surveillance

Mice of different genotypes were incorporated into the survival study as they became available. The mice were housed in polycarbonate cages in ventilated racks (Thoren Caging Systems, Inc., Hazelton, PA) on a 14-h light and 10-h dark cycle, with the temperature maintained between 21 and 23 °C. The mice were allowed free access to water and standard chow (Mouse Diet 9F, Purina Mills, St. Louis), which contains 38.5% starch, 9% fat, 20% protein, and 3% fiber. To investigate the possible impact of a ketogenic diet on survival, mice were provided a TestDiet 8053 (Purina Mills), which contains 0.0% carbohydrate, 70% fat, 13.6% protein, and 8.3% fiber. For the survival study, the date and proximate cause of death of the animals were determined by close monitoring by facility staff and by video surveillance of home cages outfitted with infra-red CCTV cameras to continuously monitor the mice therein (ProVideo CVC-320WP, Amityville, NY). Signals were processed into a quad format with an EverPlex 4BQ (EverFocus Electronics Corp., San Marino, CA) and recorded with a time lapse video cassette recorder (HS-1280U, Mitsubishi Digital Electronics America, Inc., Irvine, CA). If a mouse was found dead in its cage, the previous 24 h of video were reviewed to determine the proximate cause of death. In 19 of 21 deaths recorded, seizures were found to immediately precede death. In the few cases in which animals were euthanized for humane reasons (e.g. severe dermatitis), mice were not included in the calculations. Survival curves were plotted, and median life spans and 95% confidence intervals (CIs)³ were calculated for each genotype. To determine the effects of genotype or sex differences on life span, a univariate analysis was performed using JMP 6.0 (SAS Institute, Carey, NC).

Electroencephalography

As shown by video surveillance, Gng3^−/−Gng7^−/− double knock-out mice experienced tonic-clonic seizures immediately preceding death. To look for neurological abnormalities associated with seizure activity, we performed electroencephalography on double knock-out, single knock-out, and wild type littermates. Under anesthesia, mice (12–16 weeks old) were implanted with epidural screw electrodes (Plastics One, Roanoke, VA) in five locations, frontopolar, right and left frontal, and right and left posterior, on the mouse skull that were connected to an electrode pedestal. The frontal electrodes were 1–1.5 mm anterior to the coronal suture; the mean distance between the frontal and parietal electrodes was 5.1 mm, and the distance between the frontal electrodes was 3.6 mm. After a 2-week period for recovery, electroencephalography (EEG) recordings were made on mice at varying intervals over the next 16 weeks, using a Nicolet Bravo electro-encephalograph (Nicolet, Madison, WI). Over the course of the study, 9 of 15 Gng3^−/−Gng7^−/− double knock-out mice died compared with 0 of 8 wild type mice, 0 of 9 Gng3^−/− single knock-out mice, and 0 of 14 Gng7^−/− single knock-out mice.

All EEG recordings were retained in digital format. Prior to analyses, EEG recordings were filtered with a 1 Hz high pass filter, a 35 Hz low pass filter, and a 60 Hz notch filter. Multiple analyses were performed. First, the average spike rate was computed for each mouse from the right frontal-right posterior and left frontal-left posterior derivations, although the other derivations were used to help identify artifacts. Spike counts were measured in multiple 30-min blocks, and the results were averaged to obtain a single mean spike rate from each mouse. The Kruskal-Wallis test was used to determine whether there was a difference between the mean spike rates among mice from different genotypes. Second, the power spectrum at each frequency was evaluated and averaged from at least three 30-min EEG segments for each mouse. The mean power for each frequency (<1, 1–2, 2–6, 6–10, and >10 Hz) was then determined. A repeated measures ANOVA test was used to determine whether there was any difference in the power spectrum among mice of different genotypes. Third, the interhemispheric coherence, which is a measure of the degree of synchrony between electrical activity in the right and left hemispheres, was also computed. For each 1-s epoch, the product of the Fourier transform of the EEG activity at each frequency from the right and left hemisphere (right frontal-right posterior and left frontal-left posterior) was computed and averaged over all epochs in the 30-min file. The coherence measure for each frequency was then calculated as the square of the average product divided by the product of the mean power in each derivation. The coherence value ranges between 0, when there is no synchrony, to 1, when there is complete synchrony. Finally, GABA_B agonist-induced EEG changes were assessed among mice of different genotypes by quantifying the power in each frequency band described above for 5-min clips taken at base line, 20 and 40 min, and 7 and 10 h post-injection (10 mg/kg intraperitoneal injection of baclofen). A repeated measures ANOVA test was performed, using genotype as the between subjects variable and the mean power and post-injection time as within subjects variables.

Brain Dissection

All dissections were completed within 5–10 min of death (9, 10) by making a vertical slice 0.5–1 mm caudal to the olfactory bulbs and a second vertical slice just rostral to the optic chiasm. The intervening section was placed with the caudal face up, and the nuclei accumbens was dissected with a 1-mm micropunch (Fine Science Tools, Foster City, CA) centered over each anterior commissure. The prefrontal cortex was dissected superior to the corpus callosum near the midline, and the caudate nuclei were dissected with a 2-mm micropunch inferior to the corpus callosum, bilaterally. After removal of the hypothalamus with tweezers, the caudal portion of the brain was placed dorsal side up. The cerebellum and pons were removed by a vertical slice between the superior and inferior colliculi. A slice was then made at a 45° angle from the dorsal caudal end down toward the ventral rostral end. The enterorhinocortical regions of the ventral portion were dissected. Finally, the ventral midbrain was isolated from the remaining ventral portion, by trimming the dorsal midbrain with a transverse slice. The various brain regions were placed in individual tubes, frozen immediately with liquid nitrogen, and stored at 80 °C until used for RNA or protein analyses described below.

RNA Analyses

The distribution of Gng3 and Gng7 transcripts was determined by RT-PCR analysis. The Mouse Multiple Tissue cDNA Panel (Clontech, Palo Alto, CA) was used as a PCR template to amplify Gng3, Gng7, and Gapdh, using the indicated primers shown in supplemental Table 1. PCRs were performed using the Advantaq Plus PCR kit (Clontech). The cycling conditions were 38 cycles of 94 °C for 30 s and 68 °C for 2 min, followed by a final extension of 68 °C for 5 min. Reactions were run in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA). Aliquots were removed from each sample at 22, 24, 26, 30, 34, and 38 cycles and visualized on 2% agarose gels containing ethidium bromide.

