Prenatal Cocaine Exposure Uncouples Mglur1 from Homer1 and Gq Proteins

Prenatal Cocaine Exposure Uncouples mGluR1 from Homer1 and Gq Proteins

Kalindi Bakshi., Raminder Parihar., Satindra K. Goswami, Melissa Walsh, Eitan Friedman, Hoau- Yan Wang* Departments of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, The City University of New York Medical School, New York, New York, United States of America

Abstract Cocaine exposure during gestation causes protracted neurobehavioral changes consistent with a compromised glutamatergic system. Although cocaine profoundly disrupts glutamatergic neurotransmission and in utero cocaine exposure negatively affects metabotropic glutamate receptor-type 1 (mGluR1) activity, the effect of prenatal cocaine exposure on mGluR1 signaling and the underlying mechanism responsible for the prenatal cocaine effect remain elusive. Using brains of the 21-day-old (P21) prenatal cocaine-exposed rats, we show that prenatal cocaine exposure uncouples mGluR1s from their associated synaptic anchoring protein, Homer1 and signal transducer, Gq/11 proteins leading to markedly reduced mGluR1-mediated phosphoinositide hydrolysis in frontal cortex (FCX) and hippocampus. This prenatal cocaine-induced effect is the result of a sustained protein kinase C (PKC)-mediated phosphorylation of mGluR1 on the serine residues. In support, phosphatase treatment of prenatal cocaine-exposed tissues restores whereas PKC-mediated phosphorylation of saline-treated synaptic membrane attenuates mGluR1 coupling to both Gq/11 and Homer1. Expression of mGluR1, Homer1 or Ga proteins was not altered by prenatal cocaine exposure. Collectively, these data indicate that prenatal cocaine exposure triggers PKC-mediated hyper-phosphorylation of the mGluR1 leading to uncoupling of mGluR1 from its signaling components. Hence, blockade of excessive PKC activation may alleviate abnormalities in mGluR1 signaling and restores mGluR1-regulated brain functions in prenatal cocaine-exposed brains.

Citation: Bakshi K, Parihar R, Goswami SK, Walsh M, Friedman E, et al. (2014) Prenatal Cocaine Exposure Uncouples mGluR1 from Homer1 and Gq Proteins. PLoS ONE 9(3): e91671. doi:10.1371/journal.pone.0091671 Editor: Ryan K. Bachtell, University of Colorado, United States of America Received June 14, 2013; Accepted February 14, 2014; Published March 13, 2014 Copyright: ß 2014 Bakshi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by public service grant, MIDARP R24-DA018055 from the National Institute on Drug Abuse (EF, HYW), CUNY collaborative grant (HYW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work.

Introduction accumbens (NAc), whereas Groups II and III mGluRs are mainly localized presynaptically on axons and axonal terminals [13], [14]. Cocaine usage during pregnancy causes protracted neurological Prenatal cocaine exposure produces long-lasting cognitive and behavioral abnormalities [1–3]. Although prenatal cocaine deficits in humans and animal models [15], [16]. Since mGluR1 exposure alters glutamatergic function in various brain regions [4– is a pivotal regulator of the glutamatergic neurotransmission and 6], the effect of gestational cocaine exposure on metabotropic cognitive processes, it is highly conceivable that prenatal cocaine glutamate receptor (mGluR) is not well understood. Recently, exposure induces cognitive defects through alterations in the prenatal cocaine exposure was shown to directly disrupt mGluR1 mGluR1 signaling. This hypothesis may be supported by the data function resulting in delays in postnatal synaptic maturation [7]. showing that repeated cocaine exposure produces a persistent The mGluRs are members of the G-protein coupled receptor reduction in mGluR function in the NAc after a three week (GPCR) superfamily that modulate neuronal excitability and withdrawal period [17]. This cocaine-mediated effect was attrib- development, synaptic plasticity and neurotransmitter release uted to a decrease in, or complete deletion of Homer1, a pivotal underlying optimal cognitive function [8]. There are eight synaptic anchoring protein for group 1 mGluRs that modulates members in the mGluR family that are divided into three groups: 2+ IP3 production and intracellular Ca mobilization [12], [17], group I – mGluR1 and 5, group II – mGluR2 and 3, group III – [18]. In line with the notion that prenatal cocaine exposure affects mGluR [4,] [6–9]. The group I mGluRs (mGluR1 and mGluR5) mGluR1 function, delay in synaptic maturation in prenatal are coupled exclusively to Gq/11 to regulate phospholipase C and cocaine exposed brains was attributed in part to the impaired inositol 1,4,5-tris phosphate (IP3) receptors through their associ- mGluR1 function [7]. Together, these data lead us to thoroughly ation with a constitutively expressed synaptic scaffolding protein, study the effect of in utero cocaine exposure on mGluR1-mediated Homer1 [10–12]. Group 1 mGluRs are predominantly expressed signaling in frontal cortex (FCX) and hippocampus, two brain in the postsynaptic somatodendritic regions, especially in brain regions vital for cognitive function, in the rat prenatal cocaine areas highly responsive to psychostimulants including Nucleus model. We further identify the underlying mechanism through

