Mice with Deficiency of G Protein {gamma}3 Are Lean and Have Seizures

Emerging evidence suggests that the ${gamma}$ subunit composition ofan individual G protein contributes to the specificity of thehundreds of known receptor signaling pathways. Among the twelve ${gamma}$ subtypes, ${gamma}$ ₃ is abundantly and widely expressed in the brain.To identify specific functions and associations for ${gamma}$ ₃, a gene-targetingapproach was used to produce mice lacking the Gng3 gene (Gng3^–/–).Confirming the efficacy and specificity of gene targeting, Gng3^–/–mice show no detectable expression of the Gng3 gene, but expressionof the divergently transcribed Bscl2 gene is not affected. Suggestingunique roles for ${gamma}$ ₃ in the brain, Gng3^–/– mice displayincreased susceptibility to seizures, reduced body weights,and decreased adiposity compared to their wild-type littermates.Predicting possible associations for ${gamma}$ ₃, these phenotypic changesare associated with significant reductions in ß₂ and ${alpha}$ _i3 subunit levels in certain regions of the brain. The findingthat the Gng3^–/– mice and the previously reportedGng7^–/– mice display distinct phenotypes and different ${alpha}$ ß ${gamma}$ subunit associations supports the notion that evenclosely related ${gamma}$ subtypes, such as ${gamma}$ ₃ and ${gamma}$ ₇, perform unique functionsin the context of the organism.

INTRODUCTION

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Introduction

The G protein ß ${gamma}$ dimer performs numerous roles in thesignal transduction process, from membrane targeting of the ${alpha}$ subunit (12), to recognition of receptors (23), to activationof effectors (7), to modulation of various proteins affectingthe signal intensity or duration (24). In fact, there is potentialfor a large number of different ß ${gamma}$ dimers arising fromcombinatorial association of the 5 ß and 12 ${gamma}$ subtypes.The current challenge is to identify which of these ß ${gamma}$ dimers actually exist in vivo and to establish their roles inparticular signaling pathways and biological processes. Althoughtransfection and reconstitution provide valuable informationon the potential interactions of the ß and ${gamma}$ subtypes,these strategies fall short of identifying their actual associationsand functions in the context of the organism. By contrast, agene deletion approach represents a powerful method for determiningthis information by analyzing the resulting phenotype in knockoutmice.

The ${gamma}$ ₃ subtype has characteristic features that led us to believethat mice with a targeted disruption of Gng3 would display adistinctive phenotype. Gng3 is predominantly expressed in thebrain (1, 5, 17), where its expression increases during postnataldevelopment (1, 34). Within the brain, ${gamma}$ ₃ is widely expressedin neurons rather than glial cells (2, 22, 33). In vitro RNAsuppression studies predict a specific function for ${gamma}$ ₃ in couplingvarious receptors to changes in calcium channel activity (9,25, 26). These in vitro studies also suggested that ${gamma}$ ₃ functionsin several different heterotrimer combinations: ${alpha}$ _o2ß₁ ${gamma}$ ₃(9), ${alpha}$ ₁₃ß₁ ${gamma}$ ₃ (26), and ${alpha}$ _q/11ß_1/3 ${gamma}$ ₃ (25).

In the present study, we show that Gng3^–/– miceexhibit changes in seizure susceptibility and body weight onboth mixed and C57BL/6J genetic backgrounds. Although the receptorsignaling pathway(s) responsible for this complex phenotypeis not yet identified, we show that loss of the ${gamma}$ ₃ subunit producesconcurrent reductions in levels of the ß₂, ß₁,and ${alpha}$ _i3 subunits in certain brain regions and that a deficiencyof the ${gamma}$ ₃ subunit does not impair regulation of adenylyl cyclaseactivity. In a previous study, Schwindinger et al. generatedmice lacking the Gng7 gene, which encodes the ${gamma}$ ₇ subtype, andidentified a unique function for ${gamma}$ ₇ together with ${alpha}$ _olf in stimulationof adenylyl cyclase activity in the striatum (39). Comparisonof Gng3^–/– mice and Gng7^–/– mice revealsdifferent phenotypes, different heterotrimeric partners, anddifferent signal transduction pathways, demonstrating the uniquefunctions of these two G protein ${gamma}$ subtypes in vivo.

MATERIALS AND METHODS

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Materials and Methods

Production of mice. Targeted embryonic stem cells, chimeric mice, and F₁ heterozygoteswith a floxed Gng3 allele (Gng3^+/fl) were produced at LexiconGenetics, Inc. (The Woodlands, Tex.). To provide the potentialfor conditional inactivation of Gng3 in future studies, thetargeting vector included a 1.9-kb cassette containing a loxPsite and a PGK-neo^R selectable marker in the first intron (52bp upstream of the initiation codon), and a second, 59-bp loxPcassette in the 3' untranslated region (immediately downstreamof the termination codon). Gng3^+/fl mice were bred to mice carryingthe Cre recombinase driven by the EIIa adenovirus promoter,FVB/N-Tgn(EIIa-Cre)C5379Lmgd (Jackson Laboratory, Bar Harbor,Maine), to produce mice bearing the deleted Gng3 allele (Gng3^–).Gng3^+/– mice were then backcrossed to C57BL/6J mice (JacksonLaboratory) for 5 generations. Gng3^+/– mice were intercrossedto produce the mice used in these studies, either before (i.e.,N₀ backcross) or after (i.e., N₅ backcross) 5 backcrosses toC57BL/6J mice.

Animal care and approval. Mice were segregated by sex and group housed in ventilated racks(Thoren Caging Systems, Inc., Hazelton, Pa.). Mice were givenaccess to water and Mouse Diet 9F (Purina Mills, LLC, St. Louis,Mo.) ad libitum. Environmental factors included temperatureand humidity control and a 12-h light-dark cycle. Mice weremaintained as virus antibody free and parasite free. Animalresearch protocols were approved by the Geisinger Clinic institutionalanimal care and use committee.

