Temporal and anatomic patterns of immediate-early gene expression in the forebrain of C57BL/6 and DBA/2 mice after morphine administration.

Although morphine was previously reported to produce an instant induction of c-fos in the striatum, our recent studies have demonstrated that the expression of numerous immediate early genes (IEGs) is significantly elevated at delayed time-points (several hours) after morphine administration. To better dissect the time-course of opioid-produced IEG induction, we used in situ hybridization to examine the expression of the IEGs c-fos, zif268 and arc in the mouse forebrain at several time-points after acute morphine injection. To link drug-produced behavioral changes with the activity of specific neuronal complexes, this study was performed comparatively in the C57BL/6 and DBA/2 mouse strains, which differ markedly in their locomotor responses to opioids and opioid reward. Our study demonstrates that morphine produces two episodes of IEG induction, which are separate in time (30 min vs. 4-6 h) and which have different neuroanatomic distribution. At 30 min, one or more IEGs were induced in circumscribed subregions of the dorsal striatum (dStr) and of the nucleus accumbens (NAc) shell, as well as in the lateral septum. The observed inter-strain differences in IEG expression at 30 min support earlier proposals that activation of the dorsomedial striatum may mediate morphine-elicited locomotor stimulation (both effects were present only in the C57BL/6 strain). In contrast, NAc shell activation does not appear to be linked to morphine-elicited changes in locomotor behavior. The second IEG induction (of arc and of zif268) was more widespread, involving most of the dStr and the cortex. The second IEG induction peaked earlier in the DBA/2 mice than in the C57BL/6 mice (4 h compared with 6 h) and displayed no apparent relation to locomotor behavior. This delayed episode of IEG activation, which has largely been overlooked thus far, may contribute to the development of long-term effects of opioids such as tolerance, dependence and/or addiction.

Fos is not involved in the regulation of the proenkephalin gene by haloperidol in the mouse striatum.

The aim of the study was to determine whether the haloperidol-produced induction of the c-fos gene in the mouse striatum is the cause of the increased expression of the striatal proenkephalin (PENK) gene after repeated haloperidol administration. Mice were treated with haloperidol (1 mg/kg, i.p., once daily), MK-801 (1 mg/kg, i.p., twice daily), or with both those drugs for 9 days. Pretreatment with MK-801 prevented the haloperidol-produced induction of the striatal c-fos mRNA. In animals injected with haloperidol for 9 days, levels of the striatal PENK mRNA were increased by 100%. Coadministration of MK-801 did not reduce that increase. These results suggest that Fos is not necessary for activation of the PENK gene expression, produced by chronic haloperidol application, in the striatum.

mRNA distribution of two isoforms of somatostatin receptor 2 (mSSTR2A and mSSTR2B) in mouse brain.

The primary gene transcript of the mouse somatostatin receptor 2 is alternatively spliced giving rise to two isoforms (mSSTR2A and mSSTR2B) which differ at the C-terminus. Using reverse transcription polymerase chain reaction (RT-PCR), both mRNAs were found in the cortex, hippocampus, hypothalamus, striatum, mesencephalon, cerebellum, medulla oblongata, pituitary and in testis, however with different ratios between mSSTR2A and mSSTR2B, implicating a tissue-specific control of transcription and splicing. Among the analyzed tissues, cortex contained the highest amounts of mSSTR2A but only little mSSTR2B, whereas the pons/medulla oblongata expressed both isoforms to an equal extent. Northern blot analysis of these tissues revealed a single mRNA of about 2.4 kb using a mSSTR2A-specific hybridization probe. No additional signal was seen using a probe which hybridizes to both mSSTR2A and mSSTR2B, suggesting that the two mRNAs may be nearly identical in length. In addition, in situ hybridization indicated that mSSTR2A is predominantly expressed in mouse brain, and mSSTR2B is never expressed independently from mSSTR2A.

Evidence for Fos involvement in the regulation of proenkephalin and prodynorphin gene expression in the rat hippocampus.