In parallel, the relative abundance of Gng3 and Gng7 transcripts was assessed by qPCR analysis. Total RNA was isolated from dissected brain regions from wild type and knock-out mice using TRIzol (Invitrogen). From 1 μg of RNA, the cDNA template was prepared using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). The qPCR standards were constructed by subcloning Gng3- and Gng7-specific PCR products into PCR II Topo vector (Invitrogen), using indicated primers shown in supplemental Table 1. Plasmid standards were quantitated spectrophotometrically and were serially diluted to contain 10¹ to 10⁷ DNA molecules. Melt curve and agarose gel electrophoresis were used to confirm single product amplification. Standard curves were evaluated for linearity (r = 0.99–1.0) and amplification efficiency (>90%). For each qPCR, duplicate samples of 50 ng of total RNA equivalents of brain region cDNA or plasmid standards were amplified for 40 cycles using gene-specific primers designed to span intron junctions (supplemental Table 1). Reactions were performed using iQ SYBR Green supermix (Bio-Rad) and run on the iCycler device with version 3.1 software (Bio-Rad).

Cellular Localization Strategies

The cellular distribution of γ₃ and γ₇ reporter proteins was assessed. For this purpose, transgenic (Tg) mice, in which expression of enhanced green fluorescent protein (GFP) is driven by the Gng3 promoter (Tg(Gng3-GFP)HK208Gsat mice) or the Gng7 promoter (Tg(Gng7-GFP)FG220Gsat mice), were obtained from Mutant Mouse Regional Resource Center, University of California, Davis (stock numbers 015490-UCD and 011393-UCD). Because GFP expression in the Tg(Gng7-GFP)FG220Gsat mice was not sufficient to achieve single cell resolution, we produced a line of KI(Gng7-γ₃-IRES-GFP) mice, in which the endogenous Gng7 locus was used to independently drive γ₃ and GFP expression. For this purpose, a targeting vector was designed that contained a modified Gng7 locus replacing the protein coding exons of γ₇ with the γ₃ cDNA, an internal ribosome re-entry site (IRES), and a GFP cDNA.

To visualize cell-specific expression, Tg(Gng3-GFP)HK208Gsat and KI(Gng7-γ₃-IRES-GFP) animals were transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in PBS. Brains were removed and preserved with the same fixative at 4 °C for 24 h. Sequential coronal sections (30 μm) were made through the forebrains of 8–12-week-old mice. For these experiments, sections were washed three times for 10 min with PBS and then blocked for 30 min in 0.4% Triton X-100 in PBS containing 6% donkey or goat serum. Primary antibody incubations were performed at 4 °C overnight, using either goat anti-ChAT (ab144p; 1:100 dilution; Millipore, Billerica, MA), or anti-DARPP-32 (d1075-01; 1:100 dilution; US Biologicals, Swampscott, MA). Subsequently, sections were washed three times for 10 min with 0.4% Triton X-100 in PBS and then incubated with Rhodamine Red-X-conjugated AffiniPure donkey anti-goat IgG (705-295-147; 1:100 dilution; Jackson ImmunoResearch, West Grove, PA) or Alexa Fluor 546-conjugated goat anti-rabbit IgG (A11035; 1:250 dilution; Invitrogen) at room temperature for 1 h. Finally, sections were washed three times for 10 min with 0.4% Triton X-100 in PBS, placed on microscope slides, and sealed under glass coverslips with Prolong Gold antifade reagent (P36930; Invitrogen). For confocal imaging, a Leica TCS SP2 confocal laser scanning microscope was used for excitation of endogenous GFP, Rhodamine Red-X, or Alexa Fluor 546 fluorophores. Colocalization was determined by visualizing cell bodies of GFP⁺, Rhodamine Red-X⁺, or Alexa Fluor 546⁺ neurons.

Immunoblot Analyses

Frozen brain tissues were homogenized in HME with proteinase inhibitors (20 mm HEPES, pH 8.0, 2 mm MgCl₂, 1 mm EDTA, 1 mm benzamidine, 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 μm leupeptin, 1.4 μm pepstatin, 27 μm 1-chloro-3-tosylamido-7-amino-2-heptanone, 28 μm l-1-tosylamido-2-phenylethyl chloromethyl ketone). Membranes were obtained by centrifugation on a sucrose cushion, and membrane-associated proteins were extracted with 1% cholate at 4 °C overnight. Protein concentrations were determined using an Amido Black assay (11). Equal amounts of proteins were loaded onto 12% Nu-PAGE gels (Invitrogen) and transferred to NitroPure nitrocellulose (Osmonics, Inc., Westborough, MA), and then the blots were probed with anti-G-protein subtype-specific antibodies (12). Briefly, after blocking, the blots were incubated for 1 h in high detergent blotto (50 mm Tris-HCl, pH 8.0, 80 mm NaCl, 2 mm CaCl₂, 5% nonfat powdered milk, 2% Nonidet P-40, and 0.2% SDS) containing various dilutions of the following: anti-γ₃ rabbit polyclonal (1:200); anti-γ₇ rabbit polyclonal (1:200); anti-β₁ rabbit polyclonal (1:400); anti-β₂ rabbit polyclonal (1:200); anti-α_i3 rabbit polyclonal (1:500); anti-α_s rabbit polyclonal (1:500); anti-α_olf rabbit polyclonal (1:2000; a gift from Denis Herve); anti-GABA_BR1 and GABA_B receptor mouse monoclonal (1:200; N93A/49; NeuroMab, University of California Davis), or anti-Ras mouse monoclonal (1:2000; BD Biosciences) antibodies. After three successive washes, the blots were incubated for 1 h in high detergent blotto containing either ¹²⁵I-labeled goat anti-rabbit IgG or ¹²⁵I-labeled goat anti-mouse IgG (0.5 μCi/ml, PerkinElmer Life Sciences). After washing, the blots were imaged with a PhosphorImager, quantified with the ImageQuant software (Amersham Biosciences), and normalized to the membrane-associated Ras protein to provide an internal control for protein loading and transfer efficiency.