Genotyping. Southern blot analysis was performed on genomic tail DNA digestedwith HindIII. The probe used for this analysis was a 1-kb fragment3' of the modified Gng3 allele. Alternatively, PCR analysiswas performed using primers (Invitrogen, Rockville, Md.) shownin Table 1. Briefly, primers flanking the 3' loxP site (JR282and JR286) were used to competitively amplify the Gng3 and Gng3^flalleles, while a third primer, upstream of the 5' loxP site(JR416), was included to simultaneously amplify the Gng3^–allele. The bacteriophage P1 Cre transgene was amplified withJR353 and JR354.

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TABLE 1. Primers used in this study

Expression of the Gng3 and Bscl2 genes. PCR amplification of cDNA in the murine multiple tissue cDNApanel (BD Biosciences, Palo Alto, Calif.) was performed accordingto the manufacturer's instructions. RNA was isolated from wholebrain or adipose tissues by using TRIzol reagent (Invitrogen).First-strand cDNA was prepared from 2 µg of RNA by usingMoloney murine leukemia virus reverse transcriptase (Promega),and this was used as a template to amplify the Gng3, Bscl2,or elongation factor transcripts with the primers shown in Table1.

Audiogenic seizures. Audiogenic seizures were induced in 6- to 12-week-old mice (21).Mice were placed in a clear plastic breeding box (27 by 27 by20 cm) with a filter paper lid and were allowed to explore for1 min. Five metal keys on a ring were shaken above the box for10 s to produce a sound of 85 to 95 dB near the bottom of thebox, as measured with a sound level meter (Tandy Corp., FortWorth, Tex.). Observation was continued for at least 1 min aftercessation of the sound. All sessions were videotaped and thengraded by two observers who were unaware of the genotypes ofthe mice.

Immunoblot analysis. To examine the expression of G protein subunits in the brain,Western blot analysis was performed on cholate-solubilized membranesprepared from the cortex, hippocampus, cerebellum, striatum,or whole brain, as described previously (39). Antisera for Ras(BD Biosciences) and for the rat Na⁺/K⁺-ATPase ß subunit(Research Diagnostics, Inc., Flanders, N.J.) were used at a1:2,000 dilution. Antisera for ${alpha}$ _i1, ${alpha}$ _i3, and ${alpha}$ _q/11 (Calbiochem,La Jolla, Calif.) were used at a 1:1,000 dilution. Antiserafor ${alpha}$ _i1/i3, ${alpha}$ _i2, ${alpha}$ _o (1:500), ß₁ (1:500), ß₂(1:500), ${gamma}$ ₂, ${gamma}$ ₃, ${gamma}$ ₅ (1:100), and ${gamma}$ ₇ have been described previously(5, 13, 47) and were used at a 1:200 dilution except as indicated.Antisera for ${alpha}$ _s (a generous gift from Catherine Berlot) wereused at a 1:500 dilution, antisera for ${alpha}$ _olf (a generous giftfrom Denis Hervé) were used at 1:2,000, and antiserafor ${alpha}$ ₁₂ or ${alpha}$ ₁₃ (generous gifts from N. Dhanasekaran) were usedat 1:1,000. His-tagged G protein subunits (CytoSignal ResearchProducts, Irvine, Calif.) were used as standards for quantitativeimmunoblotting.

GTP ${gamma}$ S binding assay. Agonist-stimulated [³⁵S]GTP ${gamma}$ S autoradiography was performed aspreviously described (32, 43) with slight modifications. FollowingCO₂ euthanasia, brains were removed and frozen in isopentaneat –35°C. Coronal sections (thickness, 20 µm)at the levels of the striatum and hippocampus were cut on acryostat at –20°C and mounted on gelatin-coated slides.Slides were equilibrated in 50 mM Tris-HCl-3 mM MgCl₂-0.2 mMEGTA-100 mM NaCl (pH 7.4) (assay buffer) at 25°C for 10min and were then preincubated in assay buffer containing 2mM GDP and adenosine deaminase (9.5 mU/ml) at 25°C for 15min. Assays were conducted by incubating slides in assay bufferwith 2 mM GDP, adenosine deaminase (9.5 mU/ml), and 0.04 nM[³⁵S]GTP ${gamma}$ S (250 Ci/mmol; New England Nuclear Corp., Boston, Mass.)with (stimulated) or without (basal) agonist at 25°C for2 h. Agonists included [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin(DAMGO) (Drug Supply Program, National Institute on Drug Abuse),WIN 55,212-2, and (–)-N⁶-(2-phenylisopropyl)adenosine(PIA) (Sigma Chemical Co., St. Louis, Mo.) at a concentration(10 µM) that has been shown to produce maximal, antagonist-reversiblestimulation (32, 43). Slides were rinsed twice in 50 mM Tris-HCl,pH 7.4, at 4°C for 2 min each time and then in H₂O at 4°Cfor 30 s. Slides were dried overnight and exposed to BiomaxMR film (Eastman Kodak Co., Rochester, N.Y.) for 24 h in thepresence of ¹⁴C-labeled microscales. Films were digitized witha Sony XC-77 video camera and analyzed by using the NIH IMAGEprogram for Macintosh computers. Net agonist-stimulated [³⁵S]GTP ${gamma}$ Sbinding was calculated by subtracting basal binding from agonist-stimulatedbinding. Data are reported as means ± standard errorsfor triplicate sections of brains from six mice per group. Statisticalcomparison between wild-type and knockout mice was performedby analysis of variance followed by post hoc analysis usingthe two-tailed nonpaired Student t test.

Adenylyl cyclase assay. Membranes were prepared as previously described (39) from cerebellumsof Gng3^–/– and Gng3^+/+ mice. Adenylyl cyclase activitywas determined by incubating membranes (20 µg of protein)at 30°C for 10 min in a solution containing 0.1 ml of anagonist-specific buffer, 0.5 µCi of [ ${alpha}$ -³²P]ATP, 0.1 mMATP, 0.05 mM GTP, and an ATP regenerating system consistingof 5 mM creatine phosphate and 50 U of creatine phosphokinase/ml.For the A₁ adenosine agonist, cyclopentyl adenosine (10 to 1,000nM), the buffer consisted of 50 mM HEPES (pH 7.4), 1 mM EGTA,5 mM MgCl₂, 0.01 mM rolipram, and 1 U of adenosine deaminase/ml(30). For the cannabinoid agonist anandamide (1 to 10 µM),the buffer consisted of 50 mM Tris · HCl (pH 7.4), 2mM MgSO₄, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 0.03mM cAMP (6). Reactions were terminated by addition of 0.1 mlof 2% sodium dodecyl sulfate, 40 mM ATP, 1.4 mM cAMP, and 0.06µCi of [2,8-³H]cAMP and heating to 95°C for 3 min.The resulting [³²P]cAMP and the added [³H]cAMP were isolatedby Dowex and alumina chromatography (19) and quantified in anLS-6500 scintillation counter (Beckman) using ScinitiSafe Plus50% LSC-Cocktail (Fisher).

