Behavioral sensitization induced by methamphetamine causes differential alterations in gene expression and histone acetylation of the prefrontal cortex in rats.

BACKGROUND: Methamphetamine (METH) is one of the most widely abused illicit substances worldwide; unfortunately, its addiction mechanism remains unclear. Based on accumulating evidence, changes in gene expression and chromatin modifications might be related to the persistent effects of METH on the brain. In the present study, we took advantage of METH-induced behavioral sensitization as an animal model that reflects some aspects of drug addiction and examined the changes in gene expression and histone acetylation in the prefrontal cortex (PFC) of adult rats. METHODS: We conducted mRNA microarray and chromatin immunoprecipitation (ChIP) coupled to DNA microarray (ChIP-chip) analyses to screen and identify changes in transcript levels and histone acetylation patterns. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were performed to analyze the differentially expressed genes. We then further identified alterations in ANP32A (acidic leucine-rich nuclear phosphoprotein-32A) and POU3F2 (POU domain, class 3, transcription factor 2) using qPCR and ChIP-PCR assays. RESULTS: In the rat model of METH-induced behavioral sensitization, METH challenge caused 275 differentially expressed genes and a number of hyperacetylated genes (821 genes with H3 acetylation and 10 genes with H4 acetylation). Based on mRNA microarray and GO and KEGG enrichment analyses, 24 genes may be involved in METH-induced behavioral sensitization, and 7 genes were confirmed using qPCR. We further examined the alterations in the levels of the ANP32A and POU3F2 transcripts and histone acetylation at different periods of METH-induced behavioral sensitization. H4 hyperacetylation contributed to the increased levels of ANP32A mRNA and H3/H4 hyperacetylation contributed to the increased levels of POU3F2 mRNA induced by METH challenge-induced behavioral sensitization, but not by acute METH exposure. CONCLUSIONS: The present results revealed alterations in transcription and histone acetylation in the rat PFC by METH exposure and provided evidence that modifications of histone acetylation contributed to the alterations in gene expression caused by METH-induced behavioral sensitization.

The opioid receptor triple agonist DPI-125 produces analgesia with less respiratory depression and reduced abuse liability.

Opioid analgesics remain the first choice for the treatment of moderate to severe pain, but they are also notorious for their respiratory depression and addictive effects. This study focused on the pharmacology of a novel opioid receptor mixed agonist DPI-125 and attempted to elucidate the relationship between the δ-, µ- and κ-receptor potency ratio and respiratory depression and abuse liability. Five diarylmethylpiperazine compounds (DPI-125, DPI-3290, DPI-130, KUST202 and KUST13T02) were selected for this study. PKA fluorescence redistribution assays in CHO cells individually expressing δ-, µ- or κ-receptors were used to measure the agonist potency. The respiratory safety profiles were estimated in rats by the ratio of ED50 (pCO2 increase)/ED50 (antinociception). The abuse liability of DPI-125 was evaluated with a self-administration model in rhesus monkeys. The observed agonist potencies of DPI-125 for δ-, µ- and κ-opioid receptors were 4.29±0.36, 11.10±3.04, and 16.57±4.14 nmol/L, respectively. The other four compounds were also mixed agonists with varying potencies. DPI-125 exhibited a high respiratory safety profile, clearly related to its high δ-receptor potency. The ratio of the EC50 potencies for the µ- and δ-receptors was found to be positively correlated with the respiratory safety ratio. DPI-125 has similar potencies for µ- and κ-receptors, which is likely the reason for its reduced abuse potential. Our results demonstrate that the opioid receptor mixed agonist DPI-125 is safer and less addictive than traditional µ-agonist analgesics. These findings suggest that the development of δ>µâˆ¼κ opioid receptor mixed agonists is feasible, and such compounds could represent a promising class of potent analgesics with wider therapeutic windows.

A selective D3 receptor antagonist YQA14 attenuates methamphetamine-induced behavioral sensitization and conditioned place preference in mice.