Adenylyl Cyclase Assay

Frozen striatal punches were homogenized on ice with a motorized pestle (Kimble Chase, Vineland, NJ) in HME with proteinase inhibitors (20 mm HEPES, pH 8.0, 2 mm MgCl₂, 1 mm EDTA, 1 mm benzamidine, 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 20 μm leupeptin, 1.4 μm pepstatin, 27 μm tosyl-l-lysine chloromethyl ketone, 28 μm tosyl-l-phenylalanine chloromethyl ketone) and then repeatedly passed through a 25-gauge needle. Nuclei and unbroken cells were pelleted by low speed centrifugation (350 × g) for 5 min, and membranes were collected by ultracentrifugation at 250,000 × g for 1 h, resuspended in HME with proteinase inhibitors, and then stored at −80 °C. Protein concentrations were determined with Coomassie Plus (Pierce). Adenylyl cyclase activity was determined by incubating membrane protein (15 μg) at 30 °C for 15 min in 0.1 ml of buffer containing 10 mm imidazole, pH 7.5, 0.5 mm DTT, 0.5 mm MgCl₂, 0.2 mm EGTA, 0.1 mm ATP, 0.05 mm GTP, 1 × 10⁶ cpm of [α-³²P]ATP, 0.5 mm isobutylmethylxanthine, 5 mm creatine phosphate, 50 units/ml creatine phosphokinase, and various agonists as indicated in the text. Reactions were terminated by addition of 0.1 ml of 2% SDS, 40 mm ATP, 1.4 mm cAMP, 10,000 cpm of [³H]cAMP and heating to 100 °C for 3 min. [³²P]cAMP was isolated by chromatography on Dowex and Alumina columns, using [³H]cAMP as a recovery marker, and quantified by liquid scintillation counting.

In Vivo Responses to Baclofen

The muscle-relaxing effect of baclofen was assessed on an ENV-576 M Rota-Rod Treadmill (Med Associates, Inc., St. Albans, VT). Mice of different genotypes were placed on the Rota-Rod, which was then started at a constant 16 rpm. After acclimating mice to the instrument for 2 consecutive days, mice were given an intraperitoneal injection of saline (5 ml/kg) on the 3rd day followed by an intraperitoneal injection of a maximally effective dose of baclofen (10 mg/kg) on the 4th day. Thirty minutes after injection, mice were placed on the Rota-Rod. The results were recorded as the time spent on the instrument. Any mice that had not fallen from the Rota-Rod were removed after 5 min. The temperature lowering effect of baclofen was measured with a rectal thermometer by recording the temperature of mice before and 30 min after injection of baclofen.

Patch Clamp Electrophysiology

Gng3^+/− mice were intercrossed to prepare hippocampal neurons from individual embryonic day 18 mice (13). After plating onto 12-mm polylysine-treated coverslips, patch clamp analyses were performed on neurons in culture between 11 and 14 days without prior knowledge of the genotype. Currents from G-protein-sensitive inwardly rectifying potassium (GIRK, Kir3) channels were measured as described previously (14). Briefly, neurons were constantly voltage-clamped at −60 mV, recorded using a Multiclamp700B amplifier, digitized with a Digidata 1322B, sampled at 4 kHz, low pass-filtered at 1 kHz, and collected using pClamp9.2 (all from Molecular Devices). Series resistance and cell capacitance were automatically compensated and monitored at the beginning and end of each experiment. Potassium currents were monitored by switching from a low potassium solution containing 140 mm NaCl, 4 mm KCl, 2 mm CaCl₂, 2 mm MgCl₂, 20 mm HEPES, and 10 mm glucose, pH 7.4, to a high potassium solution containing 84 mm NaCl, 60 mm KCl, 2 mm CaCl₂, 1 mm MgCl₂, 20 mm HEPES, and 10 mm glucose. The adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA; 2 μm), the somatostatin receptor agonist (1 μm), or baclofen (100 μm) were dissolved in the bath solution and applied using an automated perfusion system. Current amplitudes were measured at −60 mV. TertiapinQ (120 nm), a specific peptide inhibitor of Kir3 channels, and barium (3 mm) were used to measure the residual inwardly rectifying current. The tertiapinQ-sensitive current is defined as basal current. Any current activated by a given receptor ligand above the basal current is defined as agonist-induced current.

Statistics

For behavioral and biochemical studies, sample statistics and Student's t tests were computed using Excel (Microsoft). Data are presented as means ± S.E. Univariate survival curves were calculated and compared with a log-rank χ² test using JMP (SAS Institute, Cary, NC). EEG results were compared with the Kruskal-Wallis test, a nonparametric ANOVA, using Statistica (StatSoft, Tulsa, OK). EEG results were also compared using one- and two-way ANOVAs with Bonferonni post-tests using Prism 5.0 (GraphPad Software, San Diego). Any significant effects have been reported in the text or the accompanying figure legend.

RESULTS

Severe Phenotype of Double Knock-out Mice

The Gng3^−/− Gng7^−/− mice were used to investigate the impact of combined loss of the γ₃ and γ₇ subunits. The effectiveness of this strategy was confirmed by immunoblot analysis of striatal membranes, revealing complete loss of the γ₃ and γ₇ proteins in brains obtained from double knock-out mice (Fig. 1A). Although born at the expected Mendelian frequency, Gng3^−/−Gng7^−/− mice showed high mortality that was not observed for either single knock-out line (Fig. 1B). In all, 51 of 52 double knock-out mice died before 1 year of age, with a median survival of 75 days (95% CI, 68–81 days). There was no significant effect of sex on life span. Because 32% of the double knock-out mice displayed handling-induced seizures at a median of 18 days before death (range, 6–130 days), we suspected that the double knock-out mice were dying as a result of recurrent seizures.

FIGURE 1.

