FIGURES AND TABLES

Figure 1.

Representative 2-DE gel images of the hippocampal proteins from mice differentially treated with saline (A) or morphine (B). Mice were treated with saline or an increasing dose (from 20 to 100 mg/kg, s.c.) of morphine for 10 days. Hippocampal protein (150 g) was separated on PH 4-7 linear IPG strips (170 cm) followed by separation on 12% SDS-PADE gels as described in Materials and Methods. The gels were visualized using silver staining. The samples for 2-DE were prepared from hippocampus of nine mice. The horizontal dimension represents a PH gradient of 4-7, and the vertical dimension is indicated by molecular markers. Labelled spots (A-C) are proteins that were differentially expressed in the hippocampus from mice differentially treated with saline or morphine.

Figure 2.

Decreased expression of Fe-S protein 1 of NADH dehydrogenase, PDHC-E2 and LDH2 in the hippocampus from mice chronically treated with morphine. (A) 2-DE images of the hippocampal proteins derived from the saline-, and chronic morphine-treated mice. (B) Quantification of the alterations of protein expression between the two groups. Mice were treated with saline or an increasing dose (from 20 to 100 mg/kg, s.c.) of morphine for 10 days. The hippocampus extracts from mice differentially treated with saline or morphine were separated by 2-DE followed by silver staining. Protein levels were quantified using PD-Quest software. Data are presented as means SEM of at least three separate experiments (n = 9). ^** p < 0.01 versus saline-treated mice.

Figure 3.

Naltrexone reversed chronic morphine-induced downregulation of Fe-S protein 1 of NADH dehydrogenase, PDHC-E2 and LDH2. (A) 2-DE images of the hippocampal proteins from chronic morphine-, and chronic morphine plus naltrexone-treated mice. (B) Quantification of the alterations of protein expression between the two groups. Mice were treated with an increasing dose (from 20 to 100 mg/kg, s.c.) of morphine alone or morphine in combination with naltrexone (4 mg/kg, i.p.) for 10 days. The hippocampus extracts from differentially treated mice were separated by 2-DE followed by silver staining. Proteins displaying altered levels were quantified using PD-Quest software. Data are presented as means SEM of at least three independent experiments (n = 9). ^** p < 0.01 versus combination-treated mice.

Figure 4.

Downregulation of mRNAs corresponding to Fe-S protein 1 of NADH dehydrogenase, PDHC-E2 and LDH2 in the hippocampus of chronic morphine-treated mice. (A) Gel electrophoresis of PCR products from mouse hippocampal cDNA. A single band of the expected size for each gene was detected on agarose gel. G1, G2, G3 and G4 represent the RT-PCR products corresponding to mRNAs of LDH2 (192 bp), Fe-S protein 1 of NADH dehydrogenase (210 bp), PDHC-E2 (141 bp) and -actin (197 bp), respectively. The marker lane shows a ladder of 14 bands from 100 to 3 000 bp (lane1). (B) Fold of reduction of mRNAs of Fe-S protein 1 of NADH dehydrogenase, PDHC-E2 and LDH2 in the hippocampus from chronic morphine-treated mice. Data are presented as means SEM of three independent experiments (n = 6). ^** p < 0.01 versus saline-treated mice.

Figure 5.

Reduction of ATP levels and impairment of glucose metabolism in the hippocampus derived from mice chronically treated with morphine. (A), Decrease of the hippocampal ATP levels by chronic but not acute morphine treatment and reversal of the chronic morphine effect by concomitant administration of naltrexone. (B), Increase of the hippocampal ATP levels by intraperitoneal injection of D-glucose (200 mg/kg, i.p.) in acute morphine-treated but not in chronic morphine-treated mice. Mice were treated either acutely with a single dose of morphine (100 mg/kg, s.c.) or chronically with an increasing dose of morphine (s.c.) alone or in combination with naltrexone (4 mg/kg, i.p.) for 10 days. ATP levels in the hippocampus from differentially treated mice were measured as described in Materials and Methods. Data are presented as means SEM of at least three independent experiments (n = 6). ^* p < 0.05 versus saline-treated mice (A) or versus acute morphine-treated mice without glucose injection (B). Sal: saline; Mor: morphine; Glu: D-glucose; mGlu: 3-O-methyl-glucose; Nal: naltrexone.

Figure 6.

Effect of D-glucose on naloxone-precipitated morphine withdrawal in acute and chronic morphine-treated mice. (A) Inhibition of naloxone-precipitated morphine withdrawal jumping by administration of D-glucose but not by 3-O-methyl-glucose in acute morphine-treated mice. (B) Failure of suppression of naloxone-precipitated withdrawal jumping by D-glucose in chronic morphine-treated mice. Mice were treated either acutely with a single dose of morphine (100 mg/kg, s.c.) or chronically with an increasing dose of morphine (s.c.) alone or in combination with naltrexone (4 mg/kg, i.p.) for 10 days. D-glucose or 3-O-methyl-glucose (8 nmol per side) was injected intrahippocampally before morphine (acute) or final morphine administration (chronic), followed by precipitation with naloxone (2 mg/kg, i.p.). Withdrawal jumping was detected as described in Materials and Methods. Data are presented as means SEM of at least three independent experiments (n = 10). ^* p < 0.05, ^** p < 0.01 versus acute morphine-treated mice (A) or versus chronic morphine-treated mice (B). Mor: morphine; Nal: naltrexone; Glu: glucose; mGlu: 3-O-methyl-glucose.

Figure 7.

Improvement of acute but not chronic morphine treatment-induced memory deficits by D-glucose. Mice were treated either acutely with a single dose of morphine (10 mg/kg, s.c.) or chronically with morphine for 10 days. Escape latency of each mouse was recorded 20 min after morphine administration with the Morris water maze test as described in Materials and Methods. D-glucose or 3-O-methyl-glucose (500 mg/kg) was given by intraperitoneal injection before morphine (acute group) or final morphine administration (chronic group). Data are presented as means SEM (n = 12). ^** p < 0.01 versus the saline group, ^## p < 0.01 versus the acute morphine group, ^* p < 0.05 versus the saline group. Mor: morphine; Nal: naltrexone; Glu: glucose; mGlu: 3-O-methyl-glucose.

Table 1 - Eight differentially expressed proteins between chronic saline- and morphine-treated mice.