For a long time Fos has been proposed to play some role in regulation of the proenkephalin (PENK) and prodynorphin (PDYN) gene expression. In recent years, however, evidence has accumulated that the transcription of both genes in several brain regions in vivo is transactivated by the transcription factor CREB rather than by Fos. In the present study, involvement of Fos in the mechanism of the PENK and PDYN gene induction in the hippocampal dentate gyrus during seizures elicited by kainic acid was studied using a knock-down technique. Pretreatment with an antisense oligonucleotide complementary to c-fos mRNA did not influence the kainic acid-elicited convulsions. It inhibited, by about 50%, the induction of Fos protein in the dentate gyrus during seizures. The subsequent induction of PENK and PDYN mRNAs was reduced by more than 60% by the c-fos antisense oligonucleotide, while constitutive expression of three other genes (alpha-tubulin, NMDA receptor-1, and GS protein alpha-subunit) was not affected. The obtained results support the view that Fos may be involved in regulation of the PENK and PDYN gene expression in the dentate gyrus during seizures, which further suggests that the mechanisms triggering the up-regulation of both these genes in the dentate gyrus may differ from these working in other brain regions, such as the striatum and hypothalamus.

Chronic morphine increases biosynthesis of nitric oxide synthase in the rat spinal cord.

The present study was undertaken to determine the influence of chronic morphine treatment on the biosynthesis of nitric oxide synthase (NOS) in the rat spinal cord using in situ hybridization and immunohistochemical methods. Repeated administration of morphine (20-100 mg/kg/day; 10 days) increased the NOS mRNA level in laminae I-IV and X 3 h after the last injection. That effect was accompanied by an increase in both the number of NOS-positive cells (24 h) and the optical density of NOS-immunoreactivity (3 and 24 h). The results indicate that repeated morphine administration increases NOS biosynthesis in the rat spinal cord, which may reflect adaptive changes accounting for development of opiate tolerance and dependence.

The NMDA receptor antagonist MK-801 markedly reduces the induction of c-fos gene by haloperidol in the mouse striatum.

The influence of the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (dizocilpine), on the haloperidol-induced increase of c-fos mRNA levels in the striata of mice was examined using the quantitative Northern blot analysis method. Administration of haloperidol (1 mg/kg. i.p.) increased striatal c-fos mRNA hybridization signal about 7-fold, as measured 30 min after injection. MK-801 (0.2-4.5 mg/kg, i.p.) dose-dependently reduced this effect of the dopamine antagonist. Higher doses of MK-801 (1.5-4.5 mg/kg) diminished c-fos mRNA increase produced by haloperidol by 80%. Such a marked inhibition of this haloperidol effect by MK-801 has not been previously reported. It suggests that the induction of c-fos gene occurring in the striatum after dopamine D2 receptor blockade is mediated in great part by endogenous glutamate acting at NMDA receptors. Since the c-fos gene protein product (Fos) is a transcription factor, our results also indicate the involvement of glutamate in the regulation of expression of other genes in the striatum.

[The effect of cadmium poisoning on glucose transformation in skeletal muscles]. / Der Einfluss der Cadmiumintoxikation auf den Glucoseumsatz in Skelettmuskeln.

Long-time effects of Cd on carbohydrate metabolism were investigated in male Wistar rats. A dose of 0.3 mg/kg body weight of Cd in acetate form was subcutaneously injected to the experimental animals twice a week through 3 months. Selected enzyme activities of glycolysis as well as concentrations of glycogen, glucose, pyruvate, lactate, triglycerides, and free fatty acids (FFA) were determined in blood serum, muscles, liver, and fatty tissues. The experimental animals differed from the control group, in that Cd intoxication was followed by decline in the blood serum only of pyruvate kinase and lactate dehydrogenase activities. In the liver, however, all enzyme activities were reduced. Glycogen glucose and FFA levels were increased. Intramuscular alterations were found to depend on the fibre type. The severest disturbance of glycolysis was recordable from red long fibres, whereas rapid white fibres were more resistant. The change in FFA concentration may be interpreted as some compensation for the impairment of carbohydrate metabolism in the energy balance.

Effect of cadmium intoxication on glucose utilization in energy metabolism of muscles.