Gene targeting strategy. A, validation of gene targeting strategy by immunoblotting cholate-extracted membrane protein from dorsal striatum of single knock-out (Gng3^−/− and Gng7^−/−), double knock-out (Gng3^−/−Gng7^−/−), and wild type (wt) mice, verifying loss of appropriate γ₃ and/or γ₇ subunit(s); Ras is shown as a loading control. B, on the regular diet, survival of Gng3^−/− mice at an N10 backcross to B6 (>1 year) is normal. In contrast, survival of Gng3^−/−Gng7^−/− double knock-out mice (75 days, 95% CI, 68–81 days) is severely reduced compared with their Gng3^+/+Gng7^−/− littermates (>1 year) (log rank χ² = 120.7, p < 0.0001). There was no significant difference between male and female double knock-out mice in terms of their reduced survival (log rank χ² = 0.3, df = 1, p = 0.6). C, on a ketogenic diet, survival of Gng3^−/−Gng7^−/− double knock-out mice (239 days, 95% CI, 152–292 days) was still reduced compared with their Gng3^+/+Gng7^−/− littermates (>1 year) (log rank χ² = 57.5, p < 0.0001). However, survival of Gng3^−/−;Gng7^−/− mice on ketogenic diet was improved relative to those on regular diet (log rank χ² = 37.8, p < 0.0001). In this case, there was a significant difference between male and female double knock-out mice in terms of overall survival (log rank χ² = 9.5, df = 1, p = 0.002).

Seizures Are the Probable Cause of Death

To examine the events surrounding their deaths, four mouse cages were subjected to continuous surveillance with infrared video cameras. From these recordings, we ascertained that 19 of 21 Gng3^−/− Gng7^−/− mice experienced seizures for ∼1 min immediately prior to their deaths. Seizure-induced deaths were observed at various times throughout the day and were not associated with any particular activity (i.e. sleeping, walking, eating, or grooming). Typically, seizures were characterized by a progression from wild running to tonic-clonic convulsion to tonic hindlimb extension that ended in death. Further supporting seizures as the proximate cause of death, administration of a ketogenic diet, which has been used as an effective treatment for refractory seizures (15–17), significantly prolonged the life span of double knock-out mice (Fig. 1C). Double knock-out mice on a ketogenic diet displayed significantly longer life spans than their wild type and single knock-out littermates on a regular diet (compare Fig. 1, B and C). However, female double knock-out mice on a ketogenic diet had a median survival of 334 days (95% CI, 239–430 days), compared with 154 days for male double knock-out mice on a ketogenic diet (95% CI, 113–185 days). Taken together, these results are most consistent with the deaths of double knock-out mice resulting from seizure activity and that administration of a ketogenic diet to suppress their seizure activity improved their viability.

Abnormal Electrical Activity in Knock-out Mice

To identify neurophysiological abnormalities in double knock-out mice, we compared video-EEG recordings from four groups of mice as follows: Gng3^−/−Gng7^−/− (n = 14); Gng3^−/− (n = 9); Gng7^−/− (n = 14); and wild type mice (n = 7). The data from three 30-min clips were analyzed for each mouse. Gng3^−/− Gng7^−/− mice exhibited several EEG abnormalities. First, representative EEG tracings (Fig. 2A) and quantitative analysis (Fig. 2B) showed that the frequency of interictal epileptiform discharges was strongly influenced by genotype, as determined by the Kruskal-Wallis test (χ² = 12.9, df = 3, p < 0.005). In particular, the interictal spike frequency was highest in the double knock-out mice, intermediate in the single knock-out mice, and lowest in the wild type mice. Because interictal spike frequency is an indicator of increased seizure risk (18), the finding that double knock-out mice exhibited more interictal discharges was consistent with their seizure phenotype. Second, double knock-out mice displayed a significantly lower inter-hemispheric coherence (Fig. 2C). Because a lower inter-hemispheric coherence is commonly observed in neurological disorders (19–21), the observation that double knock-out mice displayed reduced inter-hemispheric connectivity was also consistent with a neurological phenotype. Finally, a Spearman rank correlation analysis showed a strong inverse correlation between the inter-hemispheric coherence and the spike frequency (r = −0.38, p < 0.02). Indeed, the combination of these two EEG changes by themselves allowed the identification of double knock-out mice with 75% accuracy without prior knowledge of their genotypes.

FIGURE 2.

Comparison of EEG profiles among knock-out mice. A, representative EEG tracings, showing normal EEG in a Gng7^−/− mouse (left panel), lead placement (middle panel), and spike wave discharges in a Gng3^−/−Gng7^−/− mouse (right panel). B, mean spike rate was increased in the Gng3^−/−Gng7^−/− mice (n = 14) relative to wild type mice (n = 7), and Gng3^−/− (n = 9) and Gng7^−/− mice (n = 14) had intermediate spike rates (one-way ANOVA, F_3,3 = 4.6, p = 0.008, **, p < 0.05 for comparison with wild type in Tukey test). C, interhemispheric coherence was reduced in Gng3^−/−Gng7^−/− mice compared with all other genotypes (one-way ANOVA df = 3, F = 4.7, p = 0.007, **, p < 0.05 for comparison with all other genotypes in Tukey tests).

Functional Redundancy of Closely Related γ Subtypes within the Same Signaling Pathway as a Possible Basis for the Double Knock-out Phenotype

We first considered the possibility that the closely related γ₃ and γ₇ subtypes are substituting for one another in the same signaling pathway. Both the Gng3 and Gng7 transcripts were predominantly expressed in brain (Fig. 3A). Although the Gng3 transcript was widely expressed throughout brain, the Gng7 transcript was almost exclusively restricted to the striatum, including the caudate-putamen and nucleus accumbens, along with the enterorhinocortical region, including the hippocampus (Fig. 3B). Because their expression intersected primarily in the striatum, we focused on exploring functional interactions between the γ₃ and γ₇ subtypes in this region as the most likely basis for the double knock-out phenotype. Previously, the γ₇ subunit was shown to be required for both adenosine A_2A receptor (A_2AR) and dopamine D₁ receptor (D₁R) signaling in striatum (6, 8). Here, we demonstrated that the γ₃ subunit was not required for either of these signaling pathways. Gene targeted loss of the γ₃ protein did not affect stimulation of adenylyl cyclase by the A_2AR agonist CGS21680 (Fig. 4A) nor by dopamine or the D₁R selective agonist (±)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin hydrobromide (Fig. 4C). Likewise, combined deletion of both γ₃ and γ₇ proteins did not block activation of adenylyl cyclase by dopamine to a greater extent than loss of the γ₇ protein alone (Fig. 4C). Finally, deletion of the γ₃ subunit did not affect adenylyl cyclase stimulation by forskolin (Fig. 4B), and a combined loss of both γ₃ and γ₇ proteins subunits did not produce a greater effect than loss of the γ₇ protein alone (Fig. 4D).

FIGURE 3.

