Ilona Obara

Binge Drinking Upregulates Accumbens mGluR5–Homer2–PI3K Signaling: Functional Implications for Alcoholism

The glutamate receptor-associated protein Homer2 regulates alcohol-induced neuroplasticity within the nucleus accumbens (NAC), but the precise intracellular signaling cascades involved are not known. This study examined the role for NAC metabotropic glutamate receptor (mGluR)–Homer2–phosphatidylinositol 3-kinase (PI3K) signaling in regulating excessive alcohol consumption within the context of the scheduled high alcohol consumption (SHAC) model of binge alcohol drinking. Repeated bouts of binge drinking (∼1.5 g/kg per 30 min) elevated NAC Homer2a/b expression and increased PI3K activity in this region. Virus-mediated knockdown of NAC Homer2b expression attenuated alcohol intake, as did an intra-NAC infusion of the mGluR5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride] (0.1–1 μg/side) and the PI3K antagonist wortmannin (50 ng/side), supporting necessary roles for mGluR5/Homer2/PI3K in binge alcohol drinking. Moreover, when compared with wild-type littermates, transgenic mice with an F1128R point mutation in mGluR5 that markedly reduces Homer binding exhibited a 50% reduction in binge alcohol drinking, which was related to reduced NAC basal PI3K activity. Consistent with the hypothesis that mGluR5–Homer–PI3K signaling may be a mechanism governing excessive alcohol intake, the “anti-binge” effects of MPEP and wortmannin were not additive, nor were they observed in the mGluR5F1128R transgenic mice. Finally, mice genetically selected for a high versus low SHAC phenotype differed in NAC mGluR, Homer2, and PI3K activity, consistent with the hypothesis that augmented NAC mGluR5–Homer2–PI3K signaling predisposes a high binge alcohol-drinking phenotype. Together, these data point to an important role for NAC mGluR5–Homer2–PI3K signaling in regulating binge-like alcohol consumption that has relevance for our understanding of the neurobiology of alcoholism and its pharmacotherapy.

Local peripheral effects of μ-opioid receptor agonists in neuropathic pain in rats

Our study was designed to demonstrate peripheral antinociception of the mu-opioid receptor agonists: morphine (MF), [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) in Bennetts rat model of neuropathic pain. All the agonists were effective in antagonizing allodynia after their intraplantar (i.pl.) but not subcutaneous (s.c.) administration. Opioid peptides: DAMGO, EM-1 and EM-2 were more effective compared with corresponding doses of morphine (opioid alkaloid) in alleviating chronic pain. Peripheral mu-opioid receptors mediated the observed effects, as was evidenced by the i.pl. treatment with naloxone methiodide (active only at the site of injection) and by cyprodime, a selective mu-opioid receptor antagonist. These results have shown that opioid peptides are effective also after local treatment, and that their peripheral use may be of therapeutic interest in long-term management of chronic pain.

Extended daily access to cocaine results in distinct alterations in Homer 1b/c and NMDA receptor subunit expression within the medial prefrontal cortex

Human cocaine addicts show altered function within the basal ganglia and the medial prefrontal cortex (mPFC) and altered glutamate function within these areas is postulated to be critical for cocaine addiction. The present project utilized a highly valid animal model of cocaine addiction, to test whether excessive use of cocaine alters glutamate function within these brain areas. Rats were trained to lever‐press for i.v. saline vehicle or cocaine (0.25 mg/infusion) over seven 1‐h daily sessions, after which, saline controls and half of cocaine self‐administering animals (brief access condition) received 10 more 1‐h daily sessions, whereas the remaining cocaine animals received 10 additional 6‐h daily sessions (extended access condition). One, 14, or 60 days after the last self‐administration session, animals were sacrificed. Tissue samples from the ventral tegmental area (VTA), nucleus accumbens (N.Acc) core and shell, and mPFC were analyzed by immunoblotting for expression of Homer1b/c, Homer2a/b, mGluR1, mGluR5, NR2a, and NR2b subunits of the NMDA receptor. Brief and extended access to cocaine failed to alter protein levels within the VTA, and produced transient and similar changes in N.Acc protein expression, which were more pronounced in the core subregion. In contrast, extended access to cocaine resulted in distinct and long lasting alterations of protein expression within the mPFC that included: increased levels of Homer1b/c at 1 day, NR2b at 14 days, and NR2a at 60 days, of withdrawal. These data support the notion that altered NMDA function within the mPFC may contribute, in part, to the transition to excessive uncontrolled consumption of cocaine. Synapse 63:598–609, 2009.

