Hypoxia on the Expression of Hepatoma Upregulated Protein in Prostate Cancer Cells.

Hepatoma upregulated protein (HURP) is a multifunctional protein with clinical promise. This protein has been demonstrated to be a predictive marker for the outcome in high-risk prostate cancer (PCa) patients, besides being a resistance factor in PCa. Although changes in oxygen tension (pO2) are associated with PCa aggressiveness, the role of hypoxia in the regulation of tumor progression genes such as HURP has not yet been described. We hypothesized that pO2 alteration is involved in the regulation of HURP expression in PCa cells. In the present study, PCa cells were incubated at 2% O2 (hypoxia) and 20% O2 (normoxia) conditions. Hypoxia reduced cell growth rate of PCa cells, when compared to the growth rate of cells cultured under normoxia (p < 0.05). The decrease in cell viability was accompanied by fivefold (p < 0.05) elevated rate of vascular endothelial growth factor (VEGF) release. The expression of VEGF and the hypoxia-inducible metabolic enzyme carbonic anhydrase 9 were elevated maximally nearly 61-fold and 200-fold, respectively (p < 0.05). Noted in two cell lines (LNCaP and C4-2B) and independent of the oxygen levels, HURP expression assessed at both mRNA and protein levels was reduced. However, the decrease was more pronounced in cells cultured under hypoxia (p < 0.05). Interestingly, the analysis of patients' specimens by Western blot revealed a marked increase of HURP protein (fivefold), when compared to control (cystoprostatectomy) tissue (p < 0.05). Immunohistochemistry analysis showed an increase in the immunostaining intensity of HURP and the hypoxia-sensitive molecules, hypoxia-inducible factor 1-alpha (HIF-1α), VEGF, and heat-shock protein 60 (HSP60) in association with tumor grade. The data also suggested a redistribution of subcellular localization for HURP and HIF-1α from the nucleus to the cytoplasmic compartment in relation to increasing tumor grade. Analysis of HURP Promoter for HIF-1-binding sites revealed presence of four putative HIF binding sites on the promoter of DLGAP5/HURP gene in the non-translated region upstream from the start codon, suggesting association between HIF-1α and the regulation of HURP protein. Taken together, our findings suggest a modulatory role of hypoxia on the expression of HURP. Additionally our results provide basis for utilization of tumor-associated molecules as predictors of aggressive PCa.

Elevated Expression of Hepatoma Up-Regulated Protein Inhibits γ-Irradiation-Induced Apoptosis of Prostate Cancer Cells.

Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time that the resistance of PCa to radiation therapy is attributed to elevated expression of Hepatoma Up-Regulated Protein (HURP). In PCa cells, the induction of HURP expression suppresses γ-irradiation-induced apoptosis. γ-irradiation-induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ-irradiation-induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ-irradiation-induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio-resistance of PCa is thought to be the consequence of HURP-mediated destabilization of p53 and ATM proteins that are essential for the modulation of γ-irradiation-induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F-1 in response to the elevated expression of HURP.

Effects of oxygen on the antigenic landscape of prostate cancer cells.