RESULTS

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Results

Targeted disruption of the Gng3 gene. Mice with a targeted deletion of the Gng3 gene were producedin two steps (Fig. 1A). First, mice with a floxed Gng3 allele(Gng3^fl) were created by using a targeting vector in which thecomplete coding region of the gene was flanked by loxP sites.This provides the potential for generation of inducible or tissue-specificknockouts in future studies. Second, mice with a deleted Gng3allele (Gng3^–) were produced by crossing Gng3^fl mice withmice expressing the Cre transgene under the control of the EIIaadenovirus promoter. As shown by Southern blot analysis, thisresulted in the excision of the complete coding region of theGng3 gene (Fig. 1B). To produce the mice used in subsequentexperiments, mice carrying the Gng3^– allele were eitherintercrossed, to obtain mice on a mixed genetic background,or backcrossed for 5 generations, to produce mice on a C57BL/6Jgenetic background. PCR analysis was used to identify mice carryingthe Gng3^– allele (Fig. 1C). Heterozygous crosses producedlitters of the expected size (6.2 ± 3.3 pups) in theexpected 1:2:1 ratio (112 Gng3^+/+ pups, 225 Gng3^+/– pups,and 119 Gng3^–/– pups), indicating that disruptionof Gng3 does not affect survival to weaning. In sequential matingsof one homozygous pair, Gng3^–/– mice were fertileand weaned litters of apparently normal size (9.3 ± 1.2pups).

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FIG. 1. (A) Targeting strategy. (Top) Wild-type allele (Gng3) with exons 1 to 3 (solid boxes) and the 3' probe (shaded box). (Center) Targeted allele (Gng3^fl) with a loxP-neo^R cassette in intron 1 (arrowhead and "neo") and a loxP cassette in the 3' untranslated sequence (arrowhead). (Bottom) Deleted allele (Gng3^–) with one remaining loxP site (arrowhead) following Cre-mediated excision of the coding region of Gng3. Expected sizes of HindIII (H) fragments are indicated. (B) Southern blot of tail biopsy DNA digested with HindIII and detected with the 3' probe. Lane 1, molecular weight ladder; lanes 2 to 8, progeny of a Gng3^+/fl x TgN(EIIa-Cre) cross, with all lanes showing the 4.3-kb wild-type Gng3 allele and lanes 2, 5, and 8 also showing the 3.7-kb Gng3^– allele. A small amount of residual Gng3^fl allele can be seen in lane 8. (C) PCR products obtained with primers JR282, JR286, and JR416 of tail biopsy DNA from progeny of a Gng3^+/– x Gng3^+/– cross. Lane 1, ${phi}$ X HaeIII marker; lanes 2 and 4 to 8, wild-type mice with a single 629-bp band derived from primers JR282 and JR286; lanes 9, 10, and 13, Gng3^–/– mice with a single 755-bp band derived from primers JR282 and JR416; lanes 3, 11, and 12, heterozygous Gng3^+/– mice with both bands.

Abolished expression of the Gng3 gene. To confirm the effectiveness of the targeted gene deletion,expression of Gng3 was examined in the brain, where the genewas most highly expressed (Fig. 2A). Reverse transcription-PCR(RT-PCR) analysis demonstrated that ${gamma}$ ₃ mRNA was present at reducedlevels in brains from Gng3^+/– mice and was absent in brainsfrom Gng3^–/– mice (Fig. 2B). Likewise, Western blotanalysis with ${gamma}$ ₃-specific antisera (20) showed that the ${gamma}$ ₃ proteinwas reduced by 35% ± 5% in cholate-solubilized membraneextracts of brains from Gng3^+/– mice and was not detectedin brains from Gng3^–/– mice (Fig. 2C).

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FIG. 2. (A) RT-PCR products obtained after 30, 34, or 38 cycles of amplification with primers JR282 and JR286, showing relative expression of Gng3 in various tissues (top three panels), or products from 24 cycles with primers for glyceraldehyde-3-phosphate dehydrogenase (Gapd), confirming approximately equal amounts of cDNA in all reactions (bottom panel). (B) (Top) RT-PCR products obtained after 30 cycles of amplification with primers JR282 and JR286 for Gng3, from cDNA prepared from whole brains of three wild-type, three Gng3^+/–, and three Gng3^–/– mice, without added reverse transcriptase (–RT), or without added cDNA (–cDNA). (Bottom) RT-PCR products obtained after 29 cycles of amplification with primers JR174 and JR175 for elongation factor 1 ${alpha}$ 2 (Eef1a2), from identical aliquots of the cDNAs. (C) Western blot analysis of proteins prepared from Sf9 cells expressing ${gamma}$ ₃ (Std) or brains of three Gng3^–/–, three Gng3^+/–, and three wild-type mice. Top panel shows that ${gamma}$ ₃ is reduced in membranes from Gng3^+/– mice and absent in membranes from Gng3^–/– mice. Bottom panel shows that sodium/potassium ATPase ß-subunit levels are equal in all lanes.

Preserved expression of the Bscl2 gene. The Gng3 gene is located head-to-head with the Bscl2 gene, previouslyknown as Gng3lg (10). Because of this arrangement, these twogenes may share promoter elements. Moreover, our analysis ofexpressed sequence tags (e.g., GenBank accession no. CB566482and BI855301) reveals the existence of an alternate first exonthat places the Gng3 gene within the alternate first intronof the Bscl2 gene (Fig. 3A). To rule out the possibility thatmice carrying the Gng3^– allele may show altered expressionof the Bscl2 gene, RT-PCR analysis was performed on total RNAobtained from brains and adipose tissue. This analysis showedthat both Bscl2 mRNA transcripts were expressed at comparablelevels in brains from Gng3^+/+, Gng3^+/–, and Gng3^–/–mice (Fig. 3B). Likewise, Bscl2 mRNA transcripts were detectedat similar levels in white or brown adipose tissue from Gng3^+/+and Gng3^–/– mice (data not shown). Taken together,these results show that Gng3 deletion produces loss of ${gamma}$ ₃ mRNAand protein (Fig. 2) without affecting the expression of theclosely linked Bscl2 gene.

