Association between mu opioid receptor gene polymorphisms and Chinese heroin addicts.

Mu opioid receptor (MOR) has been shown to be associated with alcoholism and opioid dependence. The present study examined the involvement of a polymorphism in A118G in exon 1 and C1031G in intron 2 of the MOR gene in 200 Chinese heroin-dependent and 97 control subjects. Results showed a significant association for both A118G and C1031G polymorphisms and opioid dependence. The G allele is more common in the heroin-dependent group (39.5% and 30.8% for A118G and C1031G polymorphisms, respectively) when compared to the controls (29.4% and 21.1% for A118G and C1031G polymorphisms, respectively). This study suggests that the variant G allele of both A118G and C1031G polymorphisms may contribute to the vulnerability to heroin dependence.

Induction of tumor necrosis factor receptor type 2 gene expression by tumor necrosis factor-alpha in rat primary astrocytes.

Using reverse transcription-polymerase chain reaction (RT-PCR) technique, the messenger RNA (mRNA) for tumor necrosis factor receptor type 2 (TNF-R2, 75/80 kDa) was detected in rat primary astrocytes, with much lower level of expression when compared to that for tumor necrosis factor receptor type 1 (TNF-R1, 55/60 kDa). Upon exposure to TNF-alpha (100 U/ml), the TNF-R2 mRNA level was greatly enhanced at 8 h, while TNF-R1 mRNA remained unchanged even after 24 h. The induction of TNF-R2 gene expression by TNF-alpha was dose-dependent and seemed to be unique to TNF-alpha, as interleukin-6 (IL-6) had no significant effect on TNF-R2 expression. Since TNF-R2 was reported to mediate mitogenic and gene-inducing effects in many other cell types, it is likely that the reported proliferative effect of TNF-alpha on astrocytes was also mediated by this TNF receptor subtype. Upon exposure to TNF-alpha or lipopolysaccharide (LPS), the expression of TNF-alpha gene was induced, and the LPS-induced TNF-alpha seemed to selectively enhance the TNF-R2 gene expression. Collectively, our results suggest that the TNF-alpha or LPS-induced expression of both TNF-R2 and TNF-alpha may provide a positive control mechanism to further enhance the proliferative effect of TNF-alpha in astrocytes.

Effects of tumor necrosis factor alpha on taurine uptake in cultured rat astrocytes.

Taurine is known to play a major role in volume regulation in astrocytic swelling associated with stroke and brain trauma. Apart from brain edema, the severity of brain injury is related to the levels of inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha). TNFalpha had been shown to be closely associated with brain edema formation since the neutralization of TNFalpha reduced brain edema. Considering taurine has osmoregulatory functions in astrocytes, experiments were performed to study the effects of TNFalpha on taurine uptake in cultured astrocytes. Astrocytes exposed to 20 ng/ml of TNFalpha for 48 h showed a 91% increase in taurine uptake and significant increase was observed after 24 h exposure. This cytokine caused neither significant changes in cell volume nor taurine release. The increased in taurine uptake induced by TNFalpha was unlikely resulted from the modification of Na(+) movement because TNFalpha decreased tyrosine uptake, Na(+)-dependent transport system. In contrast to TNFalpha, interferon-gamma (IFNgamma) did not significantly affect taurine uptake. Taken together, our results did not support a suggestion that TNFalpha affects cell volume regulation via modulating taurine uptake in astrocytes. Increasing lines of evidence have demonstrated that taurine has anti-inflammatory and anti-oxidative effects, these findings therefore suggested that the increase in taurine uptake might be an adaptive response or a tool for astrocytes against oxidative stress.

Methamphetamine-induced oxidative stress in cultured mouse astrocytes.

Methamphetamine (METH) is a monoaminergic toxin that destroys dopamine terminals and causes astrogliosis in vivo. Oxidative stress has been shown to play an important role in the toxic effects of METH. In the present study, we sought to determine whether astrocytes are involved in METH-induced oxidative stress. Reactive oxygen species (ROS), ATP, and change in mitochondria membrane potential (delta psi(m)) were examined in cultured striatal, mesencephalic, and cortical astrocytes after 4 to 48 h of 4 mM METH treatment. Results showed that only striatal and mesencephalic astrocytes showed a significant increase in ROS formation from 8 and 12 h, respectively. At 48 h treatment, there was a 55 and 53% increase in ROS content in striatal and mesencephalic astrocytes, respectively, whereas cortical astrocytes showed only a 25% (not significant) increase. JC-1, a delta psi(m)-sensitive dye, showed a decrease in delta psi(m) at 8 h treatment for striatal and mesencephalic astrocytes and at 12 h for cortical astrocytes. Astrocytes from all three regions showed a similar pattern of initial increase followed by a decrease in ATP content, with striatal astrocytes resulting in a maximum depletion (39% of control value) at 48 h treatment. These findings showed that METH treatment resulted in the formation of ROS in the order of striatal > mesencephalic > cortical astrocytes. Although the formation of ROS did not severely interfere with ATP production, a depolarization of mitochondria was observed. The present study suggested that astrocytes may be an important element governing the selective vulnerability to the striatum to METH-induced oxidative stress.