Differential Effects of Chronic Ethanol Consumption and Withdrawal on Homer/Glutamate Receptor Expression in Subregions of the Accumbens and Amygdala of P Rats

BACKGROUND Homer proteins are constituents of scaffolding complexes that regulate the trafficking and function of central Group1 metabotropic glutamate receptors (mGluRs) and N-methyl-d-aspartate (NMDA) receptors. Research supports the involvement of these proteins in ethanol-induced neuroplasticity in mouse. In this study, we examined the effects of short versus long-term withdrawal from chronic ethanol consumption on Homer and glutamate receptor protein expression within striatal and amygdala subregions of selectively bred, alcohol-preferring P rats. METHODS For 6 months, male P rats had concurrent access to 15% and 30% ethanol solutions under intermittent (IA: 4 d/wk) or continuous (CA: 7 d/wk) access conditions in their home cage. Rats were killed 24 hours (short withdrawal: SW) or 4 weeks (long withdrawal: LW) after termination of ethanol access, subregions of interest were micropunched and tissue processed for detection of Group1 mGluRs, NR2 subunits of the NMDA receptor and Homer protein expression. RESULTS Within the nucleus accumbens (NAC), limited changes in NR2a and NR2b expression were detected in the shell (NACsh), whereas substantial changes were observed for Homer2a/b, mGluRs as well as NR2a and NR2b subunits in the core (NACc). Within the amygdala, no changes were detected in the basolateral subregion, whereas substantial changes, many paralleling those observed in the NACc, were detected in the central nucleus (CeA) subregion. In addition, most of the changes observed in the CeA, but not NACc, were present in both SW and LW rats. CONCLUSIONS Overall, these subregion specific, ethanol-induced increases in mGluR/Homer2/NR2 expression within the NAC and amygdala suggest changes in glutamatergic plasticity had taken place. This may be a result of learning and subsequent memory formation of ethanols rewarding effects in these brain structures, which may, in part, mediate the chronic relapsing nature of alcohol abuse.

Spinal and local peripheral antiallodynic activity of Ro64-6198 in neuropathic pain in the rat

&NA; The nociceptin system seems to be involved in modulation of acute nociceptive stimulation and in chronic pain processes, e.g. inflammation and neuropathy. In the present study, we examined the analgesic effect of a new opioid receptor‐like (ORL1) receptor agonist, Ro64‐6198, and compared it with the effect of endogenous ORL1 receptor agonist, nociceptin/orphanin FQ (N/OFQ), in a model of neuropathic pain in the rat. Ro64‐6198 was injected intrathecaly (i.t.), intraplantarly (i.pl.) and subcutaneously (s.c.), and responses of neuropathic rats were measured in tactile (von Frey) and thermal (cold water) allodynia tests. Ro64‐6198 did not change the pain threshold in naive animals, but exhibited antiallodynic activity in neuropathic rats. This effect was observed after i.t. and i.pl. but not after s.c. administration. Moreover, the observed antiallodynic potency of Ro64‐6198 was weaker in comparison with N/OFQ after i.t. administration of either agonist, but almost equal after i.pl. injection. Selective antagonists of the ORL1 receptor, [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 (PheΨ) and [N‐Phe1]‐NC(1‐13)NH2 (NPhe), inhibited the antiallodynic actions of Ro64‐6198 which indicated that the spinal and peripheral antinociceptive effects were mediated by ORL1 receptors. Therefore, besides spinal, also peripheral ORL1 receptors may be targeted by drugs designed for the long‐term treatment of chronic pain.