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Figure 1. The mGluR1 is associated exclusively with Gq/11 and Homer1 in the frontal cortex (FCX) and hippocampus of 10-week-old naı¨ve rats. (A) The presence of mGluR1 was examined in the immobilized anti-Ga immunoprecipitates of the solubilized FCX and hippocampal synaptosomal membranes by Western blotting. The representative Western blots show exclusive detection of mGluR1 together with Gaq/11 protein in the synaptosomes prepared from FCX and hippocampus. N = 4. (B) Basal and glutamate (1 mM)-induced mGluR1 coupling to Gq/11 and Homer1 in synaptic membranes derived from FCX and hippocampi of 10-week-old naı¨ve rats. The presence of mGluR1 was examined in immobilized anti- mGluR1 immunoprecipitates of the solubilized FCX and hippocampal synaptosomal membranes by Western blotting. The representative Western blots show Gaq/11 protein and Homer1 are present in anti-mGluR1 immunoprecipitates. Quantitative data are presented as means 6 s.e.m. of the ratios of optical intensity of indicated protein to the optical intensity of mGluR1 (2132.86150.8 and 2090.86151.8 optical intensity in FCX of Kreb’s- Ringer (K-R) and glutamate treated, respectively and 2044.56139.2 and 2000.06114.3 optical intensity in hippocampi of K-R and glutamate treated, respectively) to validate equal loading. There is no discernible difference in the level of mGluR1-associated Homer1 indicated as Homer1/mGluR1 ratios in both brain regions (0.46760.03 vs. 0.44760.03 in K-R and glutamate exposed FCX and 0.46860.03 vs. 0.48860.02 in K-R and glutamate exposed hippocampus). Incubation with 1 mM glutamate increased the level of Gq/11 associated with mGluR1 by 343.2628.9% and 332.7632.1% respectively in FCX and hippocampus. N = 4 for each group. *p,0.01 comparing the level of Gaq/11 in K-R and 1 mM glutamate exposed synaptosomal membranes by two-tailed Student’s t test. doi:10.1371/journal.pone.0091671.g001 which prenatal cocaine exposure elicits signaling dysfunction in were from Peptide International (Louisville, KY). Phorbol 12- the mGluR1 system. myristate, 13-acetate (PMA) was from Enzo Life Science (Plymouth Meeting, PA). Antibody against mGluR1 was pur- Materials and Methods chased from Transduction Laboratories (Madison, WI). (S)-3,5- dihydroxyphenylglycine (DHPG), a-Amino-5-carboxy-3-methyl- Materials and Chemicals 2-thiopeneacetic acid (3-MATIDA) were purchased from Tocris Soybean trypsin inhibitor, phenylmethylsulfonyl fluoride Bioscience (Minneapolis, MN). Antibodies against phosphotyr- [PMSF], 2-mercaptoethanol, NaF, Na2VO4, Digitonin, protein osine (SC-508), caspase-3 (SC-7272), b-actin (SC-47778), Homer1 phosphatase inhibitor I & II cocktails, recombinant cPKC, (SC-55463), Homer2 (SC-8924), Gas/olf (SC-383), Gai (SC- alkaline phosphatase, phorbol 12-myristate, 13-acetate (PMA), 7276), Gao (SC-387) or Gaq/11 (SC-392), actin (SC-1616R) were anti-phosphoserine (P3430), anti-phosphothreonine (P3555) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). purchased from Sigma (St. Louis, MO). Leupeptin and aprotinin Immobilized protein A/G-conjugated agarose, Seize-X immuno-

PLOS ONE | www.plosone.org 2 March 2014 | Volume 9 | Issue 3 | e91671 Prenatal Cocaine Attenuates mGluR1 Signaling precipitation kit, antigen elution buffer and West pico chemilu- Animals minescent reagents were purchased from Thermo Pierce (Rock- Pathogen-free, 10-week-old male and female Sprague-Dawley ford, IL). Celestrine was purchased from EMD Cal-Biochem (La rats weighing approximately 200–215 g (Taconic, Germantown, Jolla, CA). Bradford reagent, SDS-PAGE reagents, pre-stained NY) were housed individually in a 12-hr light/dark cycle with free molecular weight markers were purchased from Bio-Rad (Hercu- access to food and water. All animal procedures were in les, CA). 10-KDa cut-off filters were obtained from Cole-Palmer compliance with the National Institutes of Health Guide for Care (Vernon Hills, IL). Use of Laboratory Animals and were approved by the City College of New York Animal Care and Use Committee. The animal treatment was performed as described previously [6]. Briefly,