AIM: We have reported that a selective dopamine D3 receptor antagonist YQA14 attenuates cocaine reward and relapse to drug-seeking in mice. In the present study, we investigated whether YQA14 could inhibit methamphetamine (METH)-induced locomotor sensitization and conditioned place preference (CPP) in mice. METHODS: Locomotor activity was monitored in mice treated with METH (1 mg/kg, ip) daily on d 4-13, followed by a challenge with METH (0.5 mg/kg) on d 21. CPP was examined in mice that were administered METH (1 mg/kg) or saline alternately on each other day for 8 days (METH conditioning). YQA14 was injected intraperitoneally 20 min prior to METH or saline. RESULTS: Both repetitive (daily on d 4-13) and a single injection (on the day of challenge) of YQA14 (6.25, 12.5 and 25 mg/kg) dose-dependently inhibited the acquisition and expression of METH-induced locomotor sensitization. However, repetitive injection of YQA14 (daily during the METH conditioning) did not alter the acquisition of METH-induced CPP, whereas a single injection of YQA14 (prior to CPP test) dose-dependently attenuated the expression of METH-induced CPP. In addition, the repetitive injection of YQA14 dose-dependently facilitated the extinction and decreased the reinstatement of METH-induced CPP. CONCLUSION: Brain D3 receptors are critically involved in the reward and psychomotor-stimulating effects of METH. Thus, YQA14 deserves further study as a potential medication for METH addiction.

Y-QA31, a novel dopamine D3 receptor antagonist, exhibits antipsychotic-like properties in preclinical animal models of schizophrenia.

AIM: To investigate the potential effects of Y-QA31, a novel dopamine D3 receptor antagonist, as an antipsychotic drug. METHODS: A panel of radioligand-receptor binding assays was performed to identify the affinities of Y-QA31 for different G protein-coupled receptors. [(35)S]GTPγS-binding assays and Ca(2+) imaging were used to assess its intrinsic activities. The antipsychotic profile of Y-QA31 was characterized in mouse models for the positive symptoms and cognitive deficits of schizophrenia and extrapyramidal side effects with haloperidol and clozapine as positive controls. RESULTS: In vitro, Y-QA31 is a dopamine D3 receptor antagonist that is 186-fold more potent at the D3 receptor than at the D2 receptor. Y-QA31 also exhibits 5-HT1A receptor partial agonist and α1A adrenoceptor antagonist activities with medium affinity, whereas it exhibits very little affinity for other receptors (100-fold lower than for the D3 receptor). In vivo, Y-QA31 (10-40 mg/kg, po) significantly inhibited MK-801-induced hyperlocomotion and methamphetamine-induced prepulse inhibition disruption in a dose-dependent manner. Y-QA31 also inhibited the avoidance response and methamphetamine-induced hyperlocomotion with potency lower than haloperidol. Y-QA31 was effective in alleviating the MK-801-induced disruption of novel object recognition at a low dose (1 mg/kg, po). Moreover, Y-QA31 itself did not affect spontaneous locomotion or induce cataleptic response until its dose reached 120 mg/kg. CONCLUSION: Y-QA31 is a selective D3R antagonist that exhibits antipsychotic effects in some animal models with positive symptoms and cognitive disorder and less extrapyramidal side effects.

Intravenous ilaprazole is more potent than oral ilaprazole against gastric lesions in rats.

BACKGROUND AND AIMS: Ilaprazole is a novel proton pump inhibitor that has been marketed as an oral therapy for acid-related diseases in China and Korea. This study aimed to compare the gastroprotective effects of intravenous and enteral ilaprazole in rat models. METHODS: The rats were divided into 7-8 groups receiving vehicle, esomeprazole, and different doses of intravenous and enteral ilaprazole. The rats were then exposed to indomethacin (30 mg/kg, i.g.), or water-immersion stress and gastric lesions were examined. The effects of different treatments on histamine (10 µmol/kg/h)-induced acid secretion were also observed. RESULTS: Intravenous ilaprazole exhibited high antiulcer activity in a dose-dependent manner. Ilaprazole at a dose of 3 mg/kg decreased ulcer number and index to the same extent as 20 mg/kg esomeprazole. Moreover, the potency of intravenous ilaprazole is superior to that of intragastric ilaprazole. In anesthetized rats, the inhibitory effect of intravenous ilaprazole on histamine-induced acid secretion is faster and longer-lasting than that of intraduodenal ilaprazole. CONCLUSION: Intravenous ilaprazole is more potent than oral ilaprazole against indomethacin- or stress-induced gastric lesions, with faster and longer inhibition of acid secretion.