BACKGROUND: Use of allogeneic cancer cells-based immunotherapy for treatment of established prostate cancer (PCa) has only been marginally effective. One reason for failure could stem from the mismatch of antigenic signatures of vaccine cells and cancer in situ. Hence, it is possible that vaccine cells expressed antigens differently than tumor cells in situ. We hypothesized that cells grown in vitro at low oxygen tension (pO2) provide a better antigen match to tumors in situ and could reveal a more relevant antigenic landscape than cells grown in atmospheric pO2. METHODS: We tested this hypothesis by comparing PCa cells propagated at pO2 = 2 kPa and 20 kPa. To identify potential tumor-associated antigens (TAAs), we prepared PCa cell lysates, resolved them by two-dimensional electrophoresis and immunoblotting using spontaneous antibodies from plasma derived from PCa patients and control subjects. Antibody-labeled spots were analyzed by MALDI-TOF mass spectrometry and validated by ELISA. We selected hypoxia-regulated HSP70 and hnRNP L and hypoxia-independent HSP60 and determined the frequency of plasma samples reacting with these molecules. RESULTS: Frequency of HSP60-reactive plasma was low in healthy controls [1.3 % (1/76)], while it was elevated in PCa patients [13.0 % (7/54); p < 0.05]. These data suggest a humoral immune response to HSP60 in PCa. Levels of autoantibodies to HSP70 did not differ from healthy controls [3.7 % (2/54)] in PCa patients [5.3 % (2/38)]. Similarly, hnRNP L autoantibodies did no differ between healthy controls [6.1 % (3/49)] and PCa patients [5.3 % (2/38)]. CONCLUSIONS: Overall our results suggest the value of hypoxia as a modifier of the cellular and antigenic landscape of PCa cells. By modifying the immune reactivity of PCa cells in culture, manipulation of pO2 can be proposed as a new avenue for improving diagnosis, prognosis and immunotherapy for PCa.

Neonatal systemic exposure to lipopolysaccharide enhances susceptibility of nigrostriatal dopaminergic neurons to rotenone neurotoxicity in later life.

Brain inflammation via intracerebral injection with lipopolysaccharide (LPS) in early life has been shown to increase risks for the development of neurodegenerative disorders in adult rats. To determine if neonatal systemic LPS exposure has the same effects on enhancement of adult dopaminergic neuron susceptibility to rotenone neurotoxicity as centrally injected LPS does, LPS (2 µg/g body weight) was administered intraperitoneally into postnatal day 5 (P5) rats and when grown to P70, rats were challenged with rotenone, a commonly used pesticide, through subcutaneous minipump infusion at a dose of 1.25 mg/kg/day for 14 days. Systemically administered LPS can penetrate into the neonatal rat brain and cause acute and chronic brain inflammation, as evidenced by persistent increases in IL-1ß levels, cyclooxygenase-2 expression and microglial activation in the substantia nigra (SN) of P70 rats. Neonatal LPS exposure resulted in suppression of tyrosine hydroxylase (TH) expression, but not actual death of dopaminergic neurons in the SN, as indicated by the reduced number of TH+ cells and unchanged total number of neurons (NeuN+) in the SN. Neonatal LPS exposure also caused motor function deficits, which were spontaneously recoverable by P70. A small dose of rotenone at P70 induced loss of dopaminergic neurons, as indicated by reduced numbers of both TH+ and NeuN+ cells in the SN, and Parkinson's disease (PD)-like motor impairment in P98 rats that had experienced neonatal LPS exposure, but not in those without the LPS exposure. These results indicate that although neonatal systemic LPS exposure may not necessarily lead to death of dopaminergic neurons in the SN, such an exposure could cause persistent functional alterations in the dopaminergic system and indirectly predispose the nigrostriatal system in the adult brain to be damaged by environmental toxins at an ordinarily nontoxic or subtoxic dose and develop PD-like pathological features and motor dysfunction.

Differential action of methamphetamine on tyrosine hydroxylase and dopamine transport in the nigrostriatal pathway of µ-opioid receptor knockout mice.

Extensive anatomical and functional interactions exist between central dopaminergic and opioidergic systems and both systems are proposed to be targets for amphetamine-like drugs. We have previously reported that µ-opioid receptor (µ-OR) knockout mice are resistant to the loss of dopamine in the striatum and the development of behavioral sensitization induced by repeated methamphetamine (METH) treatment. The present study assessed whether METH-treated µ-OR knockout mice exhibit a differential response of the expression of dopamine transporter and tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis and maintaining dopamine levels. Mice daily received intraperitoneal injection of METH (0, 0.6, 2.5, or 10 mg/kg) for 7 days and sacrificed on day 11 (4 days after the last injection). The expression of TH protein in the striatum and the levels of TH mRNA and number of TH positive neurons in the substantia nigra were reduced in wild-type mice treated with METH (2.5 and 10 mg/kg), but not in the µ-OR knockout mice. In contrast, METH exposure at the highest dose (10 mg/kg) reduced dopamine transporter levels in both strains of mice. These results suggest that the µ-OR contributes to METH-induced loss of dopamine and behavioral sensitization by decreasing the expression of TH.

Methamphetamine-induced changes in the striatal dopamine pathway in µ-opioid receptor knockout mice.

BACKGROUND: Repeated exposure to methamphetamine (METH) can cause not only neurotoxicity but also addiction. Behavioral sensitization is widely used as an animal model for the study of drug addiction. We previously reported that the µ-opioid receptor knockout mice were resistant to METH-induced behavioral sensitization but the mechanism is unknown. METHODS: The present study determined whether resistance of the µ-opioid receptor (µ-OR) knockout mice to behavioral sensitization is due to differential expression of the stimulatory G protein α subunit (Gαs) or regulators of G-protein signaling (RGS) coupled to the dopamine D1 receptor. Mice received daily intraperitoneal injections of saline or METH (10 mg/kg) for 7 consecutive days to induce sensitization. On day 11(following 4 abstinent days), mice were either given a test dose of METH (10 mg/kg) for behavioral testing or sacrificed for neurochemical assays without additional METH treatment. RESULTS: METH challenge-induced stereotyped behaviors were significantly reduced in the µ-opioid receptor knockout mice when compared with those in wild-type mice. Neurochemical assays indicated that there is a decrease in dopamine D1 receptor ligand binding and an increase in the expression of RGS4 mRNA in the striatum of METH-treated µ-opioid receptor knockout mice but not of METH-treated wild-type mice. METH treatment had no effect on the expression of Gαs and RGS2 mRNA in the striatum of either strain of mice. CONCLUSIONS: These results indicate that down-regulation of the expression of the dopamine D1 receptor and up-regulation of RGS4 mRNA expression in the striatum may contribute to the reduced response to METH-induced stereotypy behavior in µ-opioid receptor knockout mice. Our results highlight the interactions of the µ-opioid receptor system to METH-induced behavioral responses by influencing the expression of RGS of dopamine D1 receptors.

Dopaminergic neuronal injury in the adult rat brain following neonatal exposure to lipopolysaccharide and the silent neurotoxicity.

Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1ß and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.

mu-Opioid receptor knockout mice are insensitive to methamphetamine-induced behavioral sensitization.

Repeated administration of psychostimulants to rodents can lead to behavioral sensitization. Previous studies, using nonspecific opioid receptor (OR) antagonists, revealed that ORs were involved in modulation of behavioral sensitization to methamphetamine (METH). However, the contribution of OR subtypes remains unclear. In the present study, using mu-OR knockout mice, we examined the role of mu-OR in the development of METH sensitization. Mice received daily intraperitoneal injection of drug or saline for 7 consecutive days to initiate sensitization. To express sensitization, animals received one injection of drug (the same as for initiation) or saline on day 11. Animal locomotor activity and stereotypy were monitored during the periods of initiation and expression of sensitization. Also, the concentrations of METH and its active metabolite amphetamine in the blood were measured after single and repeated administrations of METH. METH promoted significant locomotor hyperactivity at low doses and stereotyped behaviors at relative high doses (2.5 mg/kg and above). Repeated administration of METH led to the initiation and expression of behavioral sensitization in wild-type mice. METH-induced behavioral responses were attenuated in the mu-OR knockout mice. Haloperidol (a dopamine receptor antagonist) showed a more potent effect in counteracting METH-induced stereotypy in the mu-OR knockout mice. Saline did not induce behavioral sensitization in either genotype. No significant difference was observed in disposition of METH and amphetamine between the two genotypes. Our study indicated that the mu-opioid system is involved in modulating the development of behavioral sensitization to METH. (c) 2010 Wiley-Liss, Inc.

