Long-term post-synaptic consequences of methamphetamine on preprotachykinin mRNA expression

Exposure to repeated high doses of methamphetamine produces long-term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by approximately 20% in the middle striatum and approximately 32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by approximately 20% in the middle striatum and approximately 14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either time point. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.     

Prenatal exposure: The effects of prenatal cocaine and methamphetamine exposure on the developing child

Prenatal substance use remains a significant issue in the United States. Initial reports regarding prenatal cocaine and methamphetamine exposure suggested profound adverse effects on child development. However, subsequent prospective, longitudinal investigations have found more subtle effects. What follows is a brief review of the health, growth, behavioral, and intellectual outcomes for children exposed to prenatal cocaine and prenatal methamphetamine. Factors that may mitigate or intensify subtle adverse effects manifested in exposed children will also be discussed. Birth Defects Research (Part C) 108:142–146, 2016. © 2016 Wiley Periodicals, Inc.     

Effects of cocaine on sodium dependent dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in the presence of 1M cocaine as a function of both [DA] and [Na]. Although DA uptake is overwhelmingly dependent on sodium, it appears that a small amount of DA uptake takes place in the absence of sodium. This contrasts with a previous study of the sodium dependence of uptake without cocaine (referred to below as control), in which uptake was found to be 100% sodium dependent. The data were fitted to several rapid equilibrium models and the minimal best fit model identified. The interaction of transporter (C), DA (S), and Na⁺ (Na) in this present model is identical to the reaction scheme found previously to fit control data (no cocaine). Whereas the control model required translocation only as CNa₂S, in the presence of cocaine (I), two additional translocated species are required to fit the data (CS and CNaS). Another previous study of the interaction of carrier and cocaine at a constant [Na]₀ predicted that cocaine interacts with a transporter site other than the DA binding site and that uptake takes place as CS and CSI. The present results are consistent with the assumption that the CS and CNaS forms of the present model are actually CSI and CNaSI, since they are required to fit a model of the sodium dependence in the presence of cocaine, but are not required in the absence of cocaine.     

Contrasting responses by basal ganglia met-enkephalin systems to low and high doses of methamphetamine in a rat model

The influence of methamphetamine (METH) on basal ganglia met-enkephalin (Menk) was studied by determining levels of this peptide in striatal, pallidal and nigral regions after administering a single low (0.5 mg/kg) or high (10 mg/kg) dose of this stimulant. The Menk levels in the striatal and pallidal areas were reduced and increased after the low- and high-dose METH treatments, respectively, 12 h after drug administration in all striatal and pallidal regions examined. The low-dose effect appeared to be principally influenced by increased activation of the dopamine D2-like receptor, while the high-dose effect seemed to result from dominance of D1-like receptor activation. However, both effects required coactivation of D1- and D2-like receptors. For the most part, both low- and high-dose METH-induced changes in Menk tissue content were fully recovered by 24 h. The Menk levels were not significantly altered in the substantia nigra 3-24 h after either METH treatment. Results reported herein indicated that striatal and pallidal Menk pathways respond differently after acute treatment with low or high doses of METH.     

Extended access of cocaine self‐administration results in tolerance to the dopamine‐elevating and locomotor‐stimulating effects of cocaine

Tolerance to the neurochemical and psychoactive effects of cocaine after repeated use is a hallmark of cocaine addiction in humans. However, comprehensive studies on tolerance to the behavioral, psychoactive, and neurochemical effects of cocaine following contingent administration in rodents are lacking. We outlined the consequences of extended access cocaine self‐administration as it related to tolerance to the psychomotor activating, dopamine (DA) elevating, and DA transporter (DAT) inhibiting effects of cocaine. Cocaine self‐administration (1.5 mg/kg/inj; 40 inj; 5 days), which resulted in escalation of first hour intake, caused reductions in evoked DA release and reduced maximal rates of uptake through the DAT as measured by slice voltammetry in the nucleus accumbens core. Furthermore, we report reductions in cocaine‐induced uptake inhibition and a corresponding increase in the dose of cocaine required for 50% inhibition of DA uptake (K_i) at the DAT. Cocaine tolerance at the DAT translated to reductions in cocaine‐induced DA overflow as measured by microdialysis. In addition, cocaine‐induced elevations in locomotor activity and stereotypy were reduced, while rearing behavior was enhanced in animals with a history of cocaine self‐administration. Here, we demonstrate both neurochemical and behavioral cocaine tolerance in an extended‐access rodent model of cocaine abuse, which allows for a better understanding of the neurochemical and psychomotor tolerance that develops to cocaine in human addicts.