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FIG. 3. (A) Organization of the Bscl2 and Gng3 genes, showing the first few exons (exons 1 to 3) of Bscl2 (shaded boxes), including the alternate exon 1 (1a), and the three exons of Gng3 (solid boxes). The region replaced in the targeted allele, Gng3^–, is indicated by the double arrow. Arrows mark positions of primers used for amplification of Bscl2: the antisense primer JR443 (a), the exon 1-specific sense primer JR445 (b), and the alternate exon 1-specific sense primer JR446 (c). (B) Duplex RT-PCR of brain cDNA from three Gng3^–/– (–/–), three Gng3^+/– (+/–), and three wild-type (+/+) mice, with primers for exon 1 of Bscl2 (upper panel), the alternate exon 1 of Bscl2 (lower panel), and elongation factor Eef1a2 (both panels), showing approximately equal levels of either Bscl2 transcript across all genotypes.

Increased seizure susceptibility of Gng3^–/– mice. Gng3^–/– mice exhibit increased susceptibility toseizures, consistent with the normally high expression of Gng3in the brain. This was first observed on a mixed genetic background(i.e., N₀ backcross) when animal workers noticed that Gng3^–/–mice suffered from recurrent seizures upon cage changing. Overall,24% of Gng3^–/– mice were observed to have recurrenthandling-induced seizures, with the earliest seizure occurringat the age of 2 months. In comparison, 8% of wild-type littermateswere observed to have seizures, with the earliest occurringat 14 months. These seizures were characterized by vocalizations,excessive salivation, wild running, explosive jumping, tonic-clonicconvulsions, and tonic hindlimb extension. Moreover, the occurrenceof these seizures was associated with a reduced life span. Approximately40% of the Gng3^–/– mice died between the ages of2 months and 1 year compared to 10% of wild-type littermatesover the same observation period. Because they showed no priorclinical signs, we suspect that the Gng3^–/– miceon a mixed genetic background were dying as a result of theirrecurrent seizures.

Intriguingly, none of the Gng3^–/– mice on the C57BL/6Jbackground (i.e., N₅ backcross) were observed to have seizuresupon cage changing. Moreover, the absence of these seizureswas associated with an increased life span. Nevertheless, theGng3^–/– mice on the C57BL/6J background still showedan increased frequency and severity of seizures induced by othermeans (Fig. 4). In response to a loud noise, 90% of Gng3^–/–mice exhibited mild seizure activity that was characterizedby freezing for a mean time of 19.8 ± 6.5 s. In addition,50% of Gng3^–/– mice displayed moderate seizure activitythat was characterized by wild running and explosive jumping;15% of Gng3^–/– mice displayed severe seizure activitythat was characterized by tonic-clonic convulsions with extension;and 10% of Gng3^–/– mice died. In contrast, only25% of their wild-type littermates displayed even the mildestform of seizure activity that was characterized by freezingfor a mean time of 1.8 ± 0.8 s. Gng3^+/– mice hadan intermediate frequency and severity of audiogenic seizures.Taken together, these results indicate that Gng3^–/–mice on different genetic backgrounds show increased susceptibilityto seizures induced by various means, suggesting a role for ${gamma}$ ₃ in the regulation of neuronal excitability.

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FIG. 4. Audiogenic seizures induced by a 90-dB noise with a duration of 10 s for mice on a C57BL/6J genetic background (i.e., N₅ backcross). The fraction of mice displaying a seizure of the indicated severity is shown by genotype for Gng3^–/–, Gng3^+/–, and wild-type mice (19 mice in each group). Note that Gng3^–/– mice were more likely to demonstrate any seizure-like activity and more often progressed to more severe seizures. (Inset) Time frozen in response to auditory stimulus by genotype. Note that Gng3^–/– mice remained frozen for significantly longer than their wild-type littermates.

Reduced weight and decreased adiposity of Gng3^–/– mice. Gng3^–/– mice also displayed reduced body weights.This was first observed on a mixed genetic background and waslater confirmed on a C57BL/6J background (Fig. 5A and B). Althoughindistinguishable at weaning, the Gng3^–/– mice onthe C57BL/6J background failed to gain weight at the same rateas wild-type littermates. This difference was most pronouncedamong female Gng3^–/– mice, which weighed approximately25% less than their wild-type siblings by the age of 1 year.Female Gng3^–/– mice also showed a striking reductionin inguinal and retroperitoneal fat pad masses (Fig. 5C). Thiseffect was associated with a dramatic decrease in serum leptinlevels among female Gng3^–/– mice (Fig. 5D). Consistentwith the smaller difference in body weight, male Gng3^–/–mice showed no significant reduction in fat pad masses (Fig.5B and C). Taken together, these results indicate that Gng3^–/–mice on different genetic backgrounds exhibit reduced body weights,suggesting an additional role for ${gamma}$ ₃ in the regulation of appetiteand/or metabolism.

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FIG. 5. Lean phenotype of Gng3^–/– mice on C57BL/6J genetic background (i.e., N₅ backcross). (A) Older, female Gng3^–/– mice (open circles) weighed less than wild-type littermates (filled circles). (B) Weight differences for male Gng3^–/– mice (open squares) and wild-type littermates (filled squares) were not as pronounced. Weights were obtained at 3 to 9 irregular intervals, between the ages of 4 and 70 weeks, for 14 to 18 mice in each group. (C) Fat pad weights expressed as a percentage of body weight (% BW). Female Gng3^–/– mice had significantly reduced inguinal (Ing.) and retroperitoneal (Retro.) fat pad weights, but interscapular (Scap.) and mesenteric (Mes.) fat pad weights were unchanged. Fat pad weights of male Gng3^–/– mice were not different from those of wild-type littermates. Data are means ± standard errors for five to seven mice in each group. (D) Serum leptin levels were significantly reduced in female Gng3^–/– mice. Data are means ± standard errors for five or six mice in each group.