Expression analysis of G-protein γ₃ and γ₇ transcripts. A, RT-PCR products obtained after 34 cycles of amplification with primers JR282/JR286 (Gng3) and JR385/JR387 (Gng7), showing relative expression in various mouse tissues. RT-PCR product obtained after 24 cycles of amplification with primers Gapds/Gapdas (Gapdh), confirming similar cDNA amounts in all tissues. B, qPCR analysis showing relative number of Gng3 and Gng7 copies in different brain regions obtained from male mice between 8–12 weeks of age. PFC, prefrontal cortex; NAcc, nuclei accumbens; Caud, caudate nuclei; Hypo, hypothalamus; Cereb, cerebellum; ERC, enterorhinocortical regions; VMB, ventral midbrain.

FIGURE 4.

Comparison of signaling defects among knock-out mice. Adenylyl cyclase activity in the dorsal striatum of Gng3^−/− mice is not reduced at base line or in response to the A_2A agonist CGS-21680 (A) or the direct cyclase activator forskolin (B). In contrast, adenylyl cyclase activity in the dorsal striatum of Gng7^−/− and Gng3^−/−;Gng7^−/− mice is reduced to a similar extent at base line and in response to dopamine, the D₁ dopamine-selective agonist (6-chloro-PB) (C), or forskolin (Forsk) (D). (*, p < 0.05 for comparison with Gng3^−/− by t test, n = 6.) Immunoblot of cholate-extracted proteins from dorsal striatum normalized to Ras and expressed as percentage of levels in Gng3^−/− mice (E) shows α_olf and β₂ are markedly reduced in striatal membranes from Gng7^−/− mice and are further reduced in Gng3^−/−Gng7^−/− mice, although α_s, α_i3, and β₁ are not reduced in Gng7^−/− mice and unchanged or slightly increased in Gng3^−/−Gng7^−/− mice. (*, p < 0.05 for comparison with Gng3^−/− by t test, **, p < 0.05 for comparison with Gng7^−/− or Gng3^−/− by t test, n = 3). F shows β₂ is significantly reduced in cortical membranes from Gng3^−/− mice (*, p < 0.01 for comparison with Gng7^−/− by t test, n = 3), although α_i3 and β₁ are not changed. No further reduction of β₂ is observed in cortical membranes from Gng3^−/−Gng7^−/− mice.

Because the forskolin response is potentiated by the presence of the stimulatory G-proteins (22), we next investigated how gene targeted loss of the γ₃ or the γ₇ subunit affected levels of the G_olf or G_s protein. We showed previously that the γ₇ subunit was required for G_olf assembly in striatum (6, 8). Confirming and extending this result, gene-targeted loss of the γ₇ subunit markedly suppressed α_olf and β₂ levels that were modestly reduced further by loss of both the γ₃ and γ₇ proteins (Fig. 4E). Attesting to the specific nature of these changes, no significant reductions in α_s, α_i3, or β₁ levels were observed in either single or double knock-out mice (Fig. 4E). Likewise, we demonstrated previously that the γ₃ subunit was not required for G_olf assembly in striatum but was linked to suppression of α_i3 and β₂ levels in cortex (7). Confirming and extending these findings, gene-targeted deletion of the γ₃ subunit resulted in significant suppression of β₂ protein and a trend toward decreased α_i3 content that were not reduced further by loss of both the γ₃ and γ₇ proteins (Fig. 4F). Taken together, these results indicated the more severe phenotype of double knock-out mice was associated with circumscribed changes in the α_olf, α_i3, and β₂ levels first reported for the single knock-out mice (6–8) and was not the result of global deficits in multiple α and β proteins.

Functional Cross-talk between γ₃- and γ₇-dependent Signaling Pathways within the Same Neuronal Population as a Possible Mechanism for Double Knock-out Phenotype

Next, we considered the possibility that the γ₃ and γ₇ subtypes are acting in separate signaling pathways within the same striatal cell type. The striatum is comprised of 90% medium spiny projection neurons and 10% large aspiny interneurons (23). Employing an innovative expression profiling technique, Doyle et al. (24) showed that Gng3 and Gng7 mRNA transcripts are expressed in both striatal cell populations (Fig. 5A). Because mRNA levels of G-protein subunits might not reflect their protein levels (25), we extended this analysis to determine the cellular distribution of the γ₃ and γ₇ reporter proteins. For this purpose, corticostriatal slices from transgenic mice expressing GFP under control of the Gng3 promoter were used. To visualize GFP expressing neurons, a low magnification image of a representative slice from these mice is shown in Fig. 5B, and a higher magnification image of the same field is shown in Fig. 5C. The cortex contained numerous green cells, and the dorsal striatum (caudate-putamen) showed only a few GFP-positive neurons that represented <1% of striatal cells. The scarcity of GFP-positive neurons in dorsal striatum is suggestive of cholinergic interneurons that account for only a small fraction of striatal cells (26). To identify the cholinergic interneurons, a high magnification image of the same field stained with the ChAT antibody is shown in Fig. 5D, and the extent of overlap between GFP and ChAT expression patterns is revealed in Fig. 5E. The finding that ChAT staining showed extensive overlap with GFP expression confirmed expression of the γ₃ reporter protein in cholinergic interneurons and the surrounding neuropil (Fig. 5, B–E). These results revealed that the γ₃ reporter protein was expressed in most cortical neurons, along with striatal interneurons of the cholinergic type.

FIGURE 5.

Cellular localization of G-protein γ₃ in brain. A, levels of Gng3 and Gng7 mRNA in medium spiny neurons (Drd1 and Drd2 expressing) and cholinergic interneurons (ChAT expressing) from the striatum. Data are from Doyle et al. (24) as deposited in the NCBI GEO dataset GSE13379 (www.ncbi.nlm.nih.gov). B, in Tg(Gng3-GFP) mice, GFP is expressed in only few scattered neurons in the caudate but is expressed in the cell bodies and processes of numerous cortical neurons. C, Gng3 promoter-driven GFP expression in the caudate. D, immunofluorescence of ChAT expression in caudate. E, merge showing co-localization of GFP and ChAT in the cholinergic interneurons.