Figure 2. Prenatal cocaine exposure markedly reduces mGluR1 association with Gq/11 and Homer1 in the FCX and hippocampus of P21 rats. (A) The levels of Gq/11 and Homer1 associated with mGluR1s were examined in the immobilized anti-mGluR1 immunoprecipitates of the solubilized FCX and hippocampal synaptosomal membranes of prenatal saline- and cocaine-exposed P21 rats following incubation with Kreb’s-Ringer (K-R) or 1 mM glutamate by Western blotting. The representative Western blots show the detection of Gaq/11 protein and Homer1 in the synaptosomal membranes prepared from FCX and hippocampus. Glutamate increased mGluR1 coupling to Gq/11 but not to Homer1. N = 4. (B) Quantitative data are presented as means 6 s.e.m. of the ratios of optical intensity of indicated protein to the optical intensity of mGluR1 (1894.36122.1–2068.86348.3 optical intensity in FCX of saline and cocaine, respectively and 185.1699.9–1885.5698.3 optical intensity in hippocampi of saline and cocaine, respectively) to validate equal loading. Incubation with glutamate increased Gq/11 - mGluR1 coupling by 298.8633.9% and 387.2631.4% in FCX and hippocampus respectively. This glutamate-induced Gq/11-mGluR1 coupling was reduced by 69.0612.3% and 63.463.2% respectively in FCX and hippocampus. Glutamate did not alter Homer1-mGluR1 coupling, whereas prenatal cocaine exposure reduced Homer1-mGluR1 association by 56.064.9–57.8610.1% and 52.668.0–55.1610.4% respectively in FCX and hippocampus. N = 4 for each group. *p,0.01, **p,0.05 comparing the level of Gaq/11 in K-R and 1 mM glutamate exposed brain synaptosomal membranes by two-tailed Student’s t test. #p,0.01 comparing the Gq/11 - mGluR1 or Homer1 - mGluR1 coupling levels in cocaine- to saline-treated groups by two-tailed Student’s t test. doi:10.1371/journal.pone.0091671.g002

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Figure 3. Prenatal cocaine exposure did not alter the expression of various Ga proteins, Homer1 and mGluR1 in the FCX and hippocampus of P21 rats. (A) Representative Western blots of the Ga proteins, Homer1 and mGluR1 in the FCX and hippocampal synaptosomes of P21 rats exposed to saline or cocaine during gestation. The blots were stripped and sequentially re-probed with anti-actin (2265.56129.9 and 2378.86174.8 optical intensity in FCX of saline and cocaine, respectively and 2160.56104.8 and 2125.06131.7 optical intensity in hippocampi of saline and cocaine, respectively) to validate equal loading. (B) Densitometric quantification of the Ga proteins, Homer1 and mGluR1 levels in the FCX and hippocampus of P21 rats. Data are presented as means 6 s.e.m. of the ratios of optical intensity of indicated protein to the optical intensity of actin. N = 4 for each group. There are no discernible changes in the expression levels of any of the proteins examined in either brain regions of the prenatal cocaine exposed rats. doi:10.1371/journal.pone.0091671.g003

pregnant dams were assigned to receive daily intraperitoneal (ip) Preparation of Synaptosome and Fractionation injections from GD 8–20 of either cocaine HCl, 30 mg/kg in Synaptosomes (P2 fraction) were prepared from frontal cortices 0.9% saline or saline, 2 ml/kg. The animals were injected daily as previously described previously with few modifications [22], between 9–10 AM. Following each injection, these pregnant rats [23]. To further purify synaptosomal factions, the synaptosome- were observed for 1 hr and behavioral abnormalities recorded. enrich P2 fraction was washed twice in 5 ml of oxygenated ice- There were no discernible differences in litter size (between 7–13 cold Kreb’s-Ringer solution (K-R): 25 mM HEPES, pH 7.4; pups) and body weight of the pups at 21-day of age (47.563.1 and 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 1.3 mM CaCl2, 52.762.4 g for cocaine and saline, respectively; n = 30 each) and 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, 100 mM gender distribution (17 males/13 females and 14 males/16 females ascorbic acid, 50 mg/ml leupeptin, 10 mg/ml aprotinin, 2 mg/ml for cocaine and saline groups, respectively). There were no obvious soybean trypsin inhibitor, 0.04 mM phenylmethylsulfonyl fluoride changes in rearing behaviors. Importantly, the dose of cocaine (PMSF), 0.1 mM 2-mercaptoethanol, 10 mM NaF, 1 mM used did not induce seizure or fatality throughout the treatments. Na3VO4 and 0.5 ml/ml protein phosphatase inhibitor I & II The progenies were cross-fostered to a naı¨ve mother until cocktails (Sigma, St. Louis, MO). To obtain cytosolic and sacrificed at 21 days of age (P21). They were subjected to the membranous fractions of the synaptosomes, the washed synapto- minimum handling associated with routine animal husbandry. somes were sonicated for 10 sec on ice in 0.5 ml hypo-tonic Importantly, we did not find gender differences in our previous homogenization solution (25 mM HEPES, pH 7.4; 12 mM NaCl, studies conducted in rabbit and rats [5], [6], [19], [20], both sexes 0.5 mM KCl, 2.5 mM NaHCO3, 0.1 mM CaCl2, 0.1 mM from separate litters were employed in these experiments. To MgSO4, 0.1 mM KH2PO4, 1 mM glucose, 10 mM ascorbic acid, avoid the pitfall from oversampling [21], only one animal from 50 mg/ml leupeptin, 10 mg/ml aprotinin, 2 mg/ml soybean trypsin each litter was used in each experiment reported here. Pups were inhibitor, 0.04 mM PMSF and 0.1 mM 2-mercaptoethanol, sacrificed by rapid decapitation, the brains removed immediately 10 mM NaF, 1 mM Na2VO4 and 0.5 ml/ml protein phosphatase on ice, and coronal cuts at optic chiasm and +4 mm rostral to inhibitor I & II cocktails). Samples were then centrifuged at optic chiasm were made to dissect out the FCX and prefrontal 50,0006g for 30 min. The resultant supernatant was taken as cortex. To minimize skin lesions in the cocaine-injected rats, the cytosolic fraction and synaptic membrane pellet was resuspended injection sites were alternated every other day. in 0.5 ml of K-R solution. Protein concentrations of the synaptic