New Insights into the Role of Mild Hypoxia in Regulating Neural Stem Cell Characteristics.

The proliferation of neural stem cells (NSCs) is precisely regulated by extracellular environmental factors. In situ hypoxia, one of the key factors involved in the regulation of NSC characteristics, has attracted increasing amounts of attention. Numerous studies have demonstrated that hypoxia can significantly promote the formation of neurospheres and the proliferation of NSCs in vitro and that intermittent hypoxia can promote the proliferation of endogenous NSCs in vivo. In this article, the effects of different concentrations of oxygen on NSC proliferation and differentiation both in vivo and in vitro are reviewed, and the potential applications of hypoxia-preconditioned NSCs, as well as research progress and challenges in the treatment of central nervous system diseases, are further summarized. Here, the critical role of oxygen in the neurogenesis of NSCs is emphasized, and insights into the use of hypoxia to regulate NSC characteristics are provided.

Involvement of hippocampal phosphatidylethanolamine-binding protein in morphine dependence and withdrawal.

Drug addiction is thought to result from an intractable and aberrant learning and memory in response to drug-related stimulation, and cholinergic neurotransmission plays an important role in this process. Phosphatidylethanolamine-binding protein (PEBP) is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP), an 11 amino acid peptide that enhances the production of choline acetyltransferase (ChAT) and assists in the development of cholinergic projections from the medial septal nuclei to the hippocampus. However, whether PEBP is involved in drug addiction remains unclear. In the present study, PEBP expression in the hippocampus, as detected by proteomics analysis, was found to be dramatically up-regulated after rats received chronic morphine treatment. Western blotting analysis revealed a specific up-regulation of PEBP expression in the hippocampus but not in any other brain regions assessed. A down-regulation of hippocampal PEBP levels induced by antisense oligodeoxynucleotides resulted in aggravated morphine dependence. Together, these findings indicate that PEBP is involved in morphine dependence. Moreover, the time course of PEBP expression changes and ChAT activity was investigated during chronic morphine treatment and withdrawal. The results showed that the hippocampal PEBP levels were up-regulated during chronic morphine treatment and returned to the baseline 3 days after withdrawal, after which PEBP levels were persistently up-regulated for 28 days after withdrawal. The changes in hippocampal ChAT activity followed a pattern that was similar to that of the PEBP levels. Taken together, these results suggest that hippocampal PEBP is involved in morphine dependence and withdrawal, perhaps through modulating cholinergic transmission in the hippocampus.

[Establishment and application of a mouse model for drug-induced schizophrenia].

Schizophrenia, described as the worst disease affecting mankind, is a severe and disabling mental disorder. Schizophrenia is characterized by complicated symptoms and still lacks a diagnostic neuropathology, so developing schizophrenia animal models which have quantifiable measures tested in a similar fashion in both humans and animals will play a key role in new therapeutic approaches. According to the symptoms of cognitive impairment and emotional disorder, the N-methyl-d-aspartate (NMDA)-receptor antagonist MK-801 was applied to induce schizophrenia-like behavior in mice. Locomotor activity and prepulse inhibition (PPI) were selected as indices and the effect of clozapine was also investigated in this model. The results showed that compared with the normal group, MK-801-treated mice exhibited significantly increased locomotor activity and impaired PPI, and pre-exposure to clozapine could ameliorate the abnormality and make it back to normal level. These findings suggest that the model we established could be a useful tool for antipsychotic drug screening.

Opioid receptor trafficking and signaling: what happens after opioid receptor activation?

Prolonged opioid treatment leads to a comprehensive cellular adaptation mediated by opioid receptors, a basis to understand the development of opioid tolerance and dependence. However, the molecular mechanisms underlying opioid-induced cellular adaptation remain obscure. Recent advances in opioid receptor trafficking and signaling in cells have extensively increased our insight into the network of intracellular signal integration. This review focuses on those important intracellular biochemical processes that play critical roles in the development of opioid tolerance and dependence after opioid receptor activation, and tries to explain what happens after opioid receptor activation, and how the cellular adaptation develops from cell membrane to nucleus. Decades of research have delineated a network on opioid receptor trafficking and signaling, but the challenge remains to explain opioid tolerance and dependence from a single cellular signal network.