Possible basis for altered phenotype of Gng3^–/– mice. The existence and disparate nature of the phenotypes in Gng3^–/–mice indicate a requirement for ${gamma}$ ₃ in different brain regionsor distinct signaling pathways affecting neuronal excitabilityand body weight regulation that cannot be compensated for byrelated subtypes. Of the known ${gamma}$ subtypes, ${gamma}$ ₂, ${gamma}$ ₃, and ${gamma}$ ₇ are highlyexpressed in the brain, where they show partly overlapping patternsof expression in the cortex, striatum, hippocampus, and cerebellum(2). To determine whether loss of ${gamma}$ ₃ produces compensatory changesin the expression of other ${gamma}$ subtypes, we performed immunoblotanalysis to compare the levels of various ${gamma}$ subtypes in severalbrain regions from Gng3^+/+ and Gng3^–/– mice (Fig.6A). The nature and extent of the observed changes were foundto differ between brain regions (Fig. 6B). In this regard, ${gamma}$ ₂levels were increased slightly, by 23% ± 10%, in thecortices of Gng3^–/– mice, while ${gamma}$ ₇ levels were increasedmodestly, by 31% ± 10%, in the striatum, and the levelof ${gamma}$ ₅ was not altered in any of the brain regions examined fromthese animals. To provide a physiological context for the observedchanges, we next performed quantitative immunoblot analysisto determine the concentrations of ${gamma}$ ₂, ${gamma}$ ₃, and ${gamma}$ ₇ in these regions(Fig. 6C). In wild-type mice, ${gamma}$ ₃ and ${gamma}$ ₇ are expressed at comparablelevels in the striatum, while ${gamma}$ ₂ and ${gamma}$ ₃ are detected at roughlysimilar levels in the cortex. Thus, the relatively small increasesin ${gamma}$ ₇ subunit levels in the striatum and ${gamma}$ ₂ subunit levels inthe cortex would not be expected to fully compensate for theloss of ${gamma}$ ₃ subunit in Gng3^–/– mice.

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FIG. 6. Expression of other ${gamma}$ subtypes. (A) Representative immunoblot of cholate-extractable membrane protein (20 µg) from striata of three wild-type (+/+) and three Gng3^–/– (–/–) mice. The top portion of the blot was incubated with a Ras antiserum to correct for variations in protein loading. The bottom portion was incubated with a ${gamma}$ ₇ antiserum. Note the increased level of ${gamma}$ ₇ in the striata of Gng3^–/– mice. (B) Quantification of immunoblots for ${gamma}$ ₂ (Gng2), ${gamma}$ ₅ (Gng5), and ${gamma}$ ₇ (Gng7) in the cortex (Cor), striatum (Str), hippocampus (Hip), and cerebellum (Cer). Relative levels in Gng3^–/– mice are shown as the percent change from levels in wild-type mice. Data are means ± standard deviations for three mice in each group. *, P < 0.05 for comparison of Gng3^–/– to Gng^+/+ mice by Student's t test. (C) Concentrations of ${gamma}$ ₂, ${gamma}$ ₃, and ${gamma}$ ₇ in the cortex and striatum, showing approximately equimolar amounts of ${gamma}$ ₂ and ${gamma}$ ₃ in the cortex and of ${gamma}$ ₃ and ${gamma}$ ₇ in the striatum. Data are expressed as picomoles of ${gamma}$ subunit per microgram of membrane cholate extract and are means ± standard errors for three or four wild-type mice.

Possible G protein subunit associations revealed by Gng3^–/– mice. Under physiological conditions, G proteins function as heterotrimerswhose specific ${alpha}$ ß ${gamma}$ subunit combinations determine theirparticular roles in receptor signaling pathways (37). Increasingevidence suggests that the composition of the ${gamma}$ subunit may playa critical role in the assembly of specific ${alpha}$ ß ${gamma}$ subunitcombinations. In this regard, it was shown previously that lossof ${gamma}$ ₇ coordinately suppresses the levels of ß₁ (47)or ${alpha}$ _olf (39), suggesting a functional association between them.To determine whether loss of ${gamma}$ ₃ produces a similar effect, weperformed immunoblot analysis to compare the levels of various ${alpha}$ and ß subunits in several brain regions from Gng3^+/+and Gng3^–/– mice. The type and extent of suppressionwas found to differ between brain regions (Fig. 7). In thisregard, the level of ß₁ was reduced by 24% ±11% in the cerebellum, while levels of ß₂ were reducedby 11% ± 2%, 28% ± 16%, and 42% ± 27% inthe striatum, cortex, and cerebellum, respectively (Fig. 7B).No significant decreases in the levels of ${alpha}$ _i1, ${alpha}$ _i2, ${alpha}$ _q/11, ${alpha}$ _o, ${alpha}$ _s, ${alpha}$ _olf, ${alpha}$ ₁₂, or ${alpha}$ ₁₃ were observed in the cerebellums, cortices, hippocampi,or striata of Gng3^–/– mice (data not shown). However,small decreases of 13% ± 8% to 28% ± 16% in thelevels of ${alpha}$ _i3 were seen in cortices, hippocampi, and cerebellumsof Gng3^–/– mice, with two different antisera cross-reactingwith ${alpha}$ _i1 and ${alpha}$ _i3 showing a decrease, but an antiserum specificfor ${alpha}$ _i1 showing no difference (Fig. 7D).