The influence of cadmium intoxication on carbohydrate metabolism in skeletal muscles and liver of the male Wistar rats has been studied. Cadmium was administered as cadmium acetate in a dose of 0.3 mg Cd2+/kg body weight for three months. At the same time the control rats were injected with 0.9% NaCl. The animals were decapitated and samples of their skeletal muscles: the soleus muscle (composed mainly of red slow twitch fibers; ST) the gastrocnemius muscle containing two types of fibers (white fast twitch fibers FTb and red fast twitch fibers, FTa) and the liver were dissected out. In the samples of muscles, liver and serum contents of glycogen, glucose, pyruvate and lactate, as well as activities of hexokinase, pyruvate kinase and lactate dehydrogenase were measured. Intoxication of rats with cadmium for three months resulted in a reduction of glycolytic enzymes in the serum, ST and FTa muscle fibers and in the liver but did not change the activities of glycolytic enzymes in the FTb muscle fibers. The data obtained for the concentrations of glycogen in the liver and skeletal muscles suggest different mechanisms of cadmium influence on glycogen utilization in these organs.

The expression of proenkephalin and prodynorphin genes and the induction of c-fos gene by dopaminergic drugs are not altered in the straitum of MPTP-treated mice.

The expression of proenkephalin (PENK), prodynorphin (PDYN) and c-fos genes was studied in the striatum of C57B1/6 mice treated with 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP), which are used as a rodent model of Parkinson's disease (PD). Two weeks after systemic administration of MPTP (2 x 40 mg/kg, s.c. 18h apart), the lesion of the substantia nigra (SN) could be visualised by loss of the nigral tyrosine hydroxylase (TH) mRNA hybridization signal and by a 91% decrease in striatal dopamine levels. The levels of PENK and PDYN mRNAs were not significantly changed in the striatum of the lesioned mice, as compared to non-treated controls. The induction of the immediate early gene c-fos by the dopamine D2 receptor antagonist haloperidol was not altered, while the selective D1 receptor agonist SKF 38393 failed to induce c-fos in the striatum of MPTP-treated mice. These results are in contrast to the data concerning rats with the 6-hydroxydopamine (6-OHDA) lesion of the SN, which serve as another rodent model of PD. In the striata of 6-OHDA-lesioned rats, PENK gene is upregulated, PDYN gene is down-regulated and the induction of c-fos gene by D2 receptor antagonists is abolished, whereas selective D1 receptor agonists induce c-fos gene, which does not occur in non-lesioned rats. We presume that the lack of influence of the MPTP lesion in mice on the striatal gene expression was mainly caused by insufficient dopamine depletion in the striatum, which could not be increased in this model. The importance of the changes observed in 6-OHDA-lesioned rats has been discussed in the context of the mouse and primate MPTP models of PD.

Effects of morphine on gene expression in the rat amygdala.

Influence of morphine self-administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self-administer morphine, each having two 'yoked' control animals, receiving passive injections of either morphine or saline. After 40 sessions of self-administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real-time PCR showed significant changes in abundance of five transcripts: gamma protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self-administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, gamma1 subunit of GABAA receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC gamma, gamma1 subunit of GABAA and hsp70 genes.

Lipid peroxidation and activity of antioxidative enzymes in the rat model of ozone therapy.

Hypothetical, therapeutic effects of ozone were investigated in an animal model. One ml of oxygen or mixture of 40 micrograms ozone with oxygen were injected intraperitoneally to male rats for 10 days. Previously, rats had been poisoned with 50 ppm Cd2+ in drinking water for 12 weeks. Exhaustive treadmill running was applied to some animals before sacrification. Ozone injections increased iron-ascorbate-stimulated lipid peroxidation (LPO) in the liver and kidney, catalase (CAT) activity in the heart and glutathione S-transferase (GST) activity in the heart, kidney and liver. Oxygen increased GST activity in the brain and reduced glutathione peroxidase (GPX) activity in the kidney. Cadmium enhanced LPO in the liver and GST activity in the brain, heart, kidney and liver. In contrast to ozone, cadmium inhibited GPX activity in the brain, kidney and liver. Cadmium combined with ozone enhanced the changes of GPX activity in the kidney and liver, that of GST activity in the heart, kidney and liver as well as of CAT activity and LPO in kidney. The results suggest that ozone injections combined with tested factors may provoke an oxidative stress. The effects of ozone therapy can not be explained as the results of ozone action on the antioxidative enzymes in rat.