YQA14: a novel dopamine D3 receptor antagonist that inhibits cocaine self-administration in rats and mice, but not in D3 receptor-knockout mice.

The dopamine (DA) D3 receptor is posited to be importantly involved in drug reward and addiction, and D3 receptor antagonists have shown extraordinary promise as potential anti-addiction pharmacotherapeutic agents in animal models of drug addiction. SB-277011A is the best characterized D3 receptor antagonist in such models. However, the potential use of SB-277011A in humans is precluded by pharmacokinetic and toxicity problems. We here report a novel D3 receptor antagonist YQA14 that shows similar pharmacological properties as SB-277011A. In vitro receptor binding assays suggest that YQA14 has two binding sites on human cloned D3 receptors with K(i-High) (0.68 × 10(-4) nM) and K(i-Low) (2.11 nM), and displays > 150-fold selectivity for D3 over D2 receptors and > 1000-fold selectivity for D3 over other DA receptors. Systemic administration of YQA14 (6.25-25 mg/kg) or SB-277011A (12.5-25 mg/kg) significantly and dose-dependently reduced intravenous cocaine self-administration under both low fixed-ratio and progressive-ratio reinforcement conditions in rats, while failing to alter oral sucrose self-administration and locomotor activity, suggesting a selective inhibition of drug reward. However, when the drug dose was increased to 50 mg/kg, YQA14 and SB-277011A significantly inhibited basal and cocaine-enhanced locomotion in rats. Finally, both D3 antagonists dose-dependently inhibited intravenous cocaine self-administration in wild-type mice, but not in D3 receptor-knockout mice, suggesting that their action is mediated by D3 receptor blockade. These findings suggest that YQA14 has a similar anti-addiction profile as SB-277011A, and deserves further study and development.

Pre-treatment with Tandospirone attenuates fentanyl-induced respiratory depression without affecting the analgesic effects of fentanyl in rodents.

Opioid analgesics are widely used to treat acute, postoperative, and chronic pain. However, opioid receptor activation can result in severe respiratory depression. In this study, we demonstrated that Tandospirone (TS), a selective serotonin-1A receptor partial agonist, is effective against opioid-induced respiratory depression. Fentanyl was used to establish a respiratory depression model in rodents. We observed the effects of TS on respiratory depression in rats by using plethysmographic recordings and arterial oxygen saturation. In addition, we evaluated the effects of TS on fentanyl-induced sedation and analgesia by using the loss of righting reflex (LORR) and hot-plate tests, respectively. Rats (n = 5) were treated with TS or saline 5 min prior to fentanyl administration. TS [2 mg/kg, intravenous (i.v.)] dose-dependently attenuated fentanyl-induced respiratory depression versus saline + fentanyl group. Furthermore, pre-treatment with TS (2 mg/kg, i.v.) increased arterial oxygen saturation to 76.5 ± 2.0% at 5 min after fentanyl injection, compared with 35.9 ± 2.5% in saline pre-treated rats (P < 0.001), whereas the time to induction of LORR (P > 0.99) and duration of LORR (P = 0.95) did not differ between the "TS + fentanyl" and "saline + fentanyl" group. The antinociceptive effect of fentanyl was not affected by the administration of TS (P = 0.99) in mice (n = 10). In conclusion, we found that TS, a novel non-benzodiazepine anxiolytic/antidepressant drug, could attenuate severe fentanyl-induced respiratory depression and did not affect the analgesic/sedative effect of fentanyl. The clinical application of TS could significantly improve pain management.

Flumazenil-Insensitive Benzodiazepine Effects in Recombinant αß and Neuronal GABAA Receptors.