Methamphetamine-induced expression of zif268 mRNA is prevented by haloperidol in mice lacking mu-opioid receptor.

We investigated the role of mu-opioid receptor (mu-OR) and dopamine receptor in the modulation of methamphetamine (METH)-induced expression of zif268 mRNA in the striatum of mice. Four groups of wild-type and mu-OR knockout mice were given a single daily intraperitoneal injection of saline (control; group 1) or METH (10mg/kg; groups 2-4) for 7 consecutive days. On day 11 (after 4 abstinent days), groups 1 and 2 were challenged with saline, group 3 was challenged with METH (10mg/kg), and group 4 was challenged with dopamine receptor antagonist haloperidol (0.06 mg/kg, subcutaneous injection) plus METH (10mg/kg). Two hours after the last saline or METH injection, mouse brain tissues were taken for zif268 mRNA analysis using in situ hybridization histochemistry. In comparison to corresponding saline control group (group 1), striatal zif268 mRNA levels were unchanged in group 2 and increased in group 3 in both wild-type and mu-OR knockout mice and without genotype difference. METH challenge-enhanced expression of zif268 mRNA was completely abolished by pre-administration of haloperidol (group 4) in mu-OR knockout mice but not in wild-type mice. The results suggest a crosstalk of the two neurotransmitter systems in modulation of METH-induced IEG expression, because only in mu-OR knockout mice in which dopamine receptors were blocked were METH-induced zif268 expression abolished. METH-induced zif268 expression was not altered in mu-OR knockout mice without blockade of dopamine receptors or wild-type mice with blockade of dopamine receptors.

Gender differences in the antinociceptive effect of tramadol, alone or in combination with gabapentin, in mice.

Gender difference in the antinociceptive effect of tramadol and gabapentin (alone or in combination) were investigated in mice. For investigation of acute antinociceptive effect, tramadol and gabapentin were administered to mice by intraperitoneal injection and per os, respectively, and antinociceptive activity was measured by the tail-flick test 30 min after drug administration. For investigation of the development of antinociceptive tolerance to analgesics, mice were injected with tramadol (60 mg/kg), alone or in combination with gabapentin (75 mg/kg), twice daily for seven consecutive days and the tail-flicks were tested on experimental days 1, 3, 5 and 7. Results showed there was a lower ED(50) value of tramadol antinociception in males than in females, indicating that females were less sensitive to the drug. Gabapentin produces a limited antinociception in both males and females. The combination of gabapentin and tramadol produced synergistic effect without gender difference. Repeated administration of tramadol produced antinociceptive tolerance in both genders. Gabapentin produced synergistic effect in tramadol-tolerant mice and repeated administration of gabapentin did not alter the synergistic effect in tramadol-tolerant mice. Because females show a higher overall prevalence of pain and less sensitivity to opioids, our finding may suggest a clinical significance of combined use of the two drugs.

Methamphetamine-elicited alterations of dopamine- and serotonin-metabolite levels within mu-opioid receptor knockout mice: a microdialysis study.

mu-Opioid receptors (mu-ORs) modulate methamphetamine (MA)-induced behavioral responses, increased locomotor activity and stereotyped behavior in the mouse model. We investigated the changes in dopamine (DA) and serotonin (5-HT) metabolism in the striatum following either acute or repeated MA treatment using in vivo microdialysis. We also studied the role of mu-ORs in the modulation of MA-induced DA and 5-HT metabolism within mu-OR knockout mice. Subsequent to either acute or repeated intraperitoneal administration of MA, wild-type mice revealed decreases in extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in a dose-dependent manner. Moreover, wild-type mice had reductions in basal concentrations of DOPAC and HVA following repeated MA treatment with a higher dose. The effects of acute, repeated or challenge MA administration upon extracellular levels of DOPAC and HVA within mu-OR knockout mice significantly differed from the wild-type controls. The duration of recovery to the basal levels of extracellular DA and 5-HT metabolites induced by MA were much longer in wild-type mice than for mu-OR knockout mice. These findings suggest that mu-ORs play a modulatory role in MA-induced DA and 5-HT metabolism in the mouse striatum. This possible mechanism of MA-induced behavioral change as modulated by mu-OR merits further study.

