Regional differences in the effects of withdrawal from repeated cocaine upon Homer and glutamate receptor expression: A two-species comparison

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Regional differences in the effects of withdrawal from repeated cocaine upon Homer and glutamate receptor expression: A two-species comparison Alexis W. Ary, Karen Kathleen Szumlinski⁎ Behavioral and Neural Genetics Laboratory, Department of Psychology and the Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106-9660, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

The constitutively expressed (CC) Homer protein Homer2a/b actively regulates behavioral and

Accepted 15 September 2007

neurochemical sensitivity to cocaine in both rats and mice. The present study employed

Available online 22 September 2007

standard immunoblotting techniques to compare the effects of withdrawal from repeated cocaine (7 × 30 mg/kg) upon the protein expression of Homer2a/b with a related CC-Homer

Keywords:

protein Homer1b/c, as well as their associated glutamate receptors, within brain regions

Cocaine

implicated in cocaine addiction. To determine whether or not the observed cocaine-induced

Glutamate

changes in Homer and glutamate receptor expression generalized across mammalian species,

Homer

immunoblotting was conducted on tissue derived from both male Sprague–Dawley rats and

Metabotropic glutamate receptor

male C57BL/6J mice. In both species, withdrawal from repeated cocaine administration down-

NMDA receptor

regulated Homer1b/c and Homer2a/b within the shell, but not the core, of the nucleus

Sensitization

accumbens (NAC), and the reduced Homer levels were accompanied by decreases in mGluR1a, NR2a and NR2b. In the PFC, repeated cocaine up-regulated Homer2a/b, mGluR1 and NR2b expression, without affecting Homer1b/c levels. Cocaine-induced increases in Homer1b/c, Homer2a/b, mGluR1a and NR2a were observed in the hippocampus of both rats and mice, while in dorsal striatum, NR2a levels were elevated but Homer and Group1 mGluR levels were unchanged. Thus, withdrawal from repeated cocaine alters the expression of CC-Homer isoforms and their associated glutamate receptors in a regionally-distinct manner. As CCHomer proteins, Group1 mGluRs and NMDA receptors actively regulate cocaine-induced neuroplasticity in vivo, these data support the hypothesis that cocaine-induced changes in mGluR–Homer–NMDA signaling pathways may be important neuroadaptations mediating the enduring changes in behavior produced by repeated cocaine experience. © 2007 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Department of Psychology, University of California, Santa Barbara, Santa Barbara, CA 93106-9660, USA. E-mail address: [email protected] (K.K. Szumlinski). Abbreviations: AAV, adeno-associated virus; B6, C57BL/6J; CC, coiled-coil; cDNA, complimentary deoxyribonucleic acid; EVH1, Ena/VASP homology 1; IEG, immediate early gene; KO, knock-out; mGluR, metabotropic glutamate receptor; mRNA, messenger ribonucleic acid; NAC, nucleus accumbens; NMDA, N-methyl-D-aspartate; PFC, prefrontal cortex; WT, wild-type 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.09.035

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Introduction

Cocaine addiction is a chronic neuropsychiatric disorder characterized by persistent craving and a high propensity for relapse long after drug abstinence. Enduring alterations in both pre- and post-synaptic aspects of mesocorticolimbic glutamate transmission have been implicated in mediating behaviors produced by repeated cocaine treatment (Pierce et al., 1998; Reid and Berger, 1996; Szumlinski et al., 2004, 2006a; Williams and Steketee, 2004), including cocaine self-administration (Backstrom and Hyytia, 2007; Baker et al., 2003; Cornish and Kalivas, 2001; Di Ciano and Everitt, 2001; McFarland et al., 2004; Park et al., 2002; Pulvirenti et al., 1992). A likely molecular mediator of cocaine-induced changes in mesocorticolimbic glutamate is the Homer family of postsynaptic scaffolding proteins (for reviews, Szumlinski et al., 2006b, 2007a). Three independent genes, Homer1 (aka PSD-Zip45 or vesl-1), Homer2 (aka Cupidin or vesl-2) and Homer3 (aka vesl-3), encode this protein family (Soloviev et al., 2000), which are integral in glutamate synaptogenesis and glutamate receptor trafficking (for reviews, Bockaert et al., 2004; de Bartolomeis and Iasevoli, 2003; Duncan et al., 2005; Xiao et al., 2000). Long-form Homer proteins (Homer1b-g, Homer2a/b, and Homer3) are constitutively expressed in the brain (Klugmann et al., 2005; Shiraishi et al., 2004; Xiao et al., 1998) and share a coiled-coil (CC) structure, which allows for their multimerization (Hayashi et al., 2006), and facilitates the clustering of glutamate receptors and other proteins involved in their intracellular signaling cascades (for reviews: Bockaert et al., 2004; de Bartolomeis and Iasevoli, 2003; Duncan et al., 2005; Szumlinski et al., 2006b; Xiao et al., 2000). Additionally, Homers possess a conserved amino-terminal Ena/VASP1 homology (EVH1) domain (Brakeman et al., 1997). This domain allows Homers to bind with a proline-rich (PPXXFR;