Subsequently, corticostriatal slices from knock-in mice expressing GFP under control of the endogenous Gng7 locus were examined. Fig. 6A confirmed the expected GFP expression pattern in the striatum that recapitulated the endogenous γ₇ expression revealed by in situ hybridization (Allen Brain Atlas). To visualize GFP-expressing neurons, a low magnification image of a representative slice is shown in Fig. 6B, and a higher magnification image of the same field is shown in Fig. 6C. In contrast to the cortex that was devoid of any green cells, the dorsal striatum (caudate-putamen) contained numerous GFP-positive neurons that account for the majority of striatal cells. The preponderance of GFP-positive neurons in the dorsal striatum is indicative of medium spiny neurons that account for ∼90% of striatal neurons (27). To identify the medium spiny neurons, a high magnification view of the same field stained with dopamine- and adenosine-regulated 32-kilodalton phosphoprotein (DARPP-32) antibody is shown in Fig. 6D, and coincidence between the GFP and DARPP-32 expression patterns is revealed in Fig. 6E. The finding that GFP expression overlapped with DARPP-32 staining confirmed expression of the γ₇ reporter protein in medium spiny projections neurons and the surrounding neuropil (Fig. 6, B–E). Because medium spiny neurons receive both glutamatergic inputs from cortical neurons and cholinergic inputs from striatal interneurons, these results revealed for the first time that the γ₃ and γ₇ reporter proteins are largely segregated between different neuronal subpopulations that contact each other. Furthermore, these data argue against cross-talk between G_i/o- and G_olf-signaling pathways requiring the γ₃ and γ₇ subunits in the same neuronal population as the basis for the double knock-out phenotype.

FIGURE 6.

Cellular localization of G-protein γ₇ in striatum. A, expression of GFP in the brain of KI(Gng7-γ₃-IRES-GFP) mice is highest in the caudate and nucleus accumbens and matches the pattern of Gng7 expression determined by in situ hybridization (Allen Brain Atlas). NAcc, NAcc, nuclei accumbens. B, in KI(Gng7-γ₃-IRES-GFP) mice, GFP is expressed at high levels in the cell bodies and processes of a large number of cells in the caudate but expressed at undetectable levels in the cortex. C, Gng7 promoter driven GFP expression in the caudate. D, immunofluorescence of DARPP-32 expression in the caudate. E, merge showing colocalization of GFP and DARPP-32 in the medium spiny neurons.

Functional Interaction between Signaling Pathways in Different Neuronal Populations as the Most Likely Explanation for the Double Knock-out Phenotype

Finally, we considered the possibility that G_i/o- and G_olf-signaling pathways are acting in different neuronal populations operating within a circuit to regulate a common neurological process. Although a requirement for the γ₇ subunit in G_olf assembly and D₁R and A_2AR signaling is now established (6, 8), a role for the γ₃ subunit in a particular receptor signaling pathway has not been fully elucidated. Suggesting a role in GABA_BR signaling, the γ₃ protein is expressed in cortex, hippocampus, and striatum in a similar pattern to the GABA_B receptor (28). Moreover, gene targeted deletion of the γ₃ protein confers susceptibility to seizures in an analogous fashion to loss of the GABA_B receptor (29–31). To explore a possible role for the G-protein γ₃ subunit acting downstream of this receptor, we assessed the ability of a GABA_BR-specific agonist to induce delta waves on EEG recordings from wild type (n = 3), Gng3^−/− (n = 4), Gng7^−/− (n = 4), and Gng3^−/−Gng7^−/− mice (n = 3). Delta waves can be readily identified as slow frequency, high amplitude waves. Baclofen effectively induced delta waves on EEG recordings from wild type mice that appeared by 40 min and disappeared by 7 h post-injection (Fig. 7A). In contrast, baclofen showed an impaired ability to induce delta waves on EEG tracings from both Gng3^−/− and Gng7^−/− mice (Fig. 7A). Individual comparisons showed a significant time by band by genotype interaction (ANOVA, F_48,169 = 4.19, p < 0.00001), indicating that baclofen produced the greatest effect in wild type mice, comparably impaired effects in both Gng3^−/− mice and Gng7^−/− mice, and a significantly worsened effect in Gng3^−/−Gng7^−/− mice (Fig. 7B). The greater impairment of the delta wave response seen in double knock-out mice offers further support for a functional interaction between G-protein γ₃- and γ₇-dependent signaling pathways that modulate the delta wave response.

FIGURE 7.

EEG responses to baclofen. A, representative tracings showing EEG changes at various times following injection of baclofen (10 mg/kg) for a wild type mouse (left panel), Gng3^−/− mouse (middle panel), and Gng3^−/−Gng7^−/− mouse (right panel). B, power in the 1–2 Hz band (delta wave) of the EEG was increased in wild type mice (**, p < 0.001) and Gng7^−/− mice (*, p < 0.05) at 40 min following injection. Moreover, power in wild type mice at 40 min was significantly greater than in any other genotype (**, p < 0.001), although power in the Gng7^−/− mice at 40 min was only greater than in Gng7^−/−;Gng3^−/− mice (*, p < 0.05). Two-way ANOVA with Bonferonni post-tests: Time, df = 4, F = 16.8, p < 0.; genotype, df = 3, F = 18.1, p < 0.0001; interaction, df = 12, F = 6.5, p < 0.0001, n = 3 or 4.

To further probe a requirement for G-protein γ₃ subunit acting downstream of the GABA_B receptor, we compared additional baclofen-mediated responses (31) among Gng3^−/− and Gng7^−/− mice. To assess the muscle-relaxing effect of baclofen, we measured the time that mice spent walking on the Rota-Rod apparatus following injection of baclofen. Both wild type and Gng7^−/− mice showed the muscle-relaxing effect of baclofen, as demonstrated by decreased time spent walking on the Rota-Rod (Fig. 8A). In fact, both groups of mice employed the unusual strategy of staying on the Rota-Rod by wrapping their legs around the bar and spinning (spinning mice were given a time of 0 s). In marked contrast, Gng3^−/− mice did not show the muscle-relaxing effect of baclofen, as demonstrated by both the increased time spent walking on the Rota-Rod (Fig. 8A) and failure to exhibit the unusual spinning behavior. Next, we measured the temperature lowering effect of baclofen (31). Again, both wild type and Gng7^−/− mice exhibited the temperature lowering effect of baclofen. In contrast, this response was markedly reduced in Gng3^−/− mice (Fig. 8B). Taken together, these results revealed for the first time that loss of the G-protein γ₃ subunit was selectively associated with impaired GABA_BR responsiveness at both the neurological and behavioral levels.