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were immunoprecipitated with 1 mg immobilized anti-Gas/olf, - Gai, - Gaoor-Gaq/11 for 16 hr incubation at 4uC as described previously [24]. Following centrifugation, the obtained anti-Ga immunoprecipitates were washed three times with 1 ml of ice-cold phosphate-buffered saline, pH 7.2 (PBS), the isolated mGluR1/G protein signaling complexes were solubilized by boiling for 5 min in 100 ml of SDS-PAGE sample preparation buffer (62.5 mM Tris-HCl, pH 6.8; 10% glycerol, 2% SDS; 5% 2-mercaptoethanol, 0.1% bromophenol blue). The presence of mGluR1 was detected by Western blotting with anti-mGluR1 together with anti-Gaafter size-fractionation of the solubilized anti-Ga immunoprecipitate to confirm the presence of mGluR1 and indicated Ga proteins. In addition, synaptic membranes were incubated with 1 m M glutamate at 37uC for 5 min. The reaction was terminated by addition of ice-cold 20 mM Mg2+-containing K-R. The synaptic Figure 4. Prenatal cocaine exposure markedly reduces membranes were then collected by centrifugation and solubilized mGluR1-induced phosphoinositide hydrolysis in the FCX and as described above. The obtained membranous lysate was hippocampus of P21 rats. The levels of 1 mM DHPG induced IP3 immunoprecipitated with 1 mg of either immobilized anti- accumulation was measured using an assay kit according to manufac- mGluR1 followed by 30 ml immobilized protein A/G-conjugated turer’s instructions. Prenatal cocaine exposure did not affect the basal agarose. The resultant anti-mGluR1 immunoprecipitates was IP3 levels in FCX (12.0360.93 vs. 11.7160.93 pmol/mg protein in saline probed with anti-Gaq and Homer1 using Western blotting. and cocaine, respectively) and in hippocampus (11.9561.26 vs. 12.7161.63 pmol/mg protein in saline and cocaine, respectively). To determine the effect of prenatal cocaine exposure on the Incubation with 1 mM DHPG resulted in a 137.068.7% and a mGluR1-Gq/11-Homer1 linkages, mGluR1-Gq/11-Homer1 com- 41.2610.2% increase in IP3 levels in FCX and hippocampus, respectively. plexes in frontal cortical and hippocampal synaptic membrane Prenatal cocaine exposure markedly reduced DHPG-induced phospho- lysates from prenatal saline- and cocaine-exposed rats were inositide hydrolysis by 52.066.9–62.668.4% in both brain regions. Data isolated by immunoprecipitation for 16 hr incubation at 4uC with are presented as means 6 s.e.m. of the IP3 level in pmol/mg protein. 1 mg of immobilized anti-mGluR1- protein A-conjugated agarose N = 6 for each group. *p,0.01, **p,0.05 comparing the IP3 level in Kreb’s-Ringer and 1 mM DHPG exposed brain tissues by two-tailed beads. The resultant immunocomplexes were pelleted by centri- Student’s t test. #p,0.01 comparing DHPG-induced IP3 in cocaine to fugation at 4uC. After three washes with 1 ml of ice-cold PBS and saline-treated group by two-tailed Student’s t test. centrifugation, the isolated mGluR1 signaling complexes were doi:10.1371/journal.pone.0091671.g004 solubilized by boiling for 5 min in 100 ml of SDS-PAGE sample preparation buffer. Following brief centrifugation to remove immobilized antibody-protein A/G agarose complexes, the levels of Gq/11-mGluR1 and mGluR1-Homer1 association were esti- membranes were determined using the Bradford method (Bio- mated by the amount of Gaq/11 and Homer1 in anti-mGluR1 Rad) and used as the tissue source for determination of the levels of immunoprecipitates by Western blotting. Equal loading of the mGluR1-G protein-Homer1 coupling. anti-mGluR1 immunoprecipitates was confirmed by Western blotting that assessed the amount of mGluR1. In vitro Determination of mGluR1 - G Protein and - In the experiments where the phosphorylation state of mGluR1 Homer1 Interaction and Immunoprecipitation was assessed, frontal cortical synaptic membranes (100 mg) from To assess the effect of prenatal cocaine exposure on mGluR1 - prenatal saline- and cocaine-exposed rats were suspended in K-R G protein and -Homer1 coupling, synaptic membranes (200 mg) without protein phosphatase inhibitors. Membranes were incu- derived from frontal cortices and hippocampi of 21-day-old rats bated with 100 mg/ml alkaline phosphatase in Tris, pH 8.0, that were exposed to either saline or cocaine during gestation were 130 mM NaCl, protease inhibitors at 30uC for 20 min (total incubated with 1 mM glutamate or K-R for 5 min at 37uC (total incubation volume 100 ml). The phosphatase activity was termi- 2+ incubation volume: 250 ml). Following incubation, 750 mlCa - nated by adding 10 mM NaF/1 mM Na3VO4. To assess the effect free K-R containing 0.5 mM EGTA was added and synaptic of specific PKC-mediated phosphorylation, dephosphoryated membranes were pelleted by centrifugation. Synaptic membranes membranes from saline-treated P21 rats was incubated with were then solubilized by brief sonication (10 sec, 50% output, 0.5 mg/ml recombinant cPKC, 20 mg phosphotidylserine and Fisher Scientific) in 250 ml of immunoprecipitation buffer (25 mM 162 nM phorbol 12-myristate, 13-acetate (PMA) in the presence HEPES, pH 7.5; 200 mM NaCl, 1 mM EDTA, 50 mg/ml of 30 mM ATP in K-R at 30uC for 10 min (total incubation leupeptin, 10 mg/ml aprotinin, 2 mg/ml soybean trypsin inhibitor, volume 125 ml). The actions of PKC were terminated by addition 0.04 mM PMSF, 5 mM NaF, 1 mM sodium vanadate, 0.5 mM b- of 1 mM celestrine. Following termination of the reaction, glycerophosphate and 0.1% 2-mercaptoethanol containing 0.5% membranes were incubated with K-R or 1 mM glutamate at digitonin, 0.2% sodium cholate and 0.5% NP-40 and incubated at 37uC for 5 min. The reaction was stopped by adding 375 ml of ice- 4uC with end-to-end shaking for 1 hr. Following dilution with cold Ca2+-free K-R containing 0.5 mM EGTA and synaptic 750 ml of ice-cold immunoprecipitation buffer and centrifugation membranes were collected by centrifugation. The obtained at 4uC to remove insoluble debris, the resultant lysate was used to synaptic membrane pellet was immediately solubilized by brief identify and measure the effect of prenatal cocaine exposure on sonication in immunoprecipitation buffer with 0.5% digitonin, mGluR1-Gq/11-Homer1 interaction. 0.2% sodium cholate and 0.5% NP-40 and incubated at 4uC with To identify the G protein(s) associated with mGluR1, we used end-to-end shaking for 1 hr. Following dilution with 750 ml of ice- co-immunoprecipitation of mGluR1 with specific G protein(s). cold immunoprecipitation buffer and centrifugation at 4uCto Briefly, frontal cortical and hippocampal lysates from control rats remove insoluble debris, the mGluR1-Gq/11-Homer1 complexes