amino acid sequence) motif found in Group1 metabotropic glutamate receptors (mGluRs) (Tu et al., 1998), inositol-1,4,5triphosphate receptors (Brakeman et al., 1997; Hwang et al., 2005; Nakamura et al., 2004; Sala et al., 2005; Shin et al., 2003; Tu et al., 1998; Yuan et al., 2003), ryanodine receptors (Feng et al., 2002; Hwang et al., 2003), transient receptor potential canonical-1 ion channels (Yuan et al., 2003), and the NMDA glutamate receptor scaffolding protein Shank (Naisbitt et al., 1999; Shiraishi et al., 2003; Tu et al., 1999). The EVH1 domain on CC-Homers compete for binding with that on the truncated transcriptional variants of Homer1 (Homer1a and ania-3), which are induced as immediate early gene (IEG) products upon synaptic activity (Brakeman et al., 1997). As Homer1a lacks both the CC-domain and the ability to multimerize (Bottai et al., 2002; Xiao et al., 1998), Homer1a acts as a natural dominant negative of CC-Homer multimers and, thus, influences the functional architecture of glutamatergic terminals and synaptic plasticity (Brakeman et al., 1997; Sala et al., 2003). While a number of studies have examined for drug-induced changes in IEG Homer mRNA or protein expression within brain (for recent review, Szumlinski et al., 2007a), considerably less is known regarding the regulation of CC-Homer proteins by drugs of abuse (Kane et al., 2005; Swanson et al., 2001; Szumlinski et al., 2007b; Zhang et al., 2007a). With respect to stimulant drugs, the protein expression of IEG and CC-Homer1 isoforms is regulated differentially by cocaine within the dorsal or ventral aspects of the striatum of rats (Swanson et al., 2001; Zhang et al., 2007a). While behavioral genetics studies implicate a role for both Homer1 and Homer2 gene products in regulating behavioral and neurochemical sensitivity to cocaine (Lominac et al., 2005; Szumlinski et al., 2004, 2005a, 2006a), whether or not Homer2 protein expression is regulated within the mesocorticolimbic glutamate system by cocaine is

Fig. 1 – Repeated cocaine up-regulates Homer2a/b, mGuR1a and NR2 subunits in the PFC. Top, Summary of the mean change in protein expression observed within the PFC of rats (while bars) and mice (gray bars) at 3 weeks withdrawal from repeated saline (s) or cocaine (c) treatment (7×30 mg/kg) exhibited by rats and mice. Data were normalized to saline-treated animals (% Saline) and represent the mean± S.E.M. of 9–22 animals/group. Bottom, Representative immunoblots for the total protein levels of Homer2a/b, Homer1b/c, mGluR1a, mGluR5, NR2a and NR2b in the PFC of rats (upper row) and mice (lower row). *p<0.05 vs. respective saline control.

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Fig. 2 – Repeated cocaine down-regulates Homer2a/b, mGuR1a and NR2 subunits in the NAC shell. Top, Summary of the mean change in protein expression observed within the NAC shell of rats (while bars) and mice (gray bars) at 3 weeks withdrawal from repeated saline (s) or cocaine (c) treatment (7 × 30 mg/kg) exhibited by rats and mice. Data were normalized to saline-treated animals (% Saline) and represent the mean ± S.E.M. of 9–22 animals/group. Bottom, Representative immunoblots for the total protein levels of Homer2a/b, Homer1b/c, mGluR1a, mGluR5, NR2a and NR2b in the NAC shell of rats (upper row) and mice (lower row). *p < 0.05 vs. respective saline control.

not known. Moreover, it unknown whether or not the cocaineinduced changes in Homer2 expression relate to, or are independent of, changes in the expression of CC-Homer1 isoforms. Thus, the primary goal of the present immunoblotting study was to compare cocaine-induced changes in Homer2a/b expression with those for Homer1b/c within brain regions

implicated in mediating the psychomotor-activating and rewarding effects of cocaine (incl. prefrontal cortex, NAC, striatum, and hippocampus) (Dackis and O'Brien, 2001; Everitt and Wolf, 2002; Fuchs et al., 2007; Goldstein and Volkow, 2002; Kalivas and McFarland, 2003; Kalivas et al., 2005; Koob and Le Moal, 2001; Rogers and See, 2007). As Homer2 regulates Group1

Fig. 3 – Repeated cocaine up-regulates Homer2a/b, mGuR1a and NR2 subunits in the hippocampus. Top, Summary of the mean change in protein expression observed within the hippocampus of rats (while bars) and mice (gray bars) at 3 weeks withdrawal from repeated saline (s) or cocaine (c) treatment (7×30 mg/kg) exhibited by rats and mice. Data were normalized to saline-treated animals (% Saline) and represent the mean±S.E.M. of 9–22 animals/group. Bottom, Representative immunoblots for the total protein levels of Homer2a/b, Homer1b/c, mGluR1a, mGluR5, NR2a and NR2b in the hippocampus of rats (upper row) and mice (lower row). *p< 0.05 vs. respective saline control.

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Table 1 – Relative to repeated saline treatment, withdrawal from repeated cocaine administration produced no significant effects upon Homer or glutamate receptor expression within the NAC core of either rats or mice (t-tests; p> 0.05) Nucleus accumbens core

Dorsal striatum

Rat

Homer2a/b Homer1b/c mGluR1a mGluR5 NR2a NR2b

Mouse

Rat

Mouse

Saline

Cocaine

Saline

Cocaine

Saline

Cocaine

Saline

Cocaine

100 ± 11.0 100 ± 10.1 100 ± 12.1 100 ± 7.0 100 ± 14.6 100 ± 5.7

109 ± 9.5 98 ± 10.0 65 ± 12.7 117 ± 21.2 92 ± 13.8 114 ± 10.5

100 ± 16.1 100 ± 20.8 100 ± 16.1 100 ± 12.1 100 ± 15.8 100 ± 27.4

159 ± 34.0 105 ± 17.3 107 ± 20.6 128 ± 27.2 123 ± 23.1 87 ± 13.3

100 ± 6.2 100 ± 12.0 100 ± 8.4 100 ± 7.0 100 ± 7.1 100 ± 5.8

94 ± 9.1 126 ± 20.8 110 ± 14.1 140 ± 21.4 138 ± 13.3 112 ± 7.5

100 ± 5.9 100 ± 11.1 100 ± 12.3 100 ± 8.8 100 ± 12.5 100 ± 9.1

92 ± 5.7 99 ± 13.9 75 ± 12.5 92 ± 8.2 142 ± 12.6 97 ± 8.8

Withdrawal from repeated cocaine increased NR2a expression within the dorsal striatum of both species, but did not alter the expression of any other protein examined. Data for cocaine-treated rodents in bold italics reflect a significant difference from saline-treated controls ( p ≤ 0.05).

mGluR and NR2 expression in vivo (Szumlinski et al., 2004, 2005b, 2007b), the secondary goal of this study was to relate the cocaineinduced changes in CC-Homer expression with those of the mGluR1 and mGluR5 subtypes of Group1 mGluR and the NR2a and NR2b subunits of the NMDA receptor. To determine whether or not the cocaine-induced changes in Homer/mGluR/NMDA expression are generalizable across two mammalian species, we conducted immunoblotting in both Sprague–Dawley rats and in C57BL/6J (B6) mice.

expression was accompanied by a significant (approximately 30%) increase in the levels of mGluR1a protein (for rat: t25 =2.2, p =0.03; for mouse: t36 =2.2, p =0.04), as well as an approximately 25–50% increase in NR2b levels (for rat: t26 = 3.2, p= 0.003; for mouse: t32 = 1.9, p =0.07). As depicted in Fig. 1, species differences were observed for the rise in PFC NR2a levels, with rats exhibiting a significant 40% increase in protein expression (t16 =3.5, p= 0.003) but mice displaying unaltered NR2a levels. Neither species exhibited a change in the PFC levels of mGluR5 (Fig. 1).

2.

2.2. Withdrawal from repeated cocaine down-regulates Homers and specific glutamate receptors within the NAC shell, but not in the NAC core or dorsal striatum

Results

The data summarizing the changes in CC-Homer and glutamate receptor expression within the PFC, NAC shell and the hippocampus observed at 3 weeks withdrawal from repeated cocaine administration (7 × 30 mg/kg) are presented respectively in Figs. 1–3. The data for the NAC core and striatum are presented in Table 1 and all data are summarized in Table 2.

2.1. Withdrawal from repeated cocaine up-regulates specific CC-Homer and glutamate receptor proteins in the PFC As depicted in Fig. 1, the effects of withdrawal from repeated cocaine upon protein expression within the PFC were selective. While Homer1b/c protein levels were unaltered by repeated cocaine treatment, Homer2a/b was up-regulated by 30–50% in both species (for rat: t26 = 2.2, p= 0.04; for mouse t28 =2.0, p = 0.05). Also in both species, the cocaine-induced rise in Homer2a/b

Withdrawal from repeated cocaine down-regulated the expression of CC-Homers and certain glutamate receptors within the NAC shell (Fig. 2). In contrast, cocaine-induced decreases in protein expression were not observed in either the NAC core or in the dorsal aspect of the striatum (Table 1). Where Homer2a/b expression was selectively increased by repeated cocaine administration within the PFC (Fig. 1), the expression of both Homer1b/c (for rat: t26 = 2.8, p = 0.01; for mouse: t20 = 2.4; p = 0.03) and Homer2a/b (for rat: t26 = 2.1, p =0.04; for mouse: t20 =2.3, p = 0.03) was down-regulated significantly by cocaine withdrawal within the NAC shell (Fig. 2) (Table 1). The cocaine-induced reduction in NAC shell CC-Homer expression was paralleled in both species by a significant reduction in mGluR1a levels (for rat: t21 =4.0, p =0.001; for mouse t35 =2.5, p = 0.02), as well as in the levels of NR2a and NR2b (Fig. 2). The reductions in NR2 expression were

Table 2 – Summary of the consistent findings for cocaine-induced changes in Homer and glutamate receptor protein expression between rats and mice Brain region Prefrontal cortex Nucleus accumbens shell Nucleus accumbens core Dorsal striatum Hippocampus

Homer1b/c

Homer2a/b

mGluR1a

mGluR5

NC ↓ 40% NC NC ↑ 30–40%

↑ 30–50% ↓ 20–40% NC NC ↑ 30%

↑ 30% ↓ 30–60% NC NC

NC NC NC NC ↑ 20–70%

NR2a

NR2b ↑ 30–50%

NC ↑ 40% ↑ 30–50%

NC NC NC

Percent changes in bold reflect a statistically significant alteration in protein expression in both species after repeated cocaine withdrawal, as compared to saline controls (p ≤0.05). Percent changes in italics reflect a statistically significant change in one species and a trend in the same direction that failed to reach significance in the other species ( p=0.05–0.1). ↑ indicates increase; ↓ indicates decrease; NC indicates no change in either species.

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statistically significant in rats (for NR2a: t18 =2.3, p= 0.03; for NR2b: t13 =2.8, p =0.01); however, due to variability in the data, the approximately 20% reduction in NR2a and NR2b levels observed for cocaine-treated mice failed to reach statistical significance (p <0.1). While neither NR2 subunit was altered by repeated cocaine administration within the NAC core, withdrawal from repeated cocaine up-regulated NR2a protein levels by 30–40% in the dorsal striatum of both mice and rats (for rat: t25 = 2.6, p =0.02.; for mouse: t37 = 2.2, p = 0.04), without affecting NR2b levels (Table 1). As observed for the PFC, withdrawal from repeated cocaine did not alter the levels of mGluR5 within the NAC shell or core, or within the dorsal striatum.

2.3. Withdrawal from repeated cocaine up-regulates Homers and certain glutamate receptors within the hippocampus As illustrated in Fig. 3, the effects of withdrawal from repeated cocaine administration upon Homer and glutamate receptor protein expression within the hippocampus were more akin to those observed within the PFC (Fig. 1), than to those observed within the NAC shell (Fig. 2). As observed for the PFC, Homer2a/b expression within the hippocampus was increased significantly by approximately 30% in both species (for rat: t25 =2.8, p =0.01; for mouse: t38 =3.3, p =0.002). While the approximately 25% increase in Homer1b/c expression exhibited by cocaine-treated rats was shy of statistical significance (t24 =2.0, p =0.06), the 30% increase in Homer1b/c levels exhibited by cocaine-treated mice was significantly different from their saline controls (t29 = 2.6, p= 0.02). In both species, the rise observed for CC-Homers was accompanied by a significant increase in NR2a expression (for rat: t24 = 3.3, p =0.003; for mouse: t37 = 2.2, p =0.04), but no change in NR2b levels (Fig. 3). While cocaine-treated rats exhibited a 30% increase in mGluR1a expression (t24 =2.9, p= 0.009) and a modest rise in mGluR5 levels (t21 =1.8, p= 0.08), neither change was observed in cocaine-treated mice (Fig. 3).

3.

Discussion

Homer proteins are critically involved in regulating the functional architecture of glutamate synapses (e.g., Bockaert et al., 2004; de Bartolomeis and Iasevoli, 2003; Duncan et al., 2005; Xiao et al., 2000) and in regulating drug-induced neuroplasticity (for a recent review, Szumlinski et al., 2007a). With regards to cocaine, both Homer1b/c and Homer2a/b regulate the expression of cocaine-induced locomotor hyperactivity (Lominac et al., 2005; Szumlinski et al., 2004), as well as the expression of behavioral sensitization produced by repeated cocaine administration (Szumlinski et al., 2006a). Moreover, these CC-Homer isoforms are critically involved in maintaining both PFC and NAC levels of extracellular glutamate in vivo and in regulating the glutamate response to cocaine within these regions (Lominac et al., 2005; Szumlinski et al., 2004, 2005a, 2006a). Despite the earlier behavioral genetics data, very little is known regarding the changes in Homer protein expression in brain produced by the repeated administration of drugs of abuse (Dietrich et al., 2007; Kane et al., 2005; Swanson et al., 2001; Szumlinski et al., 2007b), as the majority of published studies to date have employed acute drug treatment regimens and/or examined exclusively for druginduced changes in mRNA expression (Ammon et al., 2003;

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Brakeman et al., 1997; Cochran et al., 2002; Fourgeaud et al., 2004; Fujiyama et al., 2003; Nichols and Sanders-Bush, 2002; Nichols et al., 2002; Zhang et al., 2007a; for detailed review, Szumlinski et al., 2007b). Moreover, with the exception of a few studies (Dietrich et al., 2007; Kane et al., 2005; Szumlinski et al., 2007b), the good majority of published in situ hybridization and immunological studies have examined for drug-induced changes in either the IEG or CC products of the Homer1 gene. To expand existing data indicating that repeated cocaine administration alters the expression of Homer1b/c and Homer3 in brain (Dietrich et al., 2007; Swanson et al., 2001), the present study employed immunoblotting to compare the effects of 3 weeks withdrawal from a sensitizing cocaine injection regimen upon the expression of Homer1b/c and Homer2a/b — the major CCHomer isoforms in forebrain (Shiraishi et al., 2004; Xiao et al., 1998). As CC-Homer proteins regulate the expression of Group1 mGluRs, as well as the NR2 subunits of the NMDA receptor in vivo (Szumlinski et al., 2004, 2005b), the cocaine-induced changes in CC-Homer expression were related to changes in the protein expression of mGluR1a, mGluR5, NR2a and NR2b. Our collection of data demonstrate that repeated cocaine administration elicits enduring, but regionally distinct, alterations in mGluR1a/Homer/ NR2 expression within the PFC, NAC shell and hippocampus and that the direction of the majority of these drug-induced alterations in protein expression are consistent across two mammalian species (Table 2).

3.1. Repeated cocaine elicits enduring, but regionally distinct, changes in CC-Homer expression Three weeks withdrawal from repeated cocaine administration produced enduring, but regionally distinct, changes in the protein expression of both Homer1b/c and Homer2a/b within several forebrain structures, including the PFC, NAC shell and hippocampus. Consistent with the results of an earlier immunoblotting study (Swanson et al., 2001), we failed to detect any significant effects of repeated cocaine treatment upon the protein expression of either Homer1b/c or Homer2a/b within the NAC core or within the more dorsal aspect of the striatum (Table 1). Also consistent with a reported decrease in Homer1b/c expression within the medial NAC (Swanson et al., 2001), we observed significant reductions in both Homer1b/c and Homer2a/b in our NAC shell samples and this effect was observed in both rats and mice (Fig. 2). The observation that CC-Homer2 isoforms are down-regulated in concert with CCHomer1 proteins within the NAC shell during cocaine withdrawal is consistent with the behavioral genetics data demonstrating an important role for both CC-Homer1 and CC-Homer2 isoforms in regulating the behavioral and NAC glutamate responses to cocaine (Szumlinski et al., 2006b, 2007a). For one, deletion of either the Homer1 or the Homer2 gene, but not the Homer3 gene, produces a “cocaine pre-sensitized” behavioral phenotype in mice and adeno-associated virus (AAV)-mediated transfection of the NAC shell with cDNA for Homer2b reverses genotypic differences in the cocaine responsiveness between WT and Homer2 KO mice (Szumlinski et al., 2004). Second, AAV-mediated transfection of the NAC with either Homer1c or Homer2b during cocaine withdrawal reverses the expression of cocaine-induced behavioral sensitization in rats (Szumlinski et al., 2007a). Third, an acute up-regulation of NAC Homer2a levels upon the local

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infusion of a TAT-Homer2a fusion protein also prevents a cocaine-sensitized behavioral response during withdrawal (Szumlinski et al., 2006a). Finally, in both the cases of the Homer2 KO mouse and the cocaine-sensitized rat, the effects of overexpressing Homer2b upon behavior were paralleled by a normalization of NAC glutamate content and a blockade in cocaine-induced glutamate sensitization (Szumlinski et al., 2004, 2007a). The present immunoblotting data confirm our earlier suggestion that cocaine down-regulates both CC-Homer1 and CC-Homer2 protein expression within the NAC shell (Szumlinski et al., 2004), lending further support to the hypothesis that a cocaine-induced reduction in both CC-Homer1 and CC-Homer2 protein expression within this region is an important neuroadaptation contributing to the enduring behavioral consequences of repeated cocaine exposure. Consistent with the earlier report by Swanson et al. (2001), PFC Homer1/c levels were unchanged at 3 weeks withdrawal from repeated cocaine treatment (Fig. 1). However, withdrawal from repeated cocaine administration up-regulated PFC Homer2a/b expression by 30–50% in both rats and mice (Fig. 1). In contrast to the NAC shell, much less is known regarding the role for PFC Homer expression in regulating cocaine-induced changes in behavior. The selective effect of repeated cocaine upon Homer2a/ b levels within the PFC is surprising in light of earlier behavioral genetic studies, which demonstrated that deletion of Homer1, but not of Homer2, produces abnormalities in PFC extracellular glutamate that include an elevation in PFC content and a reduction in cocaine-stimulated glutamate release within this region (Szumlinski et al., 2004, 2005a). The abnormalities in PFC glutamate produced by Homer1 deletion (particularly the reduction in stimulated release) have been postulated to play a role in their observed deficits in cognitive, attentional and sensorimotor processing (Jaubert et al., 2007; Lominac et al., 2005; Szumlinski et al., 2004, 2005a). However, the role of the abnormal PFC phenotype of Homer1 KO mice in regulating cocaine sensitivity is unclear, particularly when one considers the well-characterized hyper-activation of the PFC by cocaine-associated cues or cocaine imagery observed in abstinent cocaine addicts (e.g., Garavan et al., 2000; Goldstein et al., 2007; Kilts et al., 2004; Maas et al., 1998). This inconsistency, combined with the existing data indicating that withdrawal from repeated cocaine administration does not alter the PFC expression of Homer1b/c (Fig. 1; Swanson et al., 2001) argues against cocaine-induced changes in PFC Homer1 gene products in the frontal cortical abnormalities observed in cocaine addicts. While studies are on-going in our laboratory to assess the effects of Homer2 over-expression upon basal and cocainestimulated glutamate release within the PFC, PFC Homer1b/c over-expression in WT mice facilitates the capacity of cocaine to raise glutamate levels in this region (Lominac et al., 2005). If indeed CC-Homer2 over-expression augments the PFC glutamate-releasing effects of cocaine, then such data would support cocaine-induced increases in PFC Homer2a/b expression in the development of cocaine-induced frontal cortical abnormalities. This study is the first to report upon the effects of a drug of abuse upon Homer protein expression within the hippocampus, a brain region recently demonstrated to be critical for cocaine-seeking behavior in laboratory rodents (Fuchs et al., 2007; Rogers and See, 2007). As observed for the PFC, hippocampal levels of Homer2a/b were increased in both species, but in contrast to the PFC, the rise in Homer2a/b was

accompanied by a similar increase in Homer1b/c expression (Fig. 3). As no study to date has investigated the interactions between hippocampal Homer expression and cocaine or the in vivo regulation of hippocampal glutamate transmission by the various Homer proteins, it is difficult to comment upon the functional significance of the observed rise in hippocampal CC-Homer expression. However, in vitro studies conducted on hippocampal cultures indicate that CC-Homer1 or CC-Homer2 proteins are critical for: (1) regulating the morphology of dendritic spines (Sala et al., 2001, 2003); (2) the recruitment and/or clustering of synaptic proteins, including glutamate receptors, within the post-synaptic density (Kato et al., 1998; Shiraishi et al., 2003; Tadokoro et al., 1999); (3) intracellular signaling through Group1 mGluRs (Sala et al., 2005) and (4) normal whole-cell NMDA receptor current (Smothers et al., 2003, 2005). As a history of cocaine self-administration produces an enduring enhancement in hippocampal longterm potentiation that persists into withdrawal (del Olmo et al., 2006), it is tempting to speculate that cocaine-induced increases in hippocampal Homer1b/c and/or Homer2a/b expression may contribute to this form of cellular plasticity and perhaps facilitate the encoding or recall of drug-related contextual/episodic memory.

3.2. Repeated cocaine co-regulates Homer and glutamate receptor expression in forebrain Through interactions with their EVH1 domains, Homers bind directly to the mGluR1 and mGluR5 subtypes of Group1 mGluRs and indirectly, via a Shank-GKAP-PSD95 scaffold, to the NR2 subunit of the NMDA receptor (e.g., Brakeman et al., 1997; Kato et al., 1998; Naisbitt et al., 1999; Tu et al., 1998, 1999) and regulate glutamate receptor function (for reviews, Bockaert et al., 2004; de Bartolomeis and Iasevoli, 2003; Duncan et al., 2005; Fagni et al., 2004; Szumlinski et al., 2006b, 2007a; Xiao et al., 2000). While numerous studies have examined for drug-induced changes in Homer expression (particularly for that of the IEG Homer isoforms; see Szumlinski et al., 2007a for review), few studies have related changes in Homer expression to that of their glutamate receptor interacting partners (Swanson et al., 2001; Szumlinski et al., 2007b). As illustrated in Figs. 1–3, the observed changes in PFC, NAC shell and hippocampus CC-Homer expression were paralleled by changes in the levels of mGluR1a and either the NR2a and/or the NR2b subunit of the NMDA receptor. With a few exceptions (notably NR2a in the PFC and mGluR1a in the hippocampus), the magnitude of the mean changes in these glutamate receptor proteins was consistent across rats and mice, although due to variability in the mouse data, group differences were not always statistically significant (Table 2). Whether or not the species differences in cocaine's effects upon glutamate receptor expression reflect differences in the cocaine responsiveness of the mesocorticolimbic glutamate system between mouse and rat or rather, relate to the particular mouse and rat strains employed cannot be discerned from the present report. However, in support of the latter possibility, significant strain differences exist in the behavioral effects of cocaine in both rats (Glowa et al., 1994; Kosten et al., 1997) and mice (Orsini et al., 2005; Ruth et al., 1988; Wiener and Reith, 1990; Womer et al., 1994), with the majority of studies demonstrating lower cocaine behavioral responsiveness in the C57BL/6J strain.

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3.2.1. Co-regulation of Homers and mGluR1 during cocaine withdrawal In the present study, cocaine-induced changes in CC-Homer expression were coincident with alterations in the mGluR1a, but not the mGluR5, subtype of Group1 mGluR in all brain areas investigated. The coincident changes in CC-Homers and mGluR1a by cocaine are consistent with their co-regulation within the NAC of alcohol-withdrawn mice (Szumlinski et al., 2007b), as well as with the observation that Homer2 deletion reduces the total protein expression of mGluR1a, but not mGluR5, within the NAC (Szumlinski et al., 2004). However, our findings contrast with the only other comparable report in the literature (Swanson et al., 2001), which showed that 3 weeks withdrawal from repeated cocaine (Days 1 and 7: 15 mg/kg; Days 2–6: 30 mg/kg) produced a coincident decrease in Homer1b/c and mGluR5 levels within the medial NAC of male Sprague–Dawley rats, without affecting the expression of mGluR1a. It should be noted, however, that despite any observable effect of NAC mGluR1a expression, cocaine-treated rats exhibited reduced Group1 mGluR agonist-stimulated glutamate release within this region (Swanson et al., 2001). As the capacity of Group1 mGluR agonists to elicit a rise in NAC extracellular glutamate levels can be prevented by the local infusion of a selective mGluR1a antagonist (Swanson et al., 2001), these neurochemical results, coupled with the immunoblotting data collected in our laboratory (Szumlinski et al., 2007b; present study) support an important role for enduring changes in CC-Homer/mGluR1 signaling in the protracted effects of repeated drug exposure upon brain and behavior that may relate to changes in total protein expression. However, it should be noted that moderate changes in mGluR1a expression can be observed in the absence of any observable changes in Homer protein expression (as observed for the NAC core; see Table 1). This observation suggests that the co-regulation of mGluR1a and Homers by repeated cocaine is regionally selective and indicates that druginduced changes in mGluR1a expression may be independent of changes in total Homer protein expression within certain brain regions.

3.2.2. Regulation of Homers and NR2 during cocaine withdrawal The NR2 subunits of the NMDA receptor contain the glutamate binding site (e.g., Kendrick et al., 1996; Laurie and Seeburg, 1994) and regulate various aspects of NMDA receptor function, including sensitivity to cations and polyamines, as well as receptor agonists and antagonists, open probability, deactivation time, single-channel conductance and interactions with intracellular signaling molecules (e.g., Cull-Candy and Leszkiewicz, 2004; Dingledine et al., 1999; Sheng and Kim, 2002). Both NR2a and NR2b are enriched in forebrain (for reviews, Kohr, 2006; Lynch and Guttmann, 2001; Stephenson, 2001) and the Cterminus of both subunits interact with scaffolding constituents of the post-synaptic density, including Homers (e.g., Kornau et al., 1995; Muller et al., 1996; Naisbitt et al., 1999; Niethammer et al., 1998; Tu et al., 1999). While a considerable number of studies have examined for cocaine-induced changes in the obligatory NR1 subunit of the NMDA receptor, with the majority of reports indicating no significant effects on subunit expression or NMDA receptor binding (for reviews, Vanderschuren and Kalivas, 2000; Wang et al., 2006; Wolf, 1998), the number of

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studies examining for cocaine-induced changes in NR2 subunits is limited and the cocaine regimens, as well as the findings, vary considerably across studies (Hemby et al., 2005; Loftis and Janowsky, 2000; Tang et al., 2004; Yamaguchi et al., 2002; Zhang et al., 2007b). In this study, the effects of cocaine withdrawal upon NR2 expression were regionally selective and more robust in rats than mice (Table 2; see discussion above). With the exception of NR2a in the PFC of mice, the direction of the effect of cocaine withdrawal upon the expression of NR2a and/or NR2b was consistent with that observed for CC-Homers, although an increase in striatal levels of NR2a was observed in both species in the absence of any changes in CC-Homer expression (Table 1). In vivo, Homer2 gene products regulate the plasma membrane localization, but not the total protein expression of NR2 subunits (Szumlinski et al., 2004, 2005b). Consistent perhaps with a more important role for Homers in regulating NR2 trafficking, rather than the regulation of total protein expression, unpublished results from immunoblotting studies conducted in our laboratory have detected a significant effect of Homer1 deletion upon the total protein expression of NR2a within the striatum only and no effect of gene deletion upon NR2b expression in any brain region examined. Given the important roles for NR2 subunits in the biophysical and pharmacological properties of the NMDA receptor (for reviews, Kohr, 2006; Lynch and Guttmann, 2001; Stephenson, 2001), coupled with evidence indicating that Homer expression can influence the expression of NR2 subunits in brain (Szumlinski et al., 2005b, 2007b; present study), as well as NMDA receptor function (Smothers et al., 2003, 2005; Szumlinski et al., 2004, 2005a,b), further investigation is warranted to better understand the interactions between cocaine and the Homer– NMDA receptor complex.

3.3.

Conclusions

The results of the present study provide novel evidence that withdrawal from repeated cocaine administration produces enduring changes in the protein expression of both Homer2a/b and Homer1b/c within the NAC shell (down-regulation) and hippocampus (up-regulation), while cocaine withdrawal increases selectively Homer2a/b levels within the PFC. The effects of cocaine withdrawal upon CC-Homer protein expression were consistent across two mammalian species and coincided, for the most part, with changes in the protein expression of mGluR1a, NR2a and/or NR2b. As CC-Homers are critical regulators of cocaine-induced neural plasticity, these data support cocaine-induced changes in both Homer1b/c and Homer2a/b expression, and the consequent effects upon Group1 mGluR and NMDA receptor function, in the persistent abnormalities in mesocorticolimbic activity produced by repeated cocaine experience.

4.

Experimental procedures

4.1.

Subjects

Adult male Sprague–Dawley rats (weighing 200–225 g; n= 28) from Harlan (Indianapolis, IN) and adult male B6 mice (weighing 25–30 g; n =42) from Jackson Laboratories (Bar Harbor, ME) were used. All animals were pair-housed in polyethylene cages in a

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temperature (25 °C) and humidity (71%) controlled vivarium under a 12 h day/12 h light cycle (lights on: 0700 h). Experimental protocols, as well as housing and animal care, were consistent with the guidelines provided by the National Institute of Health (NIH) Guide for Care and Use of Laboratory Animals (NIH Publication NO. 80-23, revised 1996). Animals were randomly assigned to either repeated cocaine or repeated saline treatment groups. As conducted in an earlier study (Swanson et al., 2001), intraperitoneal (IP) injections of 30 mg/kg cocaine (Sigma-Aldrich, St. Louis, MO) were administered once a day for 7 consecutive days. Control animals received equivalent volumes of saline (rat= 1 ml/ kg; mouse= 0.01 ml/kg) for 7 consecutive days.

4.2.

Immunoblotting

Immunoblotting procedures were similar to those employed previously (Ary et al., 2007; Szumlinski et al., 2007a,b), originally adapted from those described in Shin et al. (2003) and Swanson et al. (2001). As conducted in an earlier study (Swanson et al., 2001), animals were decapitated 3 weeks following their last cocaine/ saline injection. The brain was sectioned along the coronal plane at the level of the PFC, NAC and striatum over an ice-cooled Plexiglas plate. The PFC (both dorsal and medial subregions combined) was dissected out using forceps, while the shell and core subregions of the NAC and the dorsal striatum were dissected using cooled micropunches. A coronal section removed the cerebellum, the brain was halved along the saggittal plane and the entire hippocampus removed using forceps. Samples were immediately frozen on dry ice and stored at −80 °C until homogenized. Samples were homogenized and protein determinations were performed as previously described in Ary et al. (2007) and Szumlinski et al. (2007b). For analysis, protein samples (30 μg for PFC, striatum and hippocampus; 10– 30 μg for NAC shell and core) were subjected to SDS-polyacrylamide gel electrophoresis. Bis–Tris gradient gels (4–12%) (Invitrogen, Carlsbad, CA) were used for separation of Homer1b/c and Homer2 proteins and Tris–Acetate gradient gels (3–8%) (Invitrogen, Carlsbad, CA) were used for separation of mGluR1a, mGluR5, NR2a, and NR2b proteins. Standard transfer and pre-blocking procedures were employed. The following rabbit polyclonal primary anti-bodies were used: anti-Homer1b/c and antiHomer2a/b (a generous gift from Dr. Paul F Worley, Johns Hopkins University School of Medicine; 1:1000 dilution), antimGluR1a and anti-mGluR5 (Upstate Cell Signaling Solutions, Lake Placid, NY; 1:1000 dilution), and anti-NR2a and anti-NR2b (Calbiochem, San Diego, CA; 1:1000 dilution). Membranes were washed, incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary anti-body (Upstate, Charlottesville, VA; 1:20,000–1:40,000 dilution) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence (ECL Plus; Amersham Biosciences, Inc., Piscataway, NJ). A rabbit anti-calnexin polyclonal primary antibody (Stressgen, Victoria, BC) was used to determine even protein transfer. For the PFC, striatum and hippocampus samples (30 μg protein loaded), statistical analysis of the density × area measurements for calnexin failed to detect sample differences (sample effect: p <0.05). This confirmed even protein loading and transfer, thus data standardization to the calnexin signal was unnecessary for these brain regions. As the total protein loaded for the NAC shell and core varied across samples (10–30 μg), data standardization

to the calnexin signal was performed for all NAC shell and core proteins. Levels of immunoreactivity were quantified using Image J (NIH, Bethesda, MD), normalized to the mean of the saline controls for each individual gel (n= 5–7/gel) (Ary et al., in press; Szumlinski et al., 2007a,b) and analyzed using Student's ttests (α= 0.05).

Acknowledgments The authors would like to thank Dr. Ilona Obara and Ms. Mimi Moras for their technical assistance and the laboratory of Dr. Paul F. Worley (The Johns Hopkins University School of Medicine) for their generous gift of the Homer1b/c and Homer2a/b primary antibodies. This project was supported by funds from the University of California at Santa Barbara to AWA and KKS and by a NARSAD Young Investigator's Award to KKS.

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