Autoradiographic analysis of GABAA receptors in mu-opioid receptor knockout mice.

Anatomical evidence indicates that gamma-aminobutyric acid (GABA)-ergic and opioidergic systems are closely linked and act on the same neurons. However, the regulatory mechanisms between GABAergic and opioidergic system have not been well characterized. In the present study, we investigated whether there are changes in GABA(A) receptors in mice lacking mu-opioid receptor gene. The GABA(A) receptor binding was carried out by autoradiography using [(3)H]-muscimol (GABA(A)), [(3)H]-flunitrazepam (FNZ, native type 1 benzodiazepine) and [(35)S]-t-butylbicyclophosphorothionate (TBPS, binding to GABA(A)-gated chloride channels) in brain slices of wild type and mu-opioid receptor knockout mice. The binding of [(3)H]-FNZ in mu-opioid receptor knockout mice was significantly higher than that of the wild type controls in most of the cortex and hippocampal CA1 and CA2 formations. mu-Opioid receptor knockout mice show significantly lower binding of [(35)S]-TBPS than that of the wild type mice in few of the cortical areas including ectorhinal cortex layers I, III, and V, but not in the hippocampus. There was no significant difference in binding of [(3)H]-muscimol between mu-opioid receptor knockout and wild type mice in the cortex and hippocampus. These data indicate that there are specific regional changes in GABA(A) receptor binding sites in mu-opioid receptor knockout mice. These data also suggest that there are compensatory up-regulation of benzodiazepine binding site of GABA(A) receptors in the cortex and hippocampus and down-regulation of GABA-gated chloride channel binding site of GABA(A) receptors in the cortex of the mu-opioid receptor knockout mice.

Role of mu-opioid receptor in modulation of preproenkephalin mRNA expression and opioid and dopamine receptor binding in methamphetamine-sensitized mice.

We examined mRNA expression of preproenkephalin (PPE), a precursor of the endogenous opioid peptide enkephalin, and ligand binding to opioid and dopamine receptors in the striatum and nucleus accumbens in methamphetamine (METH)-sensitized mu-opioid receptor (mu-OR) knockout mice and their wild-type controls. Animals received daily intraperitoneal (i.p.) injections of METH (0, 0.625, 2.5, or 10 mg/kg) for 7 consecutive days to induce sensitization. Brain tissues were taken for biochemical analysis on experimental day 11 (4 days after the last injection). Expression of PPE mRNA and ligand binding were determined by in situ hybridization and autoradiography, respectively. Results indicate that there is an increase in PPE mRNA expression and a decrease in mu-OR ligand binding in METH-sensitized wild-type mice. These changes were not detected in METH-sensitized mu-OR knockout mice. A significant increase in delta-opioid receptor (delta-OR) ligand binding was found in mu-OR knockout mice. After repeated METH exposure, striatal and nucleus accumbal dopamine D1 receptor binding was decreased in mu-OR knockout mice but was not changed in wild-type mice. D2 receptor ligand binding was increased in wild-type mice and exhibited a biphasic change, with a decrease at 0.625 and 2.5 mg/kg doses of METH and an increase with 10 mg/kg of METH, in mu-OR knockout mice. These findings suggest that the mu-OR is involved in the regulation of METH-induced changes in an endogenous opioid peptide and dopamine receptors.

Methamphetamine-induced behavioral sensitization in mice: alterations in mu-opioid receptor.