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Figure 5. Serine phosphorylation of mGluR1 is elevated in FCX and hippocampi of prenatal cocaine-exposed P21 rats. (A) Levels of Serine (S), Threonine (T) and tyrosine (Y) phosphorylation on purified native mGluR1 from prenatal saline- and cocaine-exposed P21 rats were determined in immunoprecipitates of immobilized anti-mGluR1 by Western blot using specific antibodies directed against each phospho-epitope. (B) pS-mGluR1 levels in immobilized mGluR1 immunoprecipitates of Kreb’s-Ringer (K-R) and 162 nM PMA treated FCX synaptic membrane extracts from prenatal saline- and cocaine-exposed P21 rats. The obtained protein bands were quantified by densitometric scanning of the blots. Data are represented as means 6 s.e.m. of the ratios of phosphorylated mGluR1/total mGluR1 optical intensities. n = 4 for each group. *p,0.01 comparing the pS-mGluR1 levels induced by PMA vs. K-R in each treatment group. +p,0.01 comparing the levels of pS-mGluR1 in prenatal cocaine- to saline- exposed groups by two-tailed Student’s t test. #p,0.01 comparing the levels of PMA-induced pS-mGluR1 isolated from prenatal cocaine- to saline- treated rats by two-tailed Student’s t test. No between-group differences in the levels of mGluR1were detected. doi:10.1371/journal.pone.0091671.g005