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FIG. 7. Expression of ${alpha}$ and ß subunits in Gng3^–/– brain regions. (A) Representative immunoblot of ß₂ in cerebellum from wild type (+/+) and Gng3^–/– (–/–) mice; the bottom portion was detected with Ras antisera to control for variations in protein loading. (B) Quantification of immunoblots for ß₁ (Gnb1) and ß₂ (Gnb2) in the cortex (Cor), striatum (Str), hippocampus (Hip), and cerebellum (Cer). Data are expressed as the percent change from the wild-type level in Gng3^–/– mice and are means ± standard deviations for three mice in each group. *, P < 0.05 for comparison of Gng3^–/– to Gng^+/+ mice by Student's t test. (C) Representative immunoblot of ${alpha}$ _i3 in the cerebellum for wild-type (+/+) and Gng3^–/– (–/–) mice. The top portion was detected with our ${alpha}$ _i1/i3 antisera; the bottom portion was detected with Ras antisera to control for variations in protein loading. (D) Quantification of immunoblots for ${alpha}$ _i1 (Gnai1) and ${alpha}$ _i3 in four brain regions. Two different antisera for ${alpha}$ _i3, our ${alpha}$ _i1/i3 antisera (Gnai3A) and Calbiochem ${alpha}$ _i3 antisera (Gnai3B), both cross-reacted with ${alpha}$ _i1 to varying degrees (data not shown). Note that levels of ${alpha}$ _i1/i3 were reduced in the cortex, hippocampus, and cerebellum, but levels of ${alpha}$ _i1 were unchanged in these regions, suggesting that levels of ${alpha}$ _i3 are reduced in these regions.

To provide physiological meaning for the observed changes, wenext performed quantitative immunoblot analysis on several brainregions from wild-type mice (Fig. 8). The level of ß₂was found to be approximately equal to that of ${gamma}$ ₃ in the corticesand cerebellums of wild-type mice (Fig. 8B). Thus, the 30 and40% reductions in the levels of ß₂ in the corticesand cerebellums of Gng3^–/– mice suggest that ${gamma}$ ₃ formsdimers with more than one ß subunit in these brainregions of wild-type mice and that loss of ${gamma}$ ₃ is accompaniedby loss of a ß₂ ${gamma}$ ₃ dimer in these brain regions of Gng3^–/–mice. The interpretation of the relative reductions in ${alpha}$ _i3 levelsin several brain regions is complicated by the lack of specificantisera. Two antisera, which cross-react with ${alpha}$ _i3 and ${alpha}$ _i1 tovarious degrees, gave different expression levels for ${alpha}$ _i3 inthe cortex (Fig. 8D), suggesting that the level of ${alpha}$ _i3 was eitherequal to or 3 times that of ${gamma}$ ₃. If the high-end estimate is correct,the 30% reduction in ${alpha}$ _i3 levels observed in the cortex couldbe accounted for by the loss of a unique ${alpha}$ _i3ß ${gamma}$ ₃ heterotrimer.

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FIG. 8. Quantitative immunoblots of G protein subunits. (A) Representative immunoblot showing a standard curve of the His-tagged ß₂ subunit (20, 10, 5, or 2.5 ng/lane) and 20 µg of cholate extracts of cortical membranes from three wild-type mice (mice A, B, and C). The top portion of the blot was incubated with an antiserum for ß₂; the bottom portion was incubated with a Ras antiserum to confirm equal loading of the membrane cholate extract. (B) Concentrations of ${gamma}$ ₃ and ß₂ in the cortex and cerebellum, showing approximately equimolar amounts of both subunits. Data are expressed as picomoles of subunit per microgram of membrane cholate extract and are means ± standard deviations for three mice in each group. (C) Representative immunoblot showing a standard curve of the His-tagged ${alpha}$ _i1 subunit (40, 20, and 10 ng/lane); the His-tagged ${alpha}$ _i3 subunit (20 ng/lane), showing no cross-reactivity with ${alpha}$ _i1 antisera; and 20 µg of cholate extracts of cortical membranes from three wild-type mice (mice A, B, and C). The top portion of the blot was incubated with an antiserum for ${alpha}$ _i1, and the bottom portion was incubated with a Ras antiserum. (D) Concentrations of ${gamma}$ ₃, ${alpha}$ _i1, and ${alpha}$ _i3 in the cortex. Note that both available ${alpha}$ _i3 antisera cross-reacted with ${alpha}$ _i1 (data not shown) and that the two antisera gave different results, indicating that the concentration of ${alpha}$ _i3 was either equal to or 3 times that of ${gamma}$ ₃.

Screening of possible receptor signaling pathways affected in Gng3^–/– mice. The results presented above predict the existence of a specificG_i heterotrimer composed of ${alpha}$ _i3ß ${gamma}$ ₃ subunits, whose assemblyis limited in certain brain regions of Gng3^–/– mice.This raises the possibility that one or more receptor signalingpathways whose actions are dependent on this specific G_i heterotrimermay be disrupted in these animals. To test this possibility,we used a novel technique to compare receptor activation ofG proteins in brain sections by in vitro autoradiography of[³⁵S]GTP ${gamma}$ S binding. Our preliminary screen focused on the muopioid, A₁ adenosine, and CB₁ cannabinoid receptors, since therewas some evidence in the literature to suggest their couplingto G_i proteins and their involvement in signaling pathways affectingneuronal excitability and/or body weight regulation. The agonistsDAMGO, PIA, and WIN 55,212-2 were used to visualize mu opioid-,A₁ adenosine-, and CB₁ cannabinoid receptor-stimulated [³⁵S]GTP ${gamma}$ Sbinding, respectively. Visual comparison of autoradiograms fromwild-type and Gng3^–/– mice did not reveal significantdifferences in basal or agonist-stimulated activity in any region.This observation was confirmed by densitometric analysis. Basal[³⁵S]GTP ${gamma}$ S binding did not differ between wild-type and Gng3^–/–mice in the caudate-putamen (232 ± 9 versus 219 ±7 nCi/g), anterior cingulate cortex (186 ± 8 versus 176± 6 nCi/g), hippocampus (202 ± 25 versus 187 ±5 nCi/g), or hypothalamus (313 ± 23 versus 315 ±11 nCi/g). Similarly, DAMGO-, WIN 55,212-2-, and PIA-stimulated[³⁵S]GTP ${gamma}$ S binding did not differ between wild-type and knockoutmice in any region examined (Table 2). Thus, deletion of ${gamma}$ ₃ didnot appear to affect mu opioid-, A₁ adenosine-, or CB₁ cannabinoidreceptor-mediated signaling, at least in the brain regions examined.

