Early changes in neuropeptide mRNA expression in the striatum following reserpine treatment

Chronic dopamine depletion produces neurochemical changes within the striatum as well as enhanced behavioral and metabolic responses to dopamine agonists. Changes in striatal neuropeptides have been consistently described, including increased expression of preproenkephalin mRNA and decreased expression of preprotachykinin and prodynorphin mRNA. Acute dopamine depletion following treatment with reserpine also produces enhanced behavioral and metabolic responses to agonist treatment which develop rapidly. In the present study, we used in situ hybridization histochemistry to investigate whether acute neurochemical changes occur following reserpine treatment. We evaluated neuropeptide mRNA expression in the striatum and nucleus accumbens at several time points from 6 to 120 h following single doses of reserpine and AMPT. The aim of these studies was to determine if changes in neuropeptide mRNA expression occur following acute dopamine depletion and whether such changes are specific to the striatum. Changes in striatal neuropeptide mRNA expression developed rapidly. Preproenkephalin mRNA expression by striatopallidal neurons was unchanged at 48 h, but increased by 44% at 120 h. Preprotachykinin mRNA expression in striatonigral neurons was increased at 6 h and then fell, with a maximal decrease of 45% at 48 h and partial recovery by 120 h. Prodynorphin mRNA expression was unchanged. Expression of preproenkephalin and preprotachykinin mRNA was also examined in subregions of the striatum and the nucleus accumbens. Expression of preproenkephalin mRNA was uniform in the striatum and higher in the core than the shell of the nucleus accumbens. Preprotachykinin mRNA expression in the striatum was higher in the lateral quadrants and was higher in the shell than in the core of the nucleus accumbens. The changes in neuropeptide mRNA following treatment with reserpine were only found in the striatum. These data provide further evidence for early alterations in neuronal function in the striatum following acute dopamine depletion and suggest that neuropeptide expression by striatonigral neurons may be more rapidly regulated in response to changes in dopamine levels.     

Effect of amphetamine on the expression of the metabotropic glutamate receptor 5 mRNA in developing rat brain

Mechanisms underlying the acute effects of amphetamine (AMP) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) and specific ³H-glutamate binding in the developing rat brain. Each of the postnatal day (P) 4, P21 and P60 rats received one intraperitoneal injection of AMP, 5 mg/kg or saline and were sacrificed one hour later. In situ hybridization analysis revealed that the AMP treatment raised the levels of the mGluR5 mRNA by 9–28% in the neurons of the layer 5 of motor and somatosensory cortices, whereas reduced the levels by 12–28% in the layer 5 of perirhinal cortex and the ventromedial part of caudate-putamen of the 3 ages. In the layer 2/3 neurons of cingular cortex, an 18% higher and 14% and 22% lower than control levels of the mRNA were detected in the P4 and in the P21 and P60 rats injected with AMP. Moreover, the levels of mGluR5 mