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Homers at the Interface between Reward and Pain.

Obara I, Goulding SP, Gould AT, Lominac KD, Hu JH, Zhang PW, von Jonquieres G, Dehoff M, Xiao B, Seeburg PH, Worley PF, Klugmann M, Szumlinski KK - Front Psychiatry (2013)

Bottom Line: Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs.However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI's effects) were sufficient to blunt heroin CPP in uninjured mice.However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect (intrathecal) or attenuated (NAC) heroin CPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, Neuroscience Research Institute, University of California at Santa Barbara Santa Barbara, CA, USA ; School of Medicine, Pharmacy and Health, Queen's Campus, University of Durham Stockton on Tees, UK.

ABSTRACT
Pain alters opioid reinforcement, presumably via neuroadaptations within ascending pain pathways interacting with the limbic system. Nerve injury increases expression of glutamate receptors and their associated Homer scaffolding proteins throughout the pain processing pathway. Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs. Thus, we investigated a potential role for Homers in the interactions between pain and drug reward in mice. Chronic constriction injury (CCI) of the sciatic nerve elevated Homer1b/c and/or Homer2a/b expression within all mesolimbic structures examined and for the most part, the Homer increases coincided with elevated mGluR5, GluN2A/B, and the activational state of various down-stream kinases. Behaviorally, CCI mice showed pain hypersensitivity and a conditioned place-aversion (CPA) at a low heroin dose that supported conditioned place-preference (CPP) in naïve controls. Null mutations of Homer1a, Homer1, and Homer2, as well as transgenic disruption of mGluR5-Homer interactions, either attenuated or completely blocked low-dose heroin CPP, and none of the CCI mutant strains exhibited heroin-induced CPA. However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI's effects) were sufficient to blunt heroin CPP in uninjured mice. However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect (intrathecal) or attenuated (NAC) heroin CPA. Together, these data implicate increases in glutamate receptor/Homer/kinase activity within limbic structures, perhaps outside the NAC, as possibly critical for switching the incentive motivational properties of heroin following nerve injury, which has relevance for opioid psychopharmacology in individuals suffering from neuropathic pain.

No MeSH data available.


Related in: MedlinePlus

Homer1c in the NAC bi-directionally influences neuropathic pain symptoms and heroin CPA. (A) Half coronal section of the mouse brain at the level of the NAC targeted in the AAV infusion studies. (A′) Micrograph (4×) of GFP staining within the NAC shell produced by the shRNA-Homer1c construct [see box in (A) for orientation]. (A″) Micrograph (20×) of immunostaining for the HA-tagged cDNA-Homer1c construct within NAC shell illustrating both cell body and process staining in the tissue surrounding the microinjector tip (bracket). At 3 weeks following intra-NAC infusion, mice were subjected to CCI procedures, followed by behavioral testing. (B) Relative to GFP vector controls (GFP), altering NAC Homer1c expression bi-directionally altered both mechanical hypersensitivity assessed in the von Frey test [AAV × Time ANOVA: F(2,130) = 150.6, p < 0.0001] and cold hypersensitivity assessed in the acetone test [AAV × Time ANOVA: F(2,130) = 93.27, p < 0.0001]. *p < 0.05 vs. GFP. (C) No changes in the tail-flick test were observed following intra-NAC AAV infusion (one-way ANOVA, p = 0.38). (D) An AAV × CCI interaction was observed for heroin-induced place-conditioning [F(2,52) = 3.79, p = 0.03]. In GFP controls, cDNA-Homer1c over-expression prevented heroin CPP, while shRNA-Homer1c was without effect [F(2,28) = 3.36, p = 0.04; Tukey’s post hoc tests]. T-tests confirmed the presence of a significant CPP in GFP controls [t(9) = 4.20, p = 0.002] and shRNA-infused control animals [t(9) = 3.49, p = 0.007]. In contrast, both Homer1c manipulations attenuated heroin CPA in CCI mice [F(2,24) = 7.37, p = 0.003; Tukey’s post hoc tests]. T-tests confirmed a significant CPA in scrambled controls [t(9) = 3.49, p = 0.007], but no conditioning in Homer1c-manipulated animals (t-tests, p > 0.05). *p < 0.05 Paired vs. unpaired (conditioning; t-tests); #p < 0.05 vs. scrambled AAV. The data represent the mean ± SEM of 8–11 mice/AAV/condition.
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Figure 5: Homer1c in the NAC bi-directionally influences neuropathic pain symptoms and heroin CPA. (A) Half coronal section of the mouse brain at the level of the NAC targeted in the AAV infusion studies. (A′) Micrograph (4×) of GFP staining within the NAC shell produced by the shRNA-Homer1c construct [see box in (A) for orientation]. (A″) Micrograph (20×) of immunostaining for the HA-tagged cDNA-Homer1c construct within NAC shell illustrating both cell body and process staining in the tissue surrounding the microinjector tip (bracket). At 3 weeks following intra-NAC infusion, mice were subjected to CCI procedures, followed by behavioral testing. (B) Relative to GFP vector controls (GFP), altering NAC Homer1c expression bi-directionally altered both mechanical hypersensitivity assessed in the von Frey test [AAV × Time ANOVA: F(2,130) = 150.6, p < 0.0001] and cold hypersensitivity assessed in the acetone test [AAV × Time ANOVA: F(2,130) = 93.27, p < 0.0001]. *p < 0.05 vs. GFP. (C) No changes in the tail-flick test were observed following intra-NAC AAV infusion (one-way ANOVA, p = 0.38). (D) An AAV × CCI interaction was observed for heroin-induced place-conditioning [F(2,52) = 3.79, p = 0.03]. In GFP controls, cDNA-Homer1c over-expression prevented heroin CPP, while shRNA-Homer1c was without effect [F(2,28) = 3.36, p = 0.04; Tukey’s post hoc tests]. T-tests confirmed the presence of a significant CPP in GFP controls [t(9) = 4.20, p = 0.002] and shRNA-infused control animals [t(9) = 3.49, p = 0.007]. In contrast, both Homer1c manipulations attenuated heroin CPA in CCI mice [F(2,24) = 7.37, p = 0.003; Tukey’s post hoc tests]. T-tests confirmed a significant CPA in scrambled controls [t(9) = 3.49, p = 0.007], but no conditioning in Homer1c-manipulated animals (t-tests, p > 0.05). *p < 0.05 Paired vs. unpaired (conditioning; t-tests); #p < 0.05 vs. scrambled AAV. The data represent the mean ± SEM of 8–11 mice/AAV/condition.

Mentions: The procedure for generating neurotropic chimeric AAV1/2 vectors carrying the renilla green fluorescent protein (hrGFP) cDNA or the hemagglutinin (HA) tag fused to the coding region of rat Homer1c, and Homer2b have been described in detail elsewhere (e.g., Klugmann et al., 2005) and the AAV-cDNA constructs were identical to those employed previously (e.g., Klugmann et al., 2005; Tappe et al., 2006; Cozzoli et al., 2009; Goulding et al., 2011; Ary et al., 2013). The design of the AAV constructs for expression of small hairpin RNAs (shRNA) against Homer1c were described in detail in Klugmann and Szumlinski (2008). Briefly, we used a bicistronic expression cassette entailing the human U6 promoter to drive the shRNA, followed by the hrGFP reporter under the control of the chicken-beta actin (CBA) promoter for identification of transduced neurons. The shRNA-Homer1c construct was the same as that used in a recently published report, in which we demonstrated approximately 50% protein knock-down within the brain at 3 weeks post-infusion (Ary et al., 2013). AAV-shEGFP-CBA-hrGFP was used as a generic control (GFP) in our AAV studies. The surgical procedures for intra-NAC AAV infusion (0.5 μl/side) were identical to those used in previous studies (e.g., Cozzoli et al., 2009) and resulted in placement of microinjectors within the boundaries of the NAC (see Figure 5A). Studies examining behavioral response in heroin-induced place-preference test after intrathecal AAV infusion employed mice whose neuropathic pain symptoms and AAV transduction patterns within spinal cord were described before (Obara et al., 2013). Following either intracranial or intrathecal infusion, animals were left undisturbed for 3 weeks when AAV-mediated transgene expression peaks to remain at maximally stable levels prior to behavioral testing (e.g., Klugmann et al., 2005; Klugmann and Szumlinski, 2008). Sample sizes employed in the statistical analyses of the data ranged from 8 to 11 mice/group/AAV for both the NAC and spinal cord study.


Homers at the Interface between Reward and Pain.

Obara I, Goulding SP, Gould AT, Lominac KD, Hu JH, Zhang PW, von Jonquieres G, Dehoff M, Xiao B, Seeburg PH, Worley PF, Klugmann M, Szumlinski KK - Front Psychiatry (2013)

Homer1c in the NAC bi-directionally influences neuropathic pain symptoms and heroin CPA. (A) Half coronal section of the mouse brain at the level of the NAC targeted in the AAV infusion studies. (A′) Micrograph (4×) of GFP staining within the NAC shell produced by the shRNA-Homer1c construct [see box in (A) for orientation]. (A″) Micrograph (20×) of immunostaining for the HA-tagged cDNA-Homer1c construct within NAC shell illustrating both cell body and process staining in the tissue surrounding the microinjector tip (bracket). At 3 weeks following intra-NAC infusion, mice were subjected to CCI procedures, followed by behavioral testing. (B) Relative to GFP vector controls (GFP), altering NAC Homer1c expression bi-directionally altered both mechanical hypersensitivity assessed in the von Frey test [AAV × Time ANOVA: F(2,130) = 150.6, p < 0.0001] and cold hypersensitivity assessed in the acetone test [AAV × Time ANOVA: F(2,130) = 93.27, p < 0.0001]. *p < 0.05 vs. GFP. (C) No changes in the tail-flick test were observed following intra-NAC AAV infusion (one-way ANOVA, p = 0.38). (D) An AAV × CCI interaction was observed for heroin-induced place-conditioning [F(2,52) = 3.79, p = 0.03]. In GFP controls, cDNA-Homer1c over-expression prevented heroin CPP, while shRNA-Homer1c was without effect [F(2,28) = 3.36, p = 0.04; Tukey’s post hoc tests]. T-tests confirmed the presence of a significant CPP in GFP controls [t(9) = 4.20, p = 0.002] and shRNA-infused control animals [t(9) = 3.49, p = 0.007]. In contrast, both Homer1c manipulations attenuated heroin CPA in CCI mice [F(2,24) = 7.37, p = 0.003; Tukey’s post hoc tests]. T-tests confirmed a significant CPA in scrambled controls [t(9) = 3.49, p = 0.007], but no conditioning in Homer1c-manipulated animals (t-tests, p > 0.05). *p < 0.05 Paired vs. unpaired (conditioning; t-tests); #p < 0.05 vs. scrambled AAV. The data represent the mean ± SEM of 8–11 mice/AAV/condition.
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Related In: Results  -  Collection

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Figure 5: Homer1c in the NAC bi-directionally influences neuropathic pain symptoms and heroin CPA. (A) Half coronal section of the mouse brain at the level of the NAC targeted in the AAV infusion studies. (A′) Micrograph (4×) of GFP staining within the NAC shell produced by the shRNA-Homer1c construct [see box in (A) for orientation]. (A″) Micrograph (20×) of immunostaining for the HA-tagged cDNA-Homer1c construct within NAC shell illustrating both cell body and process staining in the tissue surrounding the microinjector tip (bracket). At 3 weeks following intra-NAC infusion, mice were subjected to CCI procedures, followed by behavioral testing. (B) Relative to GFP vector controls (GFP), altering NAC Homer1c expression bi-directionally altered both mechanical hypersensitivity assessed in the von Frey test [AAV × Time ANOVA: F(2,130) = 150.6, p < 0.0001] and cold hypersensitivity assessed in the acetone test [AAV × Time ANOVA: F(2,130) = 93.27, p < 0.0001]. *p < 0.05 vs. GFP. (C) No changes in the tail-flick test were observed following intra-NAC AAV infusion (one-way ANOVA, p = 0.38). (D) An AAV × CCI interaction was observed for heroin-induced place-conditioning [F(2,52) = 3.79, p = 0.03]. In GFP controls, cDNA-Homer1c over-expression prevented heroin CPP, while shRNA-Homer1c was without effect [F(2,28) = 3.36, p = 0.04; Tukey’s post hoc tests]. T-tests confirmed the presence of a significant CPP in GFP controls [t(9) = 4.20, p = 0.002] and shRNA-infused control animals [t(9) = 3.49, p = 0.007]. In contrast, both Homer1c manipulations attenuated heroin CPA in CCI mice [F(2,24) = 7.37, p = 0.003; Tukey’s post hoc tests]. T-tests confirmed a significant CPA in scrambled controls [t(9) = 3.49, p = 0.007], but no conditioning in Homer1c-manipulated animals (t-tests, p > 0.05). *p < 0.05 Paired vs. unpaired (conditioning; t-tests); #p < 0.05 vs. scrambled AAV. The data represent the mean ± SEM of 8–11 mice/AAV/condition.
Mentions: The procedure for generating neurotropic chimeric AAV1/2 vectors carrying the renilla green fluorescent protein (hrGFP) cDNA or the hemagglutinin (HA) tag fused to the coding region of rat Homer1c, and Homer2b have been described in detail elsewhere (e.g., Klugmann et al., 2005) and the AAV-cDNA constructs were identical to those employed previously (e.g., Klugmann et al., 2005; Tappe et al., 2006; Cozzoli et al., 2009; Goulding et al., 2011; Ary et al., 2013). The design of the AAV constructs for expression of small hairpin RNAs (shRNA) against Homer1c were described in detail in Klugmann and Szumlinski (2008). Briefly, we used a bicistronic expression cassette entailing the human U6 promoter to drive the shRNA, followed by the hrGFP reporter under the control of the chicken-beta actin (CBA) promoter for identification of transduced neurons. The shRNA-Homer1c construct was the same as that used in a recently published report, in which we demonstrated approximately 50% protein knock-down within the brain at 3 weeks post-infusion (Ary et al., 2013). AAV-shEGFP-CBA-hrGFP was used as a generic control (GFP) in our AAV studies. The surgical procedures for intra-NAC AAV infusion (0.5 μl/side) were identical to those used in previous studies (e.g., Cozzoli et al., 2009) and resulted in placement of microinjectors within the boundaries of the NAC (see Figure 5A). Studies examining behavioral response in heroin-induced place-preference test after intrathecal AAV infusion employed mice whose neuropathic pain symptoms and AAV transduction patterns within spinal cord were described before (Obara et al., 2013). Following either intracranial or intrathecal infusion, animals were left undisturbed for 3 weeks when AAV-mediated transgene expression peaks to remain at maximally stable levels prior to behavioral testing (e.g., Klugmann et al., 2005; Klugmann and Szumlinski, 2008). Sample sizes employed in the statistical analyses of the data ranged from 8 to 11 mice/group/AAV for both the NAC and spinal cord study.

Bottom Line: Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs.However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI's effects) were sufficient to blunt heroin CPP in uninjured mice.However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect (intrathecal) or attenuated (NAC) heroin CPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, Neuroscience Research Institute, University of California at Santa Barbara Santa Barbara, CA, USA ; School of Medicine, Pharmacy and Health, Queen's Campus, University of Durham Stockton on Tees, UK.

ABSTRACT
Pain alters opioid reinforcement, presumably via neuroadaptations within ascending pain pathways interacting with the limbic system. Nerve injury increases expression of glutamate receptors and their associated Homer scaffolding proteins throughout the pain processing pathway. Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs. Thus, we investigated a potential role for Homers in the interactions between pain and drug reward in mice. Chronic constriction injury (CCI) of the sciatic nerve elevated Homer1b/c and/or Homer2a/b expression within all mesolimbic structures examined and for the most part, the Homer increases coincided with elevated mGluR5, GluN2A/B, and the activational state of various down-stream kinases. Behaviorally, CCI mice showed pain hypersensitivity and a conditioned place-aversion (CPA) at a low heroin dose that supported conditioned place-preference (CPP) in naïve controls. Null mutations of Homer1a, Homer1, and Homer2, as well as transgenic disruption of mGluR5-Homer interactions, either attenuated or completely blocked low-dose heroin CPP, and none of the CCI mutant strains exhibited heroin-induced CPA. However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI's effects) were sufficient to blunt heroin CPP in uninjured mice. However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect (intrathecal) or attenuated (NAC) heroin CPA. Together, these data implicate increases in glutamate receptor/Homer/kinase activity within limbic structures, perhaps outside the NAC, as possibly critical for switching the incentive motivational properties of heroin following nerve injury, which has relevance for opioid psychopharmacology in individuals suffering from neuropathic pain.

No MeSH data available.


Related in: MedlinePlus