We had previously demonstrated that opioid receptors contribute to the induction and expression of behavioral sensitization induced by repeated daily injection with 2.5 mg/kg of methamphetamine for 7 days. Using the same regimen, the present study investigated the alterations in mu-opioid receptor during the induction (on days 2, 5, and 8) and expression (on days 11 and 21) periods of behavioral sensitization. Radioligand binding revealed that the maximal binding of mu-opioid receptor was not changed on days 2 and 5, but down-regulated on day 8. After cessation of drug treatment, the maximal binding of mu-opioid receptor gradually and time-dependently returned to normal level on day 11 and up-regulated on day 21. In contrast, no changes in delta- and kappa-opioid receptors were detectable on any given day examined. The potency of DAMGO for [(35)S]-GTPgammaS coupling was enhanced on days 2, 5, 11, and 21. Moreover, 1 muM of naltrexone or beta-chlornaltrexamine significantly suppressed the basal [(35)S]-GTPgammaS coupling on days 2, 11, and 21. These findings indicate enhanced responsiveness and elevated constitutive activity of mu-opioid receptor. In summary, our data clearly demonstrate that alterations in mu-opioid receptor are involved in and may contribute to the sensitization to locomotor stimulating effect of methamphetamine.

Attenuation of methamphetamine-induced behavioral sensitization in mice by systemic administration of naltrexone.

Repeated intermittent exposure to psychostimulants was found to produce behavioral sensitization. The present study was designed to establish a mouse model and by which to investigate whether opioidergic system plays a role in methamphetamine-induced behavioral sensitization. Mice injected with 2.5 mg/kg of methamphetamine once a day for 7 consecutive days showed behavioral sensitization after challenge with 0.3125 mg/kg of the drug on day 11, whereas mice injected with a lower daily dose (1.25 mg/kg) did not. Mice received daily injections with either 1.25 or 2.5 mg/kg of methamphetamine showed behavioral sensitization after challenge with 1.25 mg/kg of the drug on days 11, 21, and 28. To investigate the role of opioidergic system in the induction and expression of behavioral sensitization, long-acting but non-selective opioid antagonist naltrexone was administrated prior to the daily injections of and challenge with methamphetamine, respectively. Our results show that the expressions of behavioral sensitization were attenuated by pretreatment with 10 or 20 mg/kg of naltrexone either during the induction period or before methamphetamine challenge when they were tested on days 11 and 21. These results indicate that repeated injection with methamphetamine dose-dependently induced behavioral sensitization in mice, and suggest the involvement of opioid receptors in the induction and expression of methamphetamine-induced behavioral sensitization.

Minocycline reduces lipopolysaccharide-induced neurological dysfunction and brain injury in the neonatal rat.

Preferential brain white matter injury and hypomyelination induced by intracerebral administration of the endotoxin lipopolysaccharide (LPS) in the neonatal rat brain has been characterized as associated with the activation of microglia. To examine whether inhibition of microglial activation might provide protection against LPS-induced brain injury and behavioral deficits, minocycline (45 mg/kg) was administered intraperitoneally 12 hr before and immediately after an LPS (1 mg/kg) intracerebral injection in postnatal day 5 (P5) Sprague-Dawley rats and then every 24 hr for 3 days. Brain injury and myelination were examined on postnatal day 21 and the tests for neurobehavioral toxicity were carried out from P3 to P21. LPS administration resulted in severe white matter injury, enlarged ventricles, deficits in the hippocampus, loss of oligodendrocytes and tyrosine hydroxylase neurons, damage to axons and dendrites, and impaired myelination as indicated by the decrease in myelin basic protein immunostaining in the P21 rat brain. LPS administration also significantly affected physical development (body weight) and neurobehavioral performance, such as righting reflex, wire hanging maneuver, cliff avoidance, locomotor activity, gait analysis, and responses in the elevated plus-maze and passive avoidance task. Treatment with minocycline significantly attenuated the LPS-induced brain injury and improved neurobehavioral performance. The protective effect of minocycline was associated with its ability to attenuate LPS-induced microglial activation. These results suggest that inhibition of microglial activation by minocycline may have long-term protective effects in the neonatal brain on infection-induced brain injury and associated neurologic dysfunction in the rat.