     

Distribution of the beta-2 adrenergic receptor messenger RNA in the rat brain by in situ hybridization histochemistry: Effects of chronic reserpine treatment

We studied the distribution of the rat brain beta-2 adrenergic receptor (AR) mRNA, and the effects of monoamine depletions by chronic reserpine treatment using in situ hybridization histochemistry. In the control group, high level signals of beta-2 AR mRNA were observed in the parietal, frontal and piriform cortices, the medial septal nuclei, the olfactory tubercle, and the midbrain. Moderate signals were found in the striatum, the retrosplenial cortex, the hippocampus, and the thalamic nuclei. After chronic reserpine treatment, beta-2 AR mRNA levels were increased in many brain regions. The large increases were seen in the hippocampus, all thalamic nuclei, the amygdaloid nuclei, and the midbrain, followed by the striatum and the occipital cortex. The receptor up-regulation resulting from chronic monoamine depletion may be due to these increases in beta-2 AR mRNA, indicating that this up-regulation may be caused by increased receptor production rather than decreased receptor degradation.     

Neuropeptide gene expression and neural activity: Assessing a working hypothesis in nucleus caudalis and dorsal horn neurons expressing preproenkephalin and preprodynorphin

1. The working hypothesis that neuropeptide gene expression in a neuron is an indicator of that neuron's physiological activity is discussed. 2. Representative examples from the literature are presented to support the hypothesis. 3. Further, we discuss the regulation of expression of two opioid peptides, preproenkephalin and preprodynorphin, in laminae I and II of the spinal cord and in nucleus caudalis of the trigeminal nuclear complex, where they may play a role in pain modulation. 4. The expression of the opioid peptide genes can be induced by both painful and nonnoxious stimuli in neurons in time-dependent and sensory-specific fashions.     

Amphetamine-evoked c-fos mRNA expression in the caudate-putamen: the effects of DA and NMDA receptor antagonists vary as a function of neuronal phenotype and environmental context

Dopamine (DA) and glutamate neurotransmission is thought to be critical for psychostimulant drugs to induce immediate early genes (IEGs) in the caudate-putamen (CPu). We report here, however, that the ability of DA and glutamate NMDA receptor antagonists to attenuate amphetamine-evoked c-fos mRNA expression in the CPu depends on environmental context. When given in the home cage, amphetamine induced c-fos mRNA expression predominately in preprodynorphin and preprotachykinin mRNA-containing neurons (Dyn-SP+ cells) in the CPu. In this condition, all of the D1R, D2R and NMDAR antagonists tested dose-dependently decreased c-fos expression in Dyn-SP+ cells. When given in a novel environment, amphetamine induced c-fos mRNA in both Dyn-SP+ and preproenkephalin mRNA-containing neurons (Enk+ cells). In this condition, D1R and non-selective NMDAR antagonists dose-dependently decreased c-fos expression in Dyn-SP+ cells, but neither D2R nor NR2B-selective NMDAR antagonists had no effect. Furthermore, amphetamine-evoked c-fos expression in Enk+ cells was most sensitive to DAR and NMDAR antagonism; the lowest dose of every antagonist tested significantly decreased c-fos expression only in these cells. Finally, novelty-stress also induced c-fos expression in both Dyn-SP+ and Enk+ cells, and this was relatively resistant to all but D1R antagonists. We suggest that the mechanism(s) by which amphetamine evokes c-fos expression in the CPu varies depending on the stimulus (amphetamine vs. stress), the striatal cell population engaged (Dyn-SP+ vs. Enk+ cells), and environmental context (home vs. novel cage).     