Dopamine uptake blockers nullify methamphetamine-induced decrease in dopamine uptake and plasma membrane potential in rat striatal synaptosomes.

Rat striatal synaptosomes showed a reduced capacity to generate a membrane potential after being exposed to methamphetamine (METH) for 1 h. As a consequence, the dopamine (DA) synaptosomes were impeded in their electrogenic-dependent reuptake of dopamine. The capacity for METH-exposed nerve terminals to generate a membrane potential may contribute to the ability of METH to destroy dopaminergic neurons. DA uptake inhibitors (DAUIs) were found to counteract the METH-induced decrease in synaptosomal [3H]DA Vmax by stablizing METH-induced reductions in PMP. Because DAUIs showed the same effects as a Na+-channel blocker, DAUIs may prevent METH-induced destruction of dopaminergic neurons by raising plasma membrane potential.

Prenatal heroin exposure. Effects on development, acoustic startle response, and locomotion in weanling rats.

The purpose of this study was to investigate the effects of prenatal heroin exposure on the offspring in postnatal behavioral development. Pregnant Sprague-Dawley rats were injected daily (s.c.) with 10mg/kg of heroin from gestational day 8 to 20. The control dam received saline injections and the pair-fed dam received saline and was yoked to a weight-matched heroin-treated dam. Litters were culled to eight to ten pups and weighed at postnatal day (PND) 1, 8, 15, and 22. Acoustic prepulse inhibition and habituation were parameters used for evaluating the sensorimotor gating and simple form of learning respectively. Locomotor activity and rearing were assessed using the photobeam activity system. All behavioral tests were performed on the offspring at PND 21 to 23. Results showed that heroin treatment significantly reduced maternal food intake, water consumption, and weight gain. Both heroin-exposed and pair-fed groups showed a marked reduction in birth weight in both male and female pups when compared with controls; however the postnatal weight gain in heroin-exposed pups was significantly lower than the pair-fed group by 3 weeks postnatally, particularly in the female pups. These female pups also showed a significant increase in ambulation and rearing when compared to the pair-fed pups. The habituation rate in both types of behavioral tests was also decreased in these female pups as compared to control and pair-fed groups. The present study indicated that prenatal heroin exposure could result in a marked retardation of postnatal development and learning. These effects are sex related.

Oxidative stress induced by MPTP and MPP(+): selective vulnerability of cultured mouse astrocytes.

Oxidative stress has been implicated in the pathogenesis of Parkinson's disease. In the present study, reactive oxygen species (ROS) formation and antioxidant enzyme superoxide dismutase (SOD) activities were examined in cultured cortical, striatal and mesencephalic mouse astrocytes after 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP(+)) treatment. Linear regression analysis showed that control mesencephalic (slope coefficient=0.01) astrocytes had a three-fold (F-test, p<0.05) greater rate of change in ROS production when compared to cortical (0.003) or striatal (0.003) astrocytes. However, when treated with 500 microM MPTP for 120 min, mesencephalic and striatal astrocytes demonstrated a decreased and increased rate of change in ROS production respectively. On the other hand, when treated with 10 microM MPP(+), a significant increase in the rate of change in ROS formation was observed in both mesencephalic and striatal astrocytes, with mesencephalic astrocytes producing a four-fold greater increase when compared to striatal astrocytes. Cortical astrocytes did not show any significant changes in ROS production when treated with MPTP or MPP(+). When astrocytes were treated with MPTP over a 24 h period, striatal astrocytes demonstrated significant increases in SOD activity to 12 h, followed by a return towards control levels after 8 h treatment. In contrast, mesencephalic astrocytes showed trends for a decrease in SOD production as well as a significant decrease in ATP levels by 24 h MPTP treatment. The present results suggested that mesencephalic astrocytes are more vulnerable to oxidative stress when compared to striatal astrocytes, given their greater rates of ROS production at basal and MPP(+) conditions. Striatal astrocytes, on the other hand, may have a more protective capacity against oxidative stress by producing greater SOD activities.

A selective regional response of cultured astrocytes to methamphetamine.

Methamphetamine (METH) has long-lasting neurotoxic effects on the dopamine and forebrain serotonin systems. It was reported that METH would induce the release of glutamate within the striatum and that it also caused astrogliosis. The mechanisms of this release and subsequent neurotoxicity are not well defined. The aim of this study was to examine the response of cultured astrocytes after METH-induced injury. Astrocytes were cultured from neonatal C57B1/6 mice brains. Cells were obtained from the mesencephalon, striatum and cortex in order to examine any regional differences. Cells were treated with 4mM METH for 4, 8, 12, 24 and 48 hr. Lactate dehydrogenase (LDH) levels were used as a measure of cell viability. At various time points, Western blot analyses were performed to study the change in GFAP and vimentin (markers for astrogliosis) levels. Change in glutamine synthase (GS), the enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia in astrocytes, was also examined. The results showed that METH caused marked astrogliosis in striatal and mesencephalic astrocytes. Cells were transformed from protoplasmic (inactive) to fibrous (reactive) form after 48 hr treatment. There were also large amounts of vacuoles present in the cytoplasm of these cells. LDH results showed that there was only slight increase in enzyme levels after 48 hr treatment suggesting that the astrogliosis observed was not due to the decrease in cell viability. The amount of GS were depleted more rapidly in striatal astrocytes (50% of control by 8 hr treatment) followed by mesencephalic astrocytes (reaching 10% of control by 48 hr treatment). Cortical astrocytes showed only a 48% depletion by 48 hr treatment, indicating that they are more resistant to METH-induced toxicity. The rapid depletion of GS obtained in striatal and mesencephalic astrocytes suggested that astrocytes of the dopaminergic system are more sensitive to METH-induced injury. This may be due to the direct effects of METH-induced oxidative stress on the mitochondria of these cells resulting in GS depletion and astrogliosis.

The time course and regional variations of lipid peroxidation after diffuse brain injury in rats.

Free radicals are generated after head injury. These radicals rapidly react with polyunsaturated fatty acids in the cell membrane and cause membrane destruction. This process is called lipid peroxidation. Malondialdehyde (MDA) is one of the end products of lipid peroxidation, and it is a frequently used indicator of lipid peroxidation in biological tissues. Using a diffuse head injury animal model, we studied the time course of lipid peroxidation in different regions of injured rat brains. In the present study, the MDA levels were 36.7%, 41.8%, and 35.1% greater than sham at one hour after injury at the frontal, parietal, and brain stem, respectively (p < 0.0001). The MDA levels in these regions continued to increase and peaked a 4 hours after the injury. The levels slowly decreased, and by 24 hours, they were still significantly higher than the sham control's. The elevation of MDA levels was less in the striatum and the temporal regions at one hour. They were 16.9% and 13.3%, respectively (p < 0.002). The MDA levels in these two regions continued to increase even after 4 hours of injury, but the degree of elevation never exceeded 35%. The results demonstrate that there is an immediate, posttraumatic burst of MDA production, suggesting the formation of free radicals after diffuse head injury. Even though all the regions sampled show the same effect, certain regions are less affected by this diffuse head injury animal model.

Prenatal cocaine exposure revealed minimal postnatal changes in rat striatal dopamine D2 receptor sites and mRNA levels in the offspring.

It has been reported from this laboratory that prenatal cocaine exposure results in the postnatal transient alterations of rat striatal dopamine uptake sites examined from postnatal 0-32 wk. The present study aims to examine whether this will result in a direct/indirect stimulation of dopamine D2 receptors. Pregnant rats were dosed orally with cocaine hydrochloride (60 mg/kg/d) from gestational day (GD) 7-21. Control animals received an equivalent volume of water. The striatum from the offspring at postnatal 0-32 wk was examined. The radioligand [3H]sulpiride was used for the Scatchard analysis of the D2 receptors, and the changes in the levels of mRNA for the D2 receptor were studied using Northern blot analysis. Results from the present study revealed that in the control group, there was an age-dependent increase in the number of D2 receptor sites (Bmax: 44.00 +/- 2.12 to 178.00 +/- 45.10 fmol/mg protein) and in the levels of D2 mRNA from PN0-32 wk with the most rapid increase occurring during the first 4 wk of postnatal development. Prenatal cocaine exposure resulting in only a significant decrease (p < 0.001) in the number of D2 receptor sites at PN0 wk and in a 10% increase in mRNA levels at PN3, 4, and 12 wk. It was concluded from this study that prenatal cocaine exposure resulted in minimal postnatal changes in the dopamine D2 receptor.

Postnatal changes in [3H]mazindol-labelled dopamine uptake sites in the rat striatum following prenatal cocaine exposure.

The present study showed that prenatal cocaine exposure (60 mg/kg/day) has a transient effect on the [3H]mazindol-labelled dopamine uptake sites in the striatum of the rat offspring examined from postnatal week 0-32. There is a 39% and 21% decrease in the number of binding sites (Bmax) in the cocaine-exposed group at postnatal weeks 3 and 4, respectively, with a recovery to near normal values by postnatal week 8.

Binding of atrial and brain natriuretic peptides to cultured mouse astrocytes from different brain regions and effect on cyclic GMP production.

We prepared primary cultures of mouse astrocytes from the cerebral cortex, hypothalamus, and cerebellum to examine the possibility of regional disparity in binding of human atrial and porcine brain natriuretic peptides (hANP, pBNP) and their effect on cyclic guanosine monophosphate (cGMP) production. 125I-hANP and 125I-pBNP bound in a specific and saturable manner to all three regions. For both peptides, Scatchard analysis suggested a single population of binding sites on astrocytes from all three regions. No significant differences were observed in the maximal binding capacities (Bmax) or binding dissociation constants (KD) between the two peptides in the astrocyte preparations from different regions. ANP and BNP also evoked cGMP stimulation in a similar, dose-dependent fashion in astrocytes from all three regions, with maximal responses to both peptides reached at a concentration above 1 microM. While BNP elicited a greater maximal cGMP accumulation than ANP, no difference could be demonstrated in the cGMP responses to either peptide between brain regions. Thus we have been unable to demonstrate regional heterogeneity in the responsiveness of astrocytes to ANP and BNP.

Loss and recovery of activities of alpha+ and alpha isozymes of (Na(+) + K+)-ATPase in cortical focal ischemia: GM1 ganglioside protects plasma membrane structure and function.

Alterations in cellular membrane structure and the subsequent failure of its function after CNS ischemia were monitored by analyzing changes in the plasma membrane marker enzyme (Na(+) + K(+)-ATPase. The levels of two isozymes of (Na(+) + K(+)-ATPase, alpha+ and alpha, which have distinct cellular and anatomical distributions, were studied to determine if differential cellular damage occurs in primary and peri-ischemic injury areas. The efficacy of monosialoganglioside (GM1) treatment was assessed, since this glycosphingolipid has been shown to reduce ischemic injury by protecting cell membrane structure/function. Using a rat model of cortical focal ischemia, levels of both ATPase isozyme activities were assayed in total membrane fractions from primary ischemic tissue (parietal cortex) and three peri-ischemic tissue areas (frontal, occipital, and temporal cortex) at 1, 3, 5, 7, and 14 days after ischemia. No significant loss of either isozyme's activity occurred in any tissue area at 1 day after ischemia. At 5 days, in the primary ischemic area, both isozyme activity levels decreased by 70-75%. The alpha+ enzyme activity loss persisted up to 14 days, while a 17% recovery in alpha activity occurred. In the three peri-ischemic tissue areas, enzyme activity losses ranged from 42%-59% at 3 days after ischemia. A complete restoration of both isozyme activities was seen at 14 days. After three days of GM1 ganglioside treatment there was no loss of total (Na*+) + K(+)-ATPase activity in the three peri-ischemic areas, and a significantly reduced loss in the primary infarct tissue. An autoradiographic analysis of brain coronal sections using 3H-ouabain supports the enzymatic data and GM1 effects. Reductions in 3H-ouabain binding in all cortical layers at 3 days after ischemia were visualized. GM1 treatment significantly reduced these 3H-ouabain binding losses. In summary, time-dependent quantitative changes in activity levels of ATPase isozymes (alpha+ and alpha) reflect the different degree of membrane damage that occurs in primary vs. peri-ischemic tissues (e.g., irreversible vs. reversible membrane damage), and that ischemia affects cell membranes of all neural elements in a largely similar fashion. GM1 ganglioside was found to reduce plasma membrane damage in all CNS cell types.

Immunohistochemical localization of substance P, enkephalin and serotonin in the developing human retina.

The localization of substance P (SP), enkephalin (ENK) and serotonin (5-HT) in the retinae of 12 human embryos/fetuses ranging in age from 6-30 weeks was determined immunohistochemically using the PAP method. At the 6 week stage [crown rump length (CRL) unknown due to incomplete specimen], the developing retina consisted of a single undifferentiated cell mass from which immunoreactive cells were absent. By 90 mm CRL (10th week of gestation), the retina was composed of an outer neuroblastic layer, an inner plexiform layer and an innermost layer of ganglion cells. At this stage, SP, ENK and 5-HT positive cells were detected solely in the outer neuroblastic layer. By 140 mm CRL (17 weeks), the retina consisted of cell layers similar in number and type to those of the adult retina. In specimens 140-216 mm CRL (gestation ages 17-24 weeks), SP, ENK and 5-HT neurons were present in the outer nuclear, inner nuclear and inner plexiform layers. In addition, 5-HT positive neurons and fibers were evident in the outer plexiform layer. By 285-295 mm CRL (26-30 weeks), neurons in the ganglion cell layer and the fovea were also SP and ENK positive. In earlier specimens, the cell bodies alone were immunopositive and not until 142 mm CRL (17 weeks) were positive processes observed. Finally, the presence of SP, ENK and 5-HT immunopositive structures occurred in a sequence from outer to inner layers of the developing human retina.