Roles of mu-opioid receptors in development of tolerance to diisopropylfluorophosphate (DFP).

Anatomical evidence indicates that cholinergic and opioidergic systems are co-localized and acting on the same neuron. However, the regulatory mechanisms between cholinergic and opioidergic system have not been well characterized. In the present study, the potential involvement of mu-opioid receptors in mediating the changes of toxic signs and muscarinic receptor binding after administration of irreversible anti-acetylcholinesterase diisopropylfluorophosphate (DFP) was investigated. DFP (1 mg/kg/day, subcutaneous injection, s.c.)-induced tremors and chewing movements were monitored during the 28-day treatment period in mu-opioid receptor knockout and wild type mice. Autoradiographic studies of total, M1, and M2 muscarinic receptors were conducted using [(3)H]-quinuclidinyl benzilate, [(3)H]-pirenzepine, and [(3)H]-AF-DX384 as ligands, respectively. DFP-induced tremors in both mu-opioid receptor knockout and wild type mice showed tolerance development. However, DFP-induced tremors in mu-opioid receptor knockout mice showed delayed tolerance development than that of DFP-treated wild type controls. DFP-induced chewing movements in both mu-opioid receptor knockout and wild type mice failed to show development of tolerance after four weeks of treatment. M2 muscarinic receptor binding of DFP-treated mu-opioid receptor knockout mice was significantly decreased than that of the DFP-treated wild type controls in the striatum, but not in the cortex and hippocampus. However, there were no significant differences in total and M1 muscarinic receptor binding between DFP-treated mu-opioid receptor knockout and wild type mice in the cortex, striatum and hippocampus. These studies indicate that mu-opioid receptors play an important role through the striatal M2 muscarinic receptors to regulate the development of tolerance to DFP-induced tremors.

Increased diisopropylfluorophosphate-induced toxicity in mu-opioid receptor knockout mice.

The potential involvement of mu-opioid receptors in mediating the changes of toxic signs and muscarinic receptor bindings after acute administration of irreversible antiacetylcholinesterase diisopropylfluorophosphate (DFP) was investigated. DFP-induced chewing movement and tremors were monitored for a period of 180 min in mu-opioid receptor knockout and wild-type mice. The autoradiographic studies of total, M1, and M2 muscarinic receptors were conducted using [(3)H]quinuclidinyl benzilate, [(3)H]pirenzepine, and [(3)H]AF-DX384 as ligands, respectively. Saline-treated mu-opioid receptor knockout and wild-type mice did not show chewing movement or tremors. Although DFP (1, 2, or 3 mg/kg, subcutaneous injection, s.c.)-induced chewing movement and tremors were shown in a dose-dependent manner, there were no significant differences in tremors induced by 1 or 2 mg/kg of DFP between mu-opioid receptor knockout and wild-type mice. There were also no significant differences in chewing movement induced by all doses of DFP between mu-opioid receptor knockout and wild-type mice. However, DFP (3 mg/kg)-induced tremors in mu-opioid receptor knockout mice were significantly increased over those in wild-type controls. Acetylcholinesterase activity in the striatum of saline-treated mu-opioid receptor knockout mice was significantly higher than that of the wild-type controls. After administration of DFP, acetylcholinesterase activity in the striatum of both mu-opioid receptor knockout and wild-type mice was significantly decreased (more than 36%, 58%, and 94% reduced at the doses of 1, 2, and 3 mg/kg, respectively) than that of their respective saline controls. M2 muscarinic receptor binding in saline-treated mu-opioid receptor knockout mice was significantly lower than that of the wild-type controls in the striatum. However, there were no significant differences in total, M1, or M2 muscarinic receptor binding in the cortex, striatum, or hippocampus of mu-opioid receptor knockout and wild-type mice after DFP administration. Our data show increased DFP-induced tremors, compensatory up-regulation of acetylcholinesterase activity, and compensatory down-regulation of M2 muscarinic receptors in the striatum of mice lacking mu-opioid receptor gene. These results suggest that the enhancement of DFP-induced tremors may be associated with the compensatory up-regulation of acetylcholinesterase activity and compensatory down-regulation of M2 muscarinic receptors in the striatum of mu-opioid receptor knockout mice.