A Comparison of Digoxigenin and Biotin Labelled DNA and RNA Probes for in Situ Hybridization

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.     

Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus

A detailed analysis of the differential effects of estrogen (E) compared to raloxifene (Ral), a selective estrogen receptor modulator (SERM), following estrogen receptor (ER) binding in gynecological tissues was conducted using gene microarrays, Northern blot analysis, and matrix metalloproteinase (MMP) 2 activity studies. We profiled gene expression in the uterus following acute (1 day) and prolonged daily (5 wk) treatment of E and Ral in ovariectomized rats. Estrogen regulated twice as many genes as Ral, largely those associated with catalysis and metabolism, whereas Ral induced genes associated with cell death and negative cell regulation. Follow-up studies confirmed that genes associated with matrix integrity were differentially regulated by Ral and E at various time points in uterine and vaginal tissues. Additional experiments were conducted to determine the levels of MMP2 activity in uterus explants from ovariectomized rats following 2 wk of treatment with E, Ral, or one of two additional SERMs: lasofoxifene, and levormeloxifene. Both E and lasofoxifene stimulated uterine MMP2 activity to a level twofold that of Ral, whereas levormeloxifene elevated MMP2 activity to a level 12-fold that of Ral. These data show that one of the significant differences between E and Ral signaling in the uterus is the regulation of genes and proteins associated with matrix integrity. This may be a potential key difference between the action of SERMs in the uterus of postmenopausal women.     

Co‐administration of betulinic acid and methamphetamine causes toxicity to dopaminergic and serotonergic nerve terminals in the striatum of late adolescent rats

Psychostimulant methamphetamine (METH) is toxic to striatal dopaminergic and serotonergic nerve terminals in adult, but not in the adolescent, brain. Betulinic acid (BA) and its derivatives are promising anti‐HIV agents with some toxic properties. Many METH users, particularly young men, are HIV‐positive; therefore, they might be treated with BA or its derivative for HIV infection. It is not known whether BA, or any of its derivatives, are neurotoxic in combination with METH in the adolescent brain. The present study investigated the effects of BA and binge METH in the striatum of late adolescent rats. BA or METH alone did not decrease the levels of dopaminergic or serotonergic markers in the striatum whereas BA and METH together decreased these markers in a BA dose‐dependent manner. BA+METH also caused decreases in the levels of mitochondrial complex I in the same manner; BA alone only slightly decreased the levels of this enzyme in striatal synaptosomes. BA or METH alone increased cytochrome c. METH alone decreased parkin, increased complex II and striatal BA levels. These results suggest that METH in combination with BA can be neurotoxic to striatal dopaminergic and serotonergic nerve terminals in the late adolescent brain via mitochondrial dysfunction and parkin deficit.

     

RNA原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

Programmed DNA Degradation and Nucleolar Biogenesis Occur in Distinct Organelles During Macronuclear Development in Tetrahymena

ABSTRACT. Programmed DNA rearrangements, including DNA degradation, characterize the development of the soma from the germline in a number of developmental systems. Pddl p (programmed DNA degradation 1 protein), a development-specific polypeptide in Tetrahymena , is enriched in developing macronuclei (anlagen) and has been implicated in DNA elimination and nucleolar biogenesis. Here, immunocytochemistry and fluorescent in situ hybridization (FISH) were employed to follow Pddl p and two nucleolar markers (Nopp52 and rDNA) during macronuclear development. Both Pdd 1p and Nopp52 localize to subnuclear structures, each of which resemble nucleoli. However, while true nucleoli form and persist during development, Pdd 1p-positive structures are only present for a brief period of macronuclear differentiation. Accordingly, two distinct organelles can be recognized in anlagen: (1) Pdd 1p-positive structures, which lack Nopp52 and rDNA, and (2) developing nucleoli which contain rDNA and Nopp52 but lack Pdd 1p. Taken together with recent data corroborating Pdd 1p's role in DNA elimination, we favor the hypothesis that Pdd 1p structures are unique, short-lived organelles, likely to function in programmed DNA degradation and not in nucleolar biogenesis.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...