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TABLE 2. Densitometric analysis of agonist-stimulated [³⁵S]GTP ${gamma}$ S autoradiography in brain regions^a

Screening of possible effectors in Gng3^–/– mice. Given the observed reduction in ${alpha}$ _i3 levels in several brain regionsof Gng3^–/– mice, we tested the possibility thatinhibition of adenylyl cyclase by one or more G protein-coupledreceptors might be disrupted (Fig. 9). As a preliminary screen,we showed that the endogenous cannabinoid receptor agonist anandamideinhibited basal adenylyl cyclase activity equally well in cerebellarmembranes from Gng3^+/+ and Gng3^–/– mice (Fig. 9A),while the A₁ adenosine receptor agonist cyclopentyladenosineinhibited forskolin-stimulated adenylyl cyclase activity equallywell in cerebellar membranes from these two lines of mice (Fig.9B). Thus, deletion of ${gamma}$ ₃ did not appear to affect adenylyl cyclasesignaling, at least for the receptors and brain regions examined.

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FIG. 9. Adenylyl cyclase assays. (A) Inhibition of adenylyl cyclase by a cannabinoid agonist, 0.3 to 10 µM anandamide, in cerebellar membranes, expressed as percent inhibition of GTP (50 µM)-stimulated activity (119 ± 12 pmol/mg of protein/min for Gng3^+/+ mice or 125 ± 16 pmol/mg of protein/min for Gng3^–/– mice). Data are means ± standard errors for six mice in each group. (B) Inhibition adenylyl cyclase by an A₁ adenosine agonist, 10 to 1,000 nM cyclopentyladenosine (CPA), in cerebellar membranes, expressed as percent inhibition of forskolin (0.3 µM)-stimulated activity (136 ± 20 pmol/mg of protein/min for Gng3^+/+ mice or 141 ± 13 pmol/mg of protein/min for Gng3^–/– mice). Data are means ± standard errors for three mice in each group.

DISCUSSION

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Discussion

Gng3^–/– mice exhibit a complex phenotype that includesneurological and metabolic abnormalities. The complexity ofthis phenotype suggests that ${gamma}$ ₃ may function in multiple brainregions and/or signaling pathways. In future studies, the abilityto limit inactivation of the Gng3 gene to specific brain regionswill allow further dissection of the functions of this genein different neuronal populations.

Seizure-related phenotype. Mice lacking Gng3 on a mixed genetic background (i.e., FVB/N,C57BL/6, and 129 strains) experience an increased frequencyof spontaneous seizures and increased mortality rates comparedto their wild-type littermate controls. An association withincreased rates of death has been described for other knockoutmice with spontaneous seizures. For instance, mice with a deficiencyof a serotonin receptor, Htr2c, demonstrate spontaneous seizuresthat occasionally progress to respiratory arrest and death,with approximately 25% of mice dying by the age of 13 weeksand 50% of mice dying during 25 weeks of observation (46). Micewith a deficiency of the GABA_B1 receptor, Gabbr1, exhibitedrecurrent spontaneous seizures typified by wild running andlimb tonus and clonus, which frequently progressed to death,with a mean life expectancy of only 21 days (36).

Intriguingly, both the spontaneous seizure and mortality phenotypesof Gng3^–/– mice are dependent on the genetic background.After crossing onto the C57BL/6J background for 5 generations,these seizure- and mortality-related changes are no longer observed.A similar decline in spontaneous seizures and sudden death wasobserved when serotonin 5-HT_2C receptor-null mice were backcrossedto C57BL/6 mice (18). Notably, these results are consistentwith a recent study showing that seizure susceptibility is straindependent, with the C57BL/6 strain exhibiting a higher electroconvulsivethreshold than the FVB/NJ and 129S3 strains (14). To confirmthe presence of a modifying factor in the C57BL/6 genome thatattenuates the seizure phenotype of Gng3^–/– mice,we are currently in the process of backcrossing the Gng3^–/–mice to more seizure-sensitive strains.

Although they no longer suffer spontaneous seizures, mice lackingGng3 on the C57BL/6J genetic background still exhibit increasedseizure susceptibility. A number of mouse models involving disruptionof G protein-coupled receptors, their agonists, or intracellulareffectors display increased susceptibility to seizures (31).Mice with a deficiency of somatostatin (4), the D2 dopaminereceptor (3), or the neuropeptide Y5 receptor (28) all displayincreased susceptibility to kainic acid-induced seizures. TheFrings mouse displays audiogenic seizures (44). Mice with adeficiency of neuropeptide Y (11), a serotonin receptor (46),the GABA_B1 receptor (36), or the GIRK2 potassium channel (42)all exhibit spontaneous or handling-induced seizures. Thesemouse models suggest potential signaling pathways through whichdisruption of Gng3 might increase seizure susceptibility.

Weight-related phenotype. Female mice lacking Gng3 show reduced body weights, decreasedadiposity, and low leptin levels compared to their wild-typelittermate controls. Importantly, the phenotype was not dependenton genetic background. The basis for this lean phenotype iscurrently under investigation. One possibility that was discountedwas derangement of Bscl2 expression as a result of targetingof the Gng3 gene. The human BSCL2 gene has been linked to aform of congenital lipodystrophy (27). However, analysis ofGng3^–/– mice revealed no disruption of Bscl2 expressionin the brain, white fat, or brown fat. Therefore, the lean phenotypewas due to loss of Gng3 expression. Possibilities under considerationinclude a change in food consumption and/or metabolic rate.A number of mouse models involving disruption of G protein-coupledreceptors or their agonists affect body weight and adiposity.Deficiency of the melanin-concentrating hormone (MCH) prohormone,Pmch (41), or its receptor, Mch1r (29), produces a lean phenotypein mice as a result of increased metabolic rate. Mch1r is coupledto inhibition of adenylyl cyclase (35) and stimulation of calciumchannels (16). Deficiency of Npy2r, Npy4r, or a combinationof both receptors produces a lean mouse (38). Npy2r is coupledto inhibition of adenylyl cyclase and inhibition of calciumchannels through a pertussin toxin-sensitive G protein (40),most likely ${alpha}$ _i3 and unknown ß ${gamma}$ subunits (15). Particularinterest revolves around these signaling pathways, since neuropeptideY affects seizure susceptibility (11). Deficiency of the CB₁cannabinoid receptor, Cnr1, produces mice with a lean phenotypedue in part to decreased caloric intake (8). Cnr1 is coupledto inhibition of presynaptic calcium channels that regulateneurotransmitter release (45). Again, special interest is focusedon this signaling pathway, since Cnr1^–/– mice showeda mortality phenotype similar to that of our Gng3^–/–mice, with $~$ 25% of the mice dying by the age of 6 months (48).