Peripheral versus central antinociceptive actions of 6-amino acid-substituted derivatives of 14-O-methyloxymorphone in acute and inflammatory pain in the rat.

Opioid analgesics with restricted access to the central nervous system represent a new approach to the treatment of severe pain with an improved safety profile. The objective of this study was to investigate the peripheral and central components of the antinociceptive actions of the 6-amino acid conjugates (glycine, alanine, and phenylalanine) of 14-O-methyloxymorphone. Their antinociceptive activities were compared with those of the centrally penetrating μ-opioid agonists morphine, fentanyl, and 14-O-methyloxymorphone. In the tail-flick test in rats, the 6-amino acid conjugates were 45- to 1170-fold more potent than morphine after i.c.v. administration and 19- to 209-fold after s.c. administration. They showed potencies similar to fentanyl after s.c. administration and were more potent after i.c.v. application. The time course of action was different between s.c. and i.c.v. administration, with significant long-lasting effects after i.c.v. administration. Systemic administration of the peripherally selective opioid antagonist naloxone methiodide antagonized the effects after s.c. but not after i.c.v. administration in the tail-flick test. Subcutaneous 6-amino acid derivatives also elicited antihyperalgesic effects in the formalin test in rats, which were reversed by systemically administered naloxone methiodide. Although morphine exerts its analgesic effects by central and peripheral mechanisms, the investigated new opioids interact primarily with peripheral opioid receptors after s.c. administration. The present data indicate that the 6-amino acid conjugates of 14-O-methyloxymorphone have limited access to the central nervous system and can mediate antinociception at peripheral sites. Also, they might find clinical application when the central actions of opioids are unwanted.

Antagonists of the κ‐opioid receptor enhance allodynia in rats and mice after sciatic nerve ligation

The administration of κ‐opioid receptor antagonists, nor‐binaltorphimine (norBNI) and 5′‐guanidinonaltrindole (GNTI) enhanced allodynia in rats and mice after sciatic nerve ligation. In order to understand the mechanism underlying this effect, we examined the possible involvement of the endogenous ligand of κ‐opioid receptor dynorphin. The experiments were carried out on male Wistar rats and on Albino‐Swiss mice. The rats had been implanted with a catheter 7 days earlier in the subarachnoid space of the spinal cord. Intrathecal (i.t.) administrations in mice were made by lumbar puncture. The animals were i.t. injected with norBNI, GNTI (κ‐opioid receptor antagonists), dynorphin A1–17 antiserum (DYN A/S), ketamine (NMDA receptor antagonist) and their combinations. The nociceptive sensitivity was assessed using the mechanical (von Frey) and therma allodynia tests on days 2–4 and 8–10 after the sciatic nerve ligation. Both antagonists, norBNI and GNTI, significantly enhanced mechanical and therma allodynia in rats and mice with neuropathic pain. The potentiation of allodynia after the administration of norBNI or GNTI was inhibited by earlier administration of DYN A/S or by ketamine. Our results suggest that allodynia is mediated through nonopioid effect of the endogenous opioid peptide, dynorphin. The nonopioid action is potentiated by the blockade of κ‐opioid receptors, and corresponding to the elevation of prodynorphin mRNA level in neuropathic pain. Furthermore, the potentiation of allodynia after the administration of the above drugs appears to be mediated through the activation of NMDA receptors directly by dynorphin.

The role of nociceptin and dynorphin in chronic pain: implications of neuro-glial interaction.

Nociceptin-opioid peptide (NOP) receptor, also known as opioid receptor like-1 (ORL1), was identified following the cloning of the kappa-opioid peptide (KOP) receptor, and the characterization of these receptors revealed high homology. The endogenous ligand of NOP, nociceptin (NOC), which shares high homology to dynorphin (DYN), was discovered shortly thereafter, and since then, it has been the subject of several investigations. Despite the many advances in our understanding of the involvement of NOC and DYN systems in pain, tolerance and withdrawal, the precise function of these systems has not been fully characterized. Here, we review the recent literature concerning the distribution of the NOC and DYN systems in the central nervous system and the involvement of these systems in nociceptive transmission, especially under chronic pain conditions. We discuss the use of endogenous and exogenous ligands of NOP and KOP receptors in pain perception, as well as the potential utility of NOP ligands in clinical practice for pain management. We also discuss the modulation of opioid effects by NOC and DYN. We emphasize the important role of neuro-glial interactions in the effects of NOC and DYN, focusing on their presence in neuronal and non-neuronal cells and the changes associated with chronic pain conditions. We also present the dynamics of immune and glial regulation of neuronal functions and the importance of this regulation in the roles of NOC and DYN under conditions of neuropathic pain and in the use of drugs that alter these systems for better control of neuropathic pain.

Accumbens Homer2-mediated signaling: a factor contributing to mouse strain differences in alcohol drinking?

Alcohol‐induced increases in nucleus accumbens glutamate actively regulate alcohol consumption, and the alcohol responsiveness of corticoaccumbens glutamate systems relates to genetic variance in alcohol reward. Here, we extend earlier data for inbred mouse strain differences in accumbens glutamate by examining for differences in basal and alcohol‐induced changes in the striatal expression of glutamate‐related signaling molecules between inbred C57BL/6J and DBA2/J mice. Repeated alcohol treatment (8 × 2 g/kg) increased the expression of Group1 metabotropic glutamate receptors, the NR2a/b subunits of the N‐methyl‐d‐aspartate receptor, Homer2a/b, as well as the activated forms of protein kinase C (PKC) epsilon and phosphoinositol‐3‐kinase within ventral, but not dorsal, striatum. Regardless of prior alcohol experience, C57BL/6J mice exhibited higher accumbens levels of mGluR1/5, Homer2a/b, NR2a and activated kinases vs. DBA2/J mice, whereas an alcohol‐induced rise in dorsal striatum mGluR1/5 expression was observed only in C57BL/6J mice. We next employed virus‐mediated gene transfer approaches to ascertain the functional relevance of the observed strain difference in accumbens Homer2 expression for B6/D2 differences in alcohol‐induced glutamate sensitization, as well as alcohol preference/intake. Manipulating nucleus accumbens shell Homer2b expression actively regulated these measures in C57BL/6J mice, whereas DBA2/J mice were relatively insensitive to the neurochemical and behavioral effects of virus‐mediated changes in Homer2 expression. These data support the over‐arching hypothesis that augmented accumbens Homer2‐mediated glutamate signaling may be an endophenotype related to genetic variance in alcohol consumption. If relevant to humans, such data pose polymorphisms affecting glutamate receptor/Homer2 signaling in the etiology of alcoholism.

Morphine and endomorphin-1 differently influence pronociceptin/orphanin FQ system in neuropathic rats.

In the present study, we investigated the influence of intrathecal (i.t.) administration of morphine and endomorphin-1 on the level of pronociceptin/orphanin FQ and opioid receptor-like 1 (ORL1) receptor mRNAs in the lumbar part of the spinal cord in the rat model of neuropathic pain. The ligation of the sciatic nerve did not change the levels of pronociceptin/orphanin FQ and ORL1 receptor mRNAs in laminae I-VI of the dorsal horn when measured by in situ hybridisation 2 and 7 days after the nerve injury, but ORL1 receptor mRNA level in the ventral horn was significantly increased. Two micro-opioid receptor agonists, morphine and endomorphin-1, whose effectiveness in neuropathic pain is different, also disparately influenced nociceptin/orphanin FQ system in this pain model, inasmuch as an increase in pronociceptin/orphanin FQ and ORL1 receptor mRNAs was observed in laminae I-VI after morphine administration (5 microg i.t.) but not after endomorphin-1 treatment (5 microg i.t.). Moreover, the injection of ORL1 receptor antagonists (PhePsi; 30 microg i.t.) before morphine potentiated the effect of morphine in neuropathic pain model. Therefore, the activation of the endogenous nociceptin/orphanin FQ system, which is known to exhibit antiopioidergic activity, apart from its analgesic action, could be the reason for lower responsiveness to morphine in neuropathic pain.