Gamma-aminobutyric acid, type A (GABAA) receptors are complex heterogeneous pentamers with various drug binding sites. Several lines of evidence suggest that benzodiazepines modulate certain GABAA receptors in a flumazenil-insensitive manner, possibly via binding sites other than the classical ones. However, GABAA receptor subtypes that contain non-classical benzodiazepine binding sites are not systemically studied. The present study investigated the high-concentration effects of three benzodiazepines and their sensitivity to flumazenil on different recombinant (α1ß2, α2ß2, α3ß2, α4ß2, α5ß2 and α1ß3) and native neuronal GABAA receptors using the whole-cell patch-clamp electrophysiology technique. The classical benzodiazepine diazepam (200 µmol/L) and midazolam (200 µmol/L) produced flumazenil-insensitive effects on α1ß2 receptor, whereas the imidazopyridine zolpidem failed to modulate the receptor. Flumazenil-insensitive effects of diazepam were also observed on the α2ß2, α3ß2 and α5ß2, but not α4ß2 receptors. Unlike ß2-containing receptors, the α1ß3 receptor was insensitive to diazepam. Moreover, the diazepam (200 µmol/L) effects on some cortical neurons could not be fully antagonized by flumazenil (200 µmol/L). These findings suggested that the non-classical (flumazenil-insensitive) benzodiazepine effects depended on certain receptor subtypes and benzodiazepine structures and may be important for designing of subtype- or binding site- specific drugs.

Wnt1/ß-catenin signaling upregulates spinal VGLUT2 expression to control neuropathic pain in mice.

Vesicular glutamate transporter 2 (VGLUT2)-which uptakes glutamate into presynaptic vesicles-is a fundamental component of the glutamate neurotransmitter system. Although several lines of evidence from genetically modified mice suggest a possible association of VGLUT2 with neuropathic pain, the specific role of VGLUT2 in the spinal cord during neuropathic pain, and its regulatory mechanism remain elusive. In this study, we report that spared nerve injury induced an upregulation of VGLUT2 in the spinal cord, and intrathecal administration of small hairpin RNAs (shRNA) against VGLUT2 before or after surgery attenuated mechanical allodynia, and pathologically-enhanced glutamate release. Meanwhile, nerve injury activated the Wnt1/ß-catenin signaling pathway in a quick-onset and sustained manner, and blocking the Wnt1 signaling with a Wnt1 targeting antibody attenuated neuropathic pain. In naïve mice, administration of a Wnt agonist or Wnt1 increased spinal VGLUT2 protein levels. Moreover, intrathecal administration of the Wnt/ß-catenin inhibitor, XAV939 attenuated mechanical allodynia, and this effect was concurrent with that of VGLUT2 downregulation. Pretreatment with VGLUT2 shRNAs abolished the allodynia induced by the Wnt agonist or Wnt1. These findings reveal a novel mechanism wherein there is Wnt1/ß-catenin-dependent VGLUT2 upregulation in neuropathic pain, thus potentiating the development of new therapeutic strategies in pain management.

The Impact and Mechanism of a Novel Allosteric AMPA Receptor Modulator LCX001 on Protection Against Respiratory Depression in Rodents.

Analgesics and sedative hypnotics in clinical use often give rise to significant side effects, particularly respiratory depression. For emergency use, specific antagonists are currently administered to counteract respiratory depression. However, antagonists are often short-lasting and eliminate drug generated analgesia. To resolve this issue, novel positive AMPA modulators, LCX001, was tested to alleviate respiratory depression triggered by different drugs. The acetic acid writhing and hot-plate test were conducted to evaluate analgesic effect of LCX001. Binding assay, whole-cell recording, live cell imaging, and Ca2+ imaging were used to clarify mechanism and impact of LCX001 on respiratory protection. Results showed that LCX001 effectively rescued and prevented opioid (fentanyl and TH-030418), propofol, and pentobarbital-induced respiratory depression by strengthening respiratory frequency and minute ventilation. The acetic acid writhing test and hot-plate test revealed potent anti-nociceptive efficacy of LCX001, in contrast to other typical ampakines that did not affect analgesia. Furthermore, LCX001 potentiated [3H]AMPA and L-glutamate binding affinity to AMPA receptors, and facilitated glutamate-evoked inward currents in HEK293 cells stably expressing GluA2(R). LCX001 had a typical positive modulatory impact on AMPAR-mediated function. Importantly, application of LCX001 generated a significant increase in GluA2(R) surface expression, and restrained opioid-induced abnormal intracellular Ca2+ load, which might participate in breathing modulation. Our study improves therapeutic interventions for the treatment of drug induced respiratory depression, and increases understanding of potential mechanism of AMPA receptor modulators.

Effect of agmatine on DAMGO-induced mu-opioid receptor down-regulation and internalization via activation of IRAS, a candidate for imidazoline I(1) receptor.