were co-immunoprecipitated using 1 mg immobilized anti- minutes in 100 ml PAGE sample buffer and then size fractionated mGluR1 protein A-agarose beads overnight at 4uC. The on 10% SDS-PAGE. Proteins were transferred to nitrocellulose immunoprecipitate was pelleted by centrifugation and washed 3 membrane and Western blotting was performed with antibodies times each with 1 ml PBS. The mGluR1-Gq/11-Homer1 com- for mGluR1, Homer-1, phosphotyrosine, phosphoserine, phos- plexes were eluted with 75 ml of antigen elution buffer (Pierce). phothreonine, Gas/olf, Gai, GaoorGaq/11. The blots were The eluate containing mGluR1-G protein-Homer1 was immedi- stripped and re-probed with anti-actin, anti- Ga (co-immunopre- ately neutralized by 10 ml 1.5 M Tris, pH 8.8 and 15 ml6X cipitation of mGluR1– G protein), anti-mGluR1 (co-immunopre- sample preparation buffer was added. After boiling for 5 min, cipitation of mGluR1 - Homer1 and -Gq/11 or phosphorylated phosphor-serine, -threonine and -tyrosine content in mGluR1 was mGluR1) to verify equal sample loading and immunoprecipitation sequentially analyzed by Western blotting with antibodies directed efficiency. against phosphoserine first (anti-phosphoserine), stripped and re- Immunoreactivity was detected by a chemiluminescent method probed three times sequentially with anti-phosphothreonine, - and visualized by immediately exposing to X-ray film for 10– phosphotyrosine and then -mGluR1. The signals were detected 30 sec. Specific protein bands were quantified by densitometric using a chemiluminescent method and visualized by exposure to x- scanning (GS-800 calibrated densitometer, Bio-Rad Laboratories). ray film for 5–30 sec. Specific protein bands were quantified by densitometric scanning (GS-800 calibrated densitometer, Bio-Rad Measurement of IP3 Accumulation Laboratories). The effect of prenatal cocaine on mGluR1-mediated IP3 accumulation was determined in frontal cortical and hippocampal Western Blotting slices from P21 prenatal cocaine- and saline-exposed rat brains To determine whether prenatal cocaine exposure influences the using an established method with slight modifications [25]. Brain expression of mGluR1, Homer1 or various Ga proteins, synaptic tissues were brought to room temperature in oxygenated K-R, membranes of frontal cortices and hippocampi from 21-day-old pH 7.4. The tissues were chopped into 3506350 mm slices, prenatal saline- and cocaine-exposed rats were boiled for 5 washed twice with K-R at room temperature and incubated for

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Figure 6. Heightened PKC-mediated serine phosphorylation of mGluR1 in prenatal cocaine- exposed brains is responsible for the reduced mGluR1 coupling to Gq/11 and Homer1. (A) Levels of mGluR1 - Gq/11 and mGluR1 - Homer1 coupling as well as serine-phosphorylated mGluR1 (pS-mGluR1) on purified mGluR1 signaling complexes. FCX synaptosomal membranes from prenatal cocaine- and saline-exposed P21 rats were incubated with either vehicle 100 mM alkaline phosphatase, 30 mM ATP containing Kreb’s-Ringer (K-R) or 162 nM PMA (20 mg phosphoatidylserine). Following completion of dephosphorylation by addition of phosphatase inhibitors, phosphate-free mGluR1 from prenatal saline-treated rats was phosphorylated by recombinant cPKC in the presence of ATP. The reaction was terminated by specific PKC inhibitor, celestrine. The resultant synaptic membranes containing differentially phosphorylated mGluR1 were then incubated with K-R or 1 mM glutamate. The mGluR1 purified by anti-mGluR1 immunoprecipitation was analyzed for phosphoserine by Western blotting. The interaction between mGluR1 and Gq/11 or Homer1 with different phosphorylation states was assessed by the levels of Gaq/11 and Homer1 in the anti-mGluR1 immunoprecipitates by Western blotting. (B) Densitometric quantification of mGluR1-associated Gaq/11 and Homer1 as well as pS-mGluR1 levels. Data are represented as means 6 s.e.m. of the ratios of Gaq/11, Homer1 or pS-mGluR1/total mGluR1 optical intensities. n = 4 for each group. *p,0.01 comparing the glutamate-induced to basal mGluR1-Gq/11 complex levels by two-tailed Student’s t test. #p,0.01 comparing the respected levels in prenatal cocaine- to saline-treated rats. No between-group differences in the levels of mGluR1were detected. doi:10.1371/journal.pone.0091671.g006

15 min in K-R followed by 30 min with 1 mM (S)-3,5-dihydrox- Data Analysis and Statistical Evaluation yphenylglycine (DHPG) or K-R at 37uC in a shaking water bath. Statistical differences between cocaine and saline groups were The slices were then washed twice with pH 7.0 hypotonic buffer assessed using the two-tailed Student’s t test. containing 20 mM Tris-HCl, 1 mM EGTA at 4uC. The slices were collected by brief centrifugation and homogenized in 1.5 ml of Results 1 M trichloroacetic acid (TCA) using 10 hand strokes of a Teflon- glass homogenizer. Homogenates were then placed on ice for Prenatal Cocaine Exposure Uncouples mGluR1 from Gq/ 15 min before centrifugation at 13,000 g for 10 min. The resultant 11 and Homer1 pellets were washed three times with distilled H2O and digested in We first assess whether mGluR1s are coupled to specific G 1 M NaOH and the protein levels were determined using protein(s). The data shown in Fig. 1A illustrates that mGluR1 is Bradford method (Bio-Rad). The specificity of DHPG was assessed coupled exclusively to Gq/11 and exposure to 1 mM glutamate by pretreatment of the slices with 10 mM a-Amino-5-carboxy-3- increases mGluR1 - Gq/11 coupling by approximately 4.5-fold in methyl-2-thiopeneacetic acid (3-MATIDA). One ml of trichloro both FCX and hippocampus. In addition, mGluR1 - Gq/11 trifluoro ethan-trioctylamine (3:1) was added to 500 ml of TCA complex is associated with scaffolding protein Homer1 but not tissue extract in a polypropylene eppendorf tube, mixed vigorously Homer2 (data not shown) as both Gaq/11 and Homer1 were for 15 sec and then centrifuged for 1 min at 10,000 g. Aliquots of detected in anti-mGluR1 immunoprecipitate in both brain regions the upper phase were taken for IP3determination according to the (Fig. 1B). Unlike the coupling of mGluR1 to Gq/11, glutamate instructions from the manufacturer of the assay kit (PerkinElmer). stimulation did not affect mGluR1-Homer1 association. The effect The IP3 concentrations are expressed as pmol/mg protein. of prenatal cocaine exposure on the association of mGluR1 with Gq/11 and Homer1 was subsequently determined by the level of Gaq/11 and Homer1 co-immunoprecipitated with mGluR1. The data summarized in Fig. 2 show that prenatal cocaine exposure