Lack of functional compensation by other ${gamma}$ subtypes. The presence of a phenotype in the Gng3^–/– micesuggests that at least some of the functions of ${gamma}$ ₃ are uniqueand cannot be replaced by other related family members. Theinability of other subunits to substitute for ${gamma}$ ₃ in the contextof the organism could be due to a distinctive structural featureor a unique expression pattern of this gene. To begin to distinguishbetween these possibilities, we examined the effect of lossof ${gamma}$ ₃ on other ${gamma}$ subtypes. We showed that loss of ${gamma}$ ₃ results ina specific increase in the level of ${gamma}$ ₂ in the cortex and thelevel of ${gamma}$ ₇ in the striatum. These results suggest that modestadaptations in ${gamma}$ subunit expression may occur in a region-specificfashion in Gng3^–/– mice. Such adaptations may reflecta compensatory change to replace the lost ${gamma}$ ₃ in the same signalingpathway. However, based on the relative levels of these ${gamma}$ subtypeswithin these brain regions, the relatively small changes in ${gamma}$ ₂ and ${gamma}$ ₇ would not be sufficient to functionally compensate forthe much larger loss of ${gamma}$ ₃. Alternatively, such changes may reflecta secondary alteration in another signaling pathway or celltype. For example, in situ hybridization studies indicate that ${gamma}$ ₃ and ${gamma}$ ₇ are expressed in different neuronal populations withinthe striatum (2). Assuming that this pattern holds true in Gng3^–/–mice, it suggests that these two ${gamma}$ subtypes normally functionin distinct signaling pathways in different cell types, withthe changes observed in Gng3^–/– mice revealing anovel, functional interaction between the two.

Heterotrimeric associations of ${gamma}$ ₃. To gain insight into possible associations of ${gamma}$ ₃, we have examinedthe effect of loss of ${gamma}$ ₃ on expression of ß₁ and ß₂in different regions of the brain. Notably, there were significantreductions in the levels of ß₂ in the cortex, striatum,and cerebellum and in the level of ß₁ in the cerebellum.In vitro studies have previously demonstrated that the ${gamma}$ subunitenhances the stability of the ß subunit; treatmentof HEK293 cells with a ribozyme directed against the ${gamma}$ ₇ subunitresults in a dramatic reduction in the half-life of the ß₁subunit (47). We also examined the effect of loss of ${gamma}$ ₃ on levelsof the various ${alpha}$ subtypes in different regions of the brain.Intriguingly, we found a small, but specific, reduction in thelevels of ${alpha}$ _i3 in the cortex, hippocampus, and cerebellum. Furtherstudies will be needed to determine if ${gamma}$ ₃ associates with additional ${alpha}$ subtypes in these brain regions.

Signal transduction pathways requiring ${gamma}$ 3. We have begun to look for possible receptor signaling pathwaysthat are responsible for the observed phenotype of Gng3^–/–mice. For this purpose, we employed a novel autoradiographictechnique that measures receptor-mediated activation of G proteinsin brain slices. Focusing on a small subset of receptors andbrain regions that could be responsible for the observed phenotype,we saw no differences in mu opioid-, A₁ adenosine-, and CB₁cannabinoid receptor-stimulated GTP ${gamma}$ S binding in selected brainregions between wild-type and Gng3^–/– mice. Obviously,we need to examine a much larger number of receptors and brainregions. Moreover, by using maximally effective concentrationsof the receptor agonist, we may have missed a modest decreasein the potency (i.e., increase in the 50% effective concentration).Nonetheless, there does not appear to be a widespread disruptionof receptor-mediated activation of G proteins in the regionsexamined to date.

Because immunoblot data revealed a small but significant reductionin the ${alpha}$ _i3 subunit levels, we also compared inhibition of adenylylcyclase activity by the CB₁ cannabinoid and A₁ adenosine receptors.No differences in adenylyl cyclase activity were observed betweenwild-type and knockout mice, but other possible effectors remainto be examined.

Contrast to Gng7^–/– mice. Initial characterization of Gng3^–/– mice revealsa complex phenotype that includes increased seizure susceptibility,decreased body weight, and reduced fat stores. This phenotypewas associated with a modest decrease in the level of ${alpha}$ _i3 andno disruption of adenylyl cyclase signaling in several brainregions of Gng3^–/– mice. Importantly, the phenotypeof Gng3^–/– mice contrasts sharply with that of Gng7^–/–mice. Gng7^–/– mice had no observable seizure activityand had normal body weight. However, they displayed an increasedacoustic startle response and a trend toward decreased exploratorylocomotor activity. This phenotype was associated with a decreasedlevel of ${alpha}$ _olf and disruption of D₁ dopamine receptor-stimulatedadenylyl cyclase signaling in the striatum (39). Because ${gamma}$ ₃ and ${gamma}$ ₇ are structurally similar and are predominantly expressed inthe brain, the finding that mice lacking one or the other subtypedisplay distinctive phenotypes provides conclusive proof thatthey perform nonredundant roles in separate signaling pathwaysor biological processes. Collectively, these results supportthe notion that the ${gamma}$ subunit composition contributes to thespecificity of signaling pathways in the context of the organism.

ACKNOWLEDGMENTS

We are indebted to the outstanding technicians in our animalcare facility: Cynthia J. Rhone, Gail L. Gregory, and ShannonWescott. We are grateful to Sigrid Wattler and Michael C. Nehlsat Lexicon Genetics, Inc., for the construction of the Gng3^+/flmice. We thank Nicole Schechter for technical assistance withthe GTP ${gamma}$ S binding.

This work was supported by NIH grants GM39867 (awarded to J.D.R.),DA-10770 and DA-05274 (awarded to D.E.S.), and DA-14277 (awardedto L.J.S.-S.).

FOOTNOTES

* Corresponding author. Mailing address: Geisinger Clinic, Weis Center for Research, 100 North Academy Ave., Danville, PA 17822. Phone: (570) 271-6684. Fax: (570) 271-6701. E-mail: jrobishaw{at}geisinger.edu.

REFERENCES

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