FIGURE 8.

Behavioral and electrophysiological responses to baclofen. A, wild type and Gng7^−/− mice showed marked impairment in their ability to walk on a Roto-Rod following injection of baclofen (10 mg/kg), although Gng3^−/− had less of an impairment. B, body temperature dropped by >3 °C in wild type mice, >2 °C in Gng7^−/− mice, but <1 °C in Gng3^−/− mice in response to baclofen (10 mg/kg). (*, p < 0.05 for comparison with wild type mice, **, p < 0.01 for comparison with wild type or Gng7^−/− mice, N ≥ 8). C, baclofen-induced GIRK currents were attenuated in neurons isolated from Gng3^−/− mice compared with those from wild type littermates (**, p < 0.01, unpaired t test). No difference was observed in basal, NECA-induced, or somatostatin-induced GIRK currents.

To directly investigate a requirement for the γ₃ subunit in GABA_BR signaling, we measured GABA_BR-mediated activation of GIRK currents in neurons from wild type mice and Gng3^−/− mice. Basal GIRK currents were only slightly reduced in Gng3^−/− mice, and the fractions of neurons exhibiting basal activity between the two genotypes were similar. Consistent with results from previous studies (32, 33), baclofen-activated GIRK channels were observed in all 27 hippocampal neurons tested from wild type mice (Fig. 8C). In contrast, baclofen-stimulated GIRK channels were seen in only in 7 of the 25 neurons from Gng3^−/− mice. The difference in the fraction of responders in wild type versus Gng3^−/− mice was significant (p = 0036, Fisher's exact test). In addition, the amplitude of baclofen-induced currents was significantly attenuated in the Gng3^−/− mice compared with the wild type mice (p < 0.01, unpaired t test). Finally attesting to the specificity of this effect, the adenosine and somatostatin receptor agonists (NECA; somatostatin) activated GIRK channels in similar proportions of neurons from wild type and Gng3^−/− mice (NECA, 11/27 for WT, and 12/25 for Gng3^−/−; somatostatin 9/26 for WT and 13/25 for Gng3^−/−). Activation of GIRK channels in a fraction of wild type neurons by these agonists is consistent with a previous report (32). In addition, the amplitudes for NECA- and somatostatin-activated currents in hippocampal neurons from the wild type and Gng3^−/− mice were not different. Collectively, these data pointed to a specific defect in a post-synaptic GABA_BR signaling pathway in a significant portion of γ₃-deficient neurons, which presumably reflects the heterogeneous nature of these cells in culture (34).

Finally, to confirm defective GABA_BR signaling was due to loss of the G-protein γ₃ and not the receptor itself, we quantified the GABA_BR levels in wild type, single, and double knock-out mice, using a monoclonal antibody that recognized both alternatively spliced forms of the R1 subunit (28). Wild type neurons express both the GABA_B1a and GABA_B1b splice variants (Fig. 9, A and B). Moreover, there were no significant differences in abundance of these forms between wild type and γ₃-deficient tissue. These data demonstrate that loss of the G-protein γ₃ subunit rather than down-regulation of the GABA_B receptor was responsible for the defective baclofen responses observed in Gng3^−/− knock-out mice. Thus, combined neurological, behavioral, and electrophysiological results point to a G_i/o protein containing γ₃ acts downstream of one or more GABA_B splice variants in a cell-specific manner (35).

FIGURE 9.

GABA_BR expression. A and B, immunoblot analysis of hippocampal membranes from control, single knock-out, and double knock-out mice (n = 3, for each genotype), using a GABA_B1 antibody. There were no significant reductions in GABA_B1 expression that could account for the impaired behavioral and electrophysiological responses to baclofen seen in Gng3^−/− and double knock-out mice.

DISCUSSION

Gng3^−/−Gng7^−/− mice show premature lethality. Because seizures trigger cardiac or respiratory arrest (36) and seizure suppression by the ketogenic diet (37–39) is very effective in prolonging their life span, we believe that the premature mortality of double knock-out mice is most likely caused by their severe seizure disorder. Thus, this mouse model offers a novel experimental system for understanding how combinatorial defects in signaling pathways can converge to produce polygenic forms of epilepsy. Furthermore, the finding that these mice display a severe seizure phenotype that is not observed for either single knock-out on the same genetic background provides strong evidence for a functional interaction between G-protein γ₃- and γ₇-dependent signaling pathways that normally limit seizure susceptibility, seizure propagation, or seizure-induced damage. Below, we compare the expression profiles, G-protein α partner preferences, and receptor requirements among the different genotypes to reveal a mechanistic explanation for the severity of the double knock-out phenotype.

Role of γ₃ in GABA_BR Signaling

The signaling pathway(s) dependent on the G-protein γ₃ subunit are not known. We show that the γ₃ reporter protein is present in cortical neurons that make glutamatergic contacts onto striatal neurons. This expression pattern mirrors that reported for the GABA_B receptor (40) responsible for inhibition of glutamate release in striatum (41). Likewise, we demonstrate that the γ₃ reporter protein is localized to striatal interneurons that integrate synaptic inputs over large areas within the striatum (42). This is consistent with the localization reported for the GABA_B receptor (40). Taken together, the similarity of their cellular expression profiles raise the possibility that a specific GABA_B receptor variant may utilize a G-protein containing the γ₃ subunit to modulate neuronal excitability and impact striatal function (35, 43).

Further supporting this possibility, Gng3^−/− mice show a similar loss-of-function phenotype to that reported for Gabbr1^−/− subunit-specific mice. In this regard, the GABA_B receptor functions as an obligate heterodimer; the GABA_B1 subunit, which is encoded by the Gabbr1 gene, contains the ligand-binding site and localization motif, although the GABA_B2 subunit, which is encoded by the Gabbr2 gene, mediates coupling to the G-protein(s) (35, 43). Complete ablation of either the Gabbr1 (29, 30) or Gabbr2 gene (31) produces a severe seizure disorder resulting in death. However, more subtle phenotypes result from individual ablation of alternatively spliced forms of the Gabbr1 gene (44, 45) that are thought to convey distinct functions through their differential subcellular localizations (44). In this regard, Gabbr1a^−/− mice, which retain predominantly post-synaptic GABA_B1b,2 receptors, display a mild seizure phenotype that does not affect viability, whereas Gabbr1b^−/− mice, which preserve mostly pre-synaptic GABA_B1a,2 receptors, exhibit no apparent seizure phenotype. Similar to the subunit-specific Gabbr1^−/− phenotypes, Gng3^−/− mice exhibit a mild seizure phenotype on the seizure-sensitive FVB background but no obvious seizure defect on the seizure-resistant B6 background.