PLOS ONE | www.plosone.org 7 March 2014 | Volume 9 | Issue 3 | e91671 Prenatal Cocaine Attenuates mGluR1 Signaling markedly reduces the levels of mGluR1 coupling to Homer1 by all of which involve excitatory synaptic transmission [26–30]. 56.064.9–57.8610.1% and glutamate-induced but not basal These data suggest that excitatory synapses including those mGluR1 coupling to Gq/11 by 69.0612.3 - 63.463.2% in both regulated by the mGluR1 are perturbed by cocaine exposure FCX and hippocampus. This prenatal cocaine effect was not during early brain development. This deficit presumably leads to caused by alteration in the mGluR1, Ga or Homer1 expression as an altered synaptic plasticity resulting in enduring changes in brain comparable levels of these proteins were noted in both brain function and abnormal behavior. In support of this possibility, our regions examined (Fig. 3). present data indicate that in utero cocaine exposure disrupts mGluR1 coupling to synaptic anchoring protein, Homer1 and Prenatal Cocaine Exposure Attenuates mGluR1-mediated signal transducer, Gq/11 protein. By associating with Homer1, mGluR1s cluster in the synaptic membrane. Hence, our data IP3 Accumulation To assess the impact of prenatal cocaine exposure on mGluR1- showing that prenatal cocaine exposure uncouple mGluR1 from Homer1 without a detectable change in mGluR1 expression mediated function, we measured the level of IP3 induced by 1 mM DHPG, a group I mGluR specific agonist. Our results summa- suggest relocation of mGluR1s away from the active glutamatergic rized in Fig. 4 indicate that prenatal cocaine reduces DHPG- synapses to parasynaptic regions. Previously, mGluR and Homer interaction was found to be essential for mGluR signaling induced IP3 accumulation in the FCX and hippocampus. Pretreatment with mGluR1-specific antagonist, 3-MATIDA mediated long term depression (LTD) [31]. In light of our finding resulted in an approximately 65–70% in both brain regions (data that Homer1 did not directly couple to Gq/11 (data not shown), not shown). In both brain areas from P21 prenatal cocaine- the reduced mGluR1 - Gq/11 coupling is responsible for the exposed rats, a 52.066.9–62.668.4% reduction in DHPG-elicited impairment of the mGluR1-mediated phosphoinositide signaling in the prenatal cocaine-exposed brain. The fact that the mGluR1 IP3 accumulation without alteration in basal IP3 level were noted (Fig. 4). specific antagonist 3-MATIDA reduced the DHPG-induced IP3 accumulation by 65–70% coupled with a 63.4–69% reduction in Prenatal Cocaine Exposure Increases Serine- mGluR1-Gq/11 association suggests that prenatal cocaine may phosphorylation of the mGluR1 also attenuate mGluR5 signaling. Concurrent with a reduced mGluR1 signaling shown here, To identify the mechanism responsible for uncoupling of the attenuated AMPAR and NMDAR function were also noted in mGluR1 from its associated G protein and Homer1, we q/11 brain tissues from in utero cocaine-exposed rodents [5–7]. The considered alteration in the phosphorylation state of the mGluR1 impaired AMPARs in the prenatal cocaine exposed rat brains was since altered phosphorylation state of the receptor can result in found to be associated with disrupted GluR2/3 - GRIP interaction receptor - G protein dissociation [26]. The data shown in Fig. 5A [6]. These findings indicate that prenatal cocaine profoundly indicate that the level of phosphoserine (pS) in mGluR1 was affects the signaling processes mediated by multiple glutamatergic increased by 150.869.2% and 143.469.5% exclusively in FCX receptors. The fact that activation of mGluR1 rescued the delayed and hippocampus of prenatal cocaine-exposed brains, respectively. synaptic maturation with impaired AMPAR and NMDAR Because mGluR1 is coupled to phospholipase C, we considered function in the prenatal cocaine-exposed brains suggests that PKC as the primary phosphorylating kinase for mGluR1. mGluR1 is a key regulator of the glutamatergic synaptic Incubation of FCX slices from prenatal saline- and cocaine- maturation during brain development [7]. Since mGluR1 is exposed P21 rats with PKC activator, phorbol 12-myristate 13- expressed abundantly in cognition- and reward-relevant brain acetate (PMA), an approximately 2.5-fold increase in pS-mGluR1 was observed in saline- but not cocaine-exposed tissues (Fig. 5B). areas including NAc and VTA, a profound reduction in the To further deduce whether the increased serine-phosphorylated mGluR1 coupling to Homer1 and Gq/11 in the prenatal cocaine- mGluR1 is responsible for the uncoupling of mGluR1 from both exposed brain coupled with attenuated signaling is likely to hamper also AMPAR and NMDAR signaling [7], leading to the Gq/11 and Homer1, synaptic membranes from prenatal saline- and cocaine-exposed rats were treated with alkaline-phosphatase. deficits in cognition and the impaired reward processing seen in Figure 6B illustrates that removal of pS from mGluR1s by alkaline animals exposed to cocaine during gestation. This impairment in phosphatase treatment in the prenatal cocaine exposed FCX reward processing is evidenced by studies showing that rabbits restored Gq/11 and Homer1 coupling to mGluRs to the level exposed to cocaine in utero are tolerant to cocaine-induced motor observed in saline-treated animals. In addition, incubation of the sensitization, which is considered a behavioral correlate of synaptic membranes from saline-treated rats with recombinant addictive process [32], [33]. cPKC in the presence 20 mg phosphatidylserine, 1 mM Ca2+/ The reduced coupling of mGluR1 to Homer1 and Gq/11 is the 162 nM PMA and de-phosphorylated mGluR1 (phosphatase result of a sustained PKC-mediated phosphorylation of mGluR1 treatment followed by 10 mM NaF and 1 mM Na vanadate), a in prenatal cocaine-exposed brains. This observation is reminis- 2.5–2.7 fold increased in serine phosphorylation of mGluR1 cent of our previous report that an increased phosphorylation of accompanied by a 44.762.8% reduction in glutamate-induced D1A dopamine receptors in prenatal cocaine-exposed rabbits results in uncoupling of D1A dopamine receptors from their signal coupling to Gq/11 and a 45.063.2–46.263.6% decrease in mGluR1- Homer1 association were noted (Fig. 6). These data transducer, Gs and Golf proteins [26]. A sustained PKC activation indicated by an overwhelming cytosol-to-membrane translocation indicate that reduction in mGluR1 coupling to Gq/11 and Homer1 in prenatal cocaine-exposed brains is the result of PKC-mediated together with markedly increased association of GRIP with mGluR1 phosphorylation at the serine sites. activated PKC-a,-c and -f as well as PKMf has been reported in prenatal cocaine-exposed brains [6]. This results in a sustained Discussion PKC- and Src-mediated phosphorylation of GRIP, the synaptic anchoring protein for GluR2- and GluR3-containing AMPARs Prenatal cocaine exposure has negative impact on cognitive thus preventing GRIP from interacting with GluR2/3 [6]. Unlike processing in both humans and animal models as illustrated by our previous finding, PKC but not Src mediates mGluR1 reduced attention processing and impairments in motor and phosphorylation since serine phosphorylation is exclusively language skills as well as associative and discrimination learning, elevated in mGluR1 of the prenatal cocaine brains. In agreement