Changes in acetylcholinesterase activity and muscarinic receptor bindings in mu-opioid receptor knockout mice.

Anatomical evidence indicates that cholinergic and opioidergic systems are co-localized and acting on the same neurons. However, the regulatory mechanisms between cholinergic and opioidergic system have not been well characterized. In the present study, we investigated whether there are compensatory changes of acetylcholinesterase activity and cholinergic receptors in mice lacking mu-opioid receptor gene. The acetylcholinesterase activity was determined by histochemistry assay. The cholinergic receptor binding was carried out by quantitative autoradiography using [3H]-quinuclidinyl benzilate (nonselective muscarinic receptors), N-[3H]-methylscopolamine (nonselective muscarinic receptors), [3H]-pirenzepine (M1 subtype muscarinic receptors) and [3H]-AF-DX384 (M2 subtype muscarinic receptors) in brain slices of wild-type and mu-opioid receptor knockout mice. The acetylcholinesterase activity of mu-opioid receptor knockout mice was higher than that of the wild-type in the striatal caudate putamen and nucleus accumbens, but not in the cortex and hippocampus areas. In addition, the bindings in N-[3H]-methylscopolamine and [3H]-AF-DX384 of mu-opioid receptor knockout mice were significantly lower when compared with that of the wild-type controls in the striatal caudate putamen and nucleus accumbens. However, there were no significant differences in bindings of [3H]-quinuclidinyl benzilate and [3H]-pirenzepine between mu-opioid receptor knockout and wild-type mice in the cortex, striatum and hippocampus. These data indicate that there are up-regulation of acetylcholinesterase activity and compensatory down-regulation of M2 muscarinic receptors in the striatal caudate putamen and nucleus accumbens of mu-opioid receptor knockout mice.

Increased dopamine D2 receptor binding and enhanced apomorphine-induced locomotor activity in mu-opioid receptor knockout mice.

Previous studies from our laboratory have indicated possible interactions between opioidergic and dopaminergic neurons in the central nervous system. In this study, apomorphine-induced locomotor activity and the D1 and D2 subtype dopamine receptor binding were examined in mice lacking the mu-opioid receptor genes. The ambulatory time, vertical time and total motor distance of locomotor activity were measured after administration of apomorphine (2mg/kg, i.p.) for a period of 90min. The autoradiographic studies of D1 and D2 dopamine receptors were conducted using [3H] SCH23390 and [3H] raclopride as ligand, respectively. In wild type mice that received apomorphine, 2mg/kg, i.p., the locomotor activity such as ambulatory time, vertical time and total motor distance were not significantly altered as compared with that of the saline control group. However, the locomotor activity measured was significantly increased in the same dose of apomorphine treated mu-opioid receptor knockout mice between 5 and 40min after administration. The results obtained also show that the binding of D2 dopamine receptor in mu-opioid receptor knockout mice was significantly higher than that of the wild type in the caudate putamen. However, the binding of the D1 dopamine receptor in mu-opioid receptor knockout mice was not significantly different from that of the wild type. It appears that the apomorphine treated mu-opioid receptor knockout mice showed enhancement in locomotor activity. The enhanced locomotor activity may be related to the compensatory up-regulation of D2 dopamine receptors in mice lacking mu-opioid receptor genes.