Confirming the G-protein γ₃ subunit is acting downstream of the GABA_B receptor, we identified defective GABA_BR signaling in Gng3^−/− mice. In the post-synaptic setting, the GABA_B1b,2 receptor reportedly couples through a G_i/o-protein to activate a specific GIRK channel (33). Pointing to a specific role in this process, baclofen-induced GIRK activation is lost in a significant proportion of hippocampal neurons derived from Gng3^−/− mice. Ruling out other possible explanations for this defect, there are no significant differences in GABA_B receptor abundance or effector regulation by other receptor agonists. Therefore, loss of the G-protein, secondary to genetic deletion of the γ₃ subunit, appears to be the most likely explanation for the defective GIRK activation seen in this study. Suggesting possible G-protein α partners, the gene-targeted loss of the γ₃ subunit produces coordinate suppression of the α_i3, α_o, and β₂ subunits in certain brain regions (7). Taken together, these results are most consistent with the post-synaptic GABA_B1b,2 receptor acting through G-protein α_i/oβ₂γ₃ trimer to open GIRK channels, causing reduced neuronal excitability. When this pathway is disrupted by either genetic inactivation of the GABA_B1 receptor (44), the G-protein γ₃ subunit (this study), or the GIRK channel (46), animals are prone to developing seizures depending on the presence or absence of other modifier genes in the strain background.

Role of γ₇ in D₁R and A_2aR Signaling

The signaling pathway(s) dependent on G-proteins containing the γ₇ subunit are now known. The γ₇ reporter protein is preferentially expressed in striatal projection neurons (also called medium spiny neurons). As shown previously (6, 8), Gng7^−/− mice lacking the γ₇ subunit exhibit impaired assembly of a specific G-protein α_olfβ₂γ₇ trimer, defective D₁R- and A_2AR-stimulated adenylyl cyclase activation, and altered locomotor behaviors (47). However, these knock-out mice do not exhibit any evidence of spontaneous seizure activity or premature lethality (6). Moreover, mice lacking either the A_2AR (48, 49) or the D₁R (50, 51) are not described as having seizures, although loss of the D₁R-expressing cells themselves has been shown to trigger a progressive seizure disorder (52). Hence, it is not clear how defective A_2AR or D₁R signaling contributes to the severe seizure phenotype of Gng3^−/− Gng7^−/− double knock-out mice. Based on the available evidence, we speculate that a G-protein α_olfβ₂γ₇ trimer acting downstream of the A_2AR or D₁R may confer a neuro-protective effect. Supporting such a possibility, seizure activity has been shown to increase adenosine levels and A_2AR signaling (53), although the consequences are controversial. Consistent with a neuroprotective effect, A_2AR stimulation attenuates brain damage induced by kanaic acid-induced excitoxicity (54) or striatal lesion (55). Conversely, arguing against a neuroprotective action, A_2AR blockade also reduces brain damage (49, 56). Clearly, more studies will be needed to resolve this issue. Providing some insight into these conflicting results, a recent paper suggests that A_2AR activation may switch between neuroprotective and neurodegenerative states depending on existing levels of glutamate (57). Alternatively, D₁R activation may be responsible for conferring a neuroprotective effect (58). Additional studies will be needed to directly investigate a possible role for the G-protein γ₇ subunit in these processes.

Convergent Roles of γ₃ and γ₇ in Separate Signaling Pathways

The severe seizures and increased interictal spiking seen in double knock-out mice point to a novel functional interaction between G_i/o- and G_olf-dependent signaling pathways requiring γ₃ and γ₇ in mediating neuronal excitability and/or protection. As a working model, we speculate that a post-synaptic GABA_B receptor utilizes a G_i/o protein containing the γ₃ subunit to regulate neuronal excitability (43), whereas the A_2A or D₁ receptor requires the G_olf protein containing the γ₇ subunit to confer a neuroprotective response (49, 54, 59). In such a scenario, simultaneous disruption of both signaling pathways could account for the strong seizure phenotype that dramatically reduces the life span of double knock-out mice to only 75 days. Based on these data, the G-protein γ₃ and γ₇ subtypes can be added to a growing list of susceptibility genes that may act synergistically to contribute to human seizure disorders of polygenic origin. In this regard, evidence showing the GABA_B, A_2A adenosine, and D₁ dopamine receptors utilize specific G-protein αβγ heterotrimers offers a new interface for more selective intervention in seizure disorders.

Likewise, the greater blockade of the delta wave response seen in double knock-out mice points to a similar functional interaction between G_i/o- and G_olf-dependent signaling pathways requiring γ₃ and γ₇ in mediating wakefulness. In this regard, GABA_BR blockade has been shown to reduce delta waves (31), providing a likely explanation for the reduced response seen in Gng3^−/− mice. Moreover, D₁R antagonists have been reported to suppress the amplitude of delta waves (60, 61), offering a possible explanation for the decreased delta wave response seen in Gng7^−/− mice. Finally, disruption of both GABA_BR and D₁R signaling would be entirely consistent with the complete blockade of the delta wave response seen in double knock-out mice. At this point, where and how these signaling pathways converge is not known.

In summary, we report a new mouse line that reveals for the first time that in vivo disruption of G_i/o and G_olf signaling pathways produces a severe seizure phenotype. This is particularly interesting in that it is one of the few mouse models that recapitulate the polygenic basis of many forms of human epilepsy. To determine the relevance of this mouse model to the clinical condition, future work will explore whether similar defects in these signaling pathways are observed in surgically resected epileptic tissue from patients undergoing treatment for refractory seizures. Notably, the demonstration that individual or combinatorial deletion of the closely related γ₃ and γ₇ subunits produces distinct and identifiable seizures reinforces the growing recognition that the nature of the γ component plays a critical role in the signal transduction process (6–8).