PLOS ONE | www.plosone.org 8 March 2014 | Volume 9 | Issue 3 | e91671 Prenatal Cocaine Attenuates mGluR1 Signaling with our theory that PKC hyperactivation plays a pivotal role in consequent uncoupling of mGluR1 from Homer1 and Gq/11 cognitive processing in prenatal cocaine-exposed individuals, elucidated here may be one of the prominent events underlying excessive PKC activation was found to dramatically impair prenatal cocaine-induced cognitive impairment. Moreover, both prefrontal cortex-mediated cognitive functions and increase our previous and current data suggest that blocking excessive PKC distractibility [34]. Future experiments are needed to determine translocation, a primary PKC activation mechanism, may be whether the reduced mGluR1 - Homer1 - Gq/11 coupling effective in preventing/reversing prenatal cocaine from promoting observed in brains of P21 prenatal cocaine-exposed rats is protracted deficits in AMPAR- and mGluR- regulated neuro- persistent or simply a transient shift in synaptic plasticity during transmission [6], [40]. In this regard, therapeutic agents such as early development. Nevertheless, previous studies conducted by us lithium and valproate that block PKC translocation without and others in rabbit indicate that such synaptic plasticity changes directly interfering with its enzymatic activity [41] may have are persistent well into adulthood [35–37]. therapeutic value in maintaining adequate mGluR1-regulated In addition to the profound downregulation of the glutamater- synaptic activity in subjects exposed to cocaine during gestation. gic mGluR1, AMPAR and NMDAR function [5], [6] prenatal cocaine exposure has been shown to affect other neurotransmitter Author Contributions systems including dopamine D1 receptor - Gs/olf coupling [19], [20], [26], GABAergic neurons [38] and the noradrenergic system Conceived and designed the experiments: H-YW. Performed the [39]. Notwithstanding these changes reported to occur in prenatal experiments: KB RP SKG MW. Analyzed the data: H-YW. Wrote the cocaine-exposed brains, we propose that hyper-activation of PKC paper: H-YW. Helped edit the manuscript: EF. resulting in sustained phosphorylation of mGluR1s and the

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