Agmatine, an endogenous ligand for imidazoline I(1) receptor, has previously been shown to prevent opioid tolerance in rats and mice, but the cellular mechanisms remain unknown. In the present study, the effects of agmatine activation on imidazoline I(1) receptor on the desensitization, down-regulation and internalization of micro opioid receptor were investigated. Two cell lines, CHO cells transfected micro opioid receptor (CHO-micro cells) and co-transfected micro opioid receptor and imidazoline I(1) receptor antisera-selected protein (IRAS) (CHO-micro/IRAS cells), were used. In both CHO-micro cells and CHO-micro/IRAS cells, agmatine (0.01-10 microM) did not affect the desensitization of micro opioid receptor induced by [d-Ala(2), N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) (10 microM) treatment for 30 min. However, agmatine (0.1-100 nM) co-pretreatment with DAMGO (1 microM) for 12 h concentration-dependently inhibited DAMGO-induced down-regulation of micro opioid receptor in CHO-micro/IRAS cells, but not in CHO-micro cells. Efaroxan, the I(1)/alpha(2)-adrenoceptors mix antagonist, completely reversed the inhibitory effect of agmatine, suggesting the participation of imidazoline I(1) receptor. In addition, agmatine (1-100 nM) inhibited DAMGO-induced internalization of micro opioid receptor in CHO-micro/IRAS cells, which was reversed by efaroxan as well. While treatment with DAMGO (1 microM) or co-treatment with agmatine (1-100 nM) for 12 h failed to affect the mRNA level of micro opioid receptor. Taken together, these results indicate that the inhibitory effect of agmatine on tolerance in vitro might be related to attenuation of the internalization and down-regulation of micro opioid receptor via activation of imidazoline I(1) receptor.

Analgesic activity of ZC88, a novel N-type voltage-dependent calcium channel blocker, and its modulation of morphine analgesia, tolerance and dependence.

ZC88 is a novel non-peptide N-type voltage-sensitive calcium channel blocker synthesized by our institute. In the present study, the oral analgesic activity of ZC88 in animal models of acute and neuropathic pain, and functional interactions between ZC88 and morphine in terms of analgesia, tolerance and dependence were investigated. In mice acetic acid writhing tests, ZC88 (10-80 mg/kg) administered by oral route showed significant antinociceptive effects in a dose-dependent manner. The ED50 values of ZC88 were 14.5 and 14.3 mg/kg in male and female mice, respectively. In sciatic nerve chronic constriction injury rats, mechanical allodynia was ameliorated by oral administration of ZC88 at doses of 14, 28 and 56 mg/kg, suggesting ZC88 relieved allodynic response of neuropathic pain. When concurrently administered with morphine, ZC88 (20-80 mg/kg) dose-dependently potentiated morphine analgesia and attenuated morphine analgesic tolerance in hot-plate tests. ZC88 also prevented chronic exposure to morphine-induced physical dependence and withdrawal, but not morphine-induced psychological dependence in conditioned place preference model. These results suggested that ZC88, a new non-peptide N-type calcium channel blocker, had notable oral analgesia and anti-allodynia for acute and neuropathic pain. ZC88 might be used in pain relief by either application alone or in combination with opioids because it enhanced morphine analgesia while prevented morphine-induced tolerance and physical dependence.

Agmatine inhibits morphine-induced drug discrimination in rats.

Our previous studies have shown that agmatine inhibited morphine-induced conditioned place preference and locomotor sensitization in rats. In the present study, we further investigated the effects of agmatine on the discriminative stimulating effects produced by morphine in rats. Agmatine, at the dose range of 10-80 mg/kg (i.g.), neither induced drug discrimination, nor substituted for morphine stimulus in rats that were previously treated with morphine, suggesting that agmatine itself has no psychomotor-stimulating potential. However, pretreatment with agmatine (40, 80 mg/kg, i.g.) significantly inhibited the acquisition, but not expression, of morphine-induced drug discrimination as assessed by the correct nose-poke response. Further, chronic administration of agmatine (40, 80 mg/kg/day x 12 days, i.g., 25 min prior to morphine) also significantly accelerated the extinction of the discrimination induced by morphine. These data suggest that agmatine inhibits the acquisition and accelerates the extinction of morphine-induced discrimination, supporting possible use of agmatine in the treatment of opioid dependence.

Effects of intragastric agmatine on morphine-induced physiological dependence in beagle dogs and rhesus monkeys.

Our previous studies demonstrated that subcutaneous injection of agmatine inhibits tolerance to and physiological dependence on morphine in mice and rats. In the present study we further evaluated the effects of intragastric (i.g.) administration of agmatine on morphine-induced physiological dependence in mice, rats, beagle dogs and rhesus monkeys. When agmatine (5-40 mg/kg, i.g.) was co-administered with morphine during the development of morphine-induced physiological dependence, it inhibited the abstinent syndrome precipitated by naloxone in mice, rats and beagle dogs. In addition, agmatine (40 mg/kg, i.g.) inhibited the abstinent syndrome precipitated by naloxone in mice when it was administered on the test day. In naloxone precipitated and naturally abstinent morphine dependent model in rhesus monkeys, agmatine (40 or 80 mg/kg, i.g.) inhibited the development of physiological dependence when it was co-administered with morphine. After the development of morphine dependence, agmatine (80 mg/kg, i.g.) inhibited the naturally abstinent syndrome during the 7-d abstinent period. All these results suggested that intragastric administration of agmatine inhibits morphine-induced physiological dependence in animal models.

Aquaporin 4 deficiency modulates morphine pharmacological actions.

Acute administration of opioids produces analgesia, while chronic administration induces tolerance and dependence. Aquaporin 4 (AQP4) is most strongly expressed in astrocytes throughout central nervous system, and plays an important role in some pathophysiological processes in brain. However, whether AQP4 modulates opioid analgesia, tolerance and dependence or not remains unknown. In the present study, the effects of AQP4 deficiency on morphine analgesia, tolerance and physical dependence were investigated. (1) In hot-plate tests, ED(50) values of morphine analgesia were 3.77 and 3.96 mg/kg in male and female AQP4 knockout mice, which were lower than that in wild-type mice (5.23 and 5.20mg/kg in males and females). (2) Repeated treatment with morphine resulted in analgesic tolerance to morphine in wild-type mice, whereas the morphine tolerance was attenuated in AQP4 knockout mice treated as the same schedule. (3) After repeated morphine administration, naloxone precipitation induced significant abstinent jumping in wild-type mice, whereas naloxone-induced abstinent jumping was not observed in AQP4 knockout mice. This suggested that AQP4 deficiency inhibited the development of morphine physical dependence. (4) Repeated morphine administration down-regulated cerebral glutamate transporter 1 (GLT-1) expression in wild-type mice. However, the down-regulation of GLT-1 expression diminished in AQP4 knockout mice. Taken together, these results demonstrated that AQP4 deficiency potentiated morphine analgesia, attenuated morphine tolerance and physical dependence. The suppression of down-regulation of cerebral GLT1 expression might mediate the attenuation of AQP4 deficiency to morphine tolerance and dependence.

Selective downregulation of vesicular glutamate transporter2 in ventral posterolateral nucleus of thalamus attenuates neuropathic mechanical allodynia in mice.

Vesicular glutamate transporters (VGLUTs) transport glutamate into synaptic vesicles prior to exocytotic release. The expression pattern of VGLUT2 and studies of genetically modified mice have revealed that VGLUT2 contributes to neuropathic pain. We previously showed that VGLUT2 is upregulated in supraspinal regions including the thalamus in mice following spared nerve injury (SNI), and blocking VGLUTs using the VGLUT inhibitor CSB6B attenuated mechanical allodynia. To further evaluate the role of VGLUT2 in neuropathic pain, in this study, we developed a lentiviral vector expressing small hairpin RNAs (shRNAs) against mouse VGLUT2, which was injected into the ventral posterolateral (VPL) nucleus of the thalamus in the presence or absence of SNI. The administration of VGLUT2 shRNAs result in downregulation of VGLUT2 mRNA and protein expression, and decreased extracellular glutamate release in primary cultured neurons. We also showed that VGLUT2 shRNAs attenuated SNI-induced mechanical allodynia, in accordance with knockdown of VGLUT2 in the VPL nucleus in mice. Accordingly, our study supports the essential role of supraspinal VGLUT2 in neuropathic pain in adult mice and, thereby, validates VGLUT2 as a potential target for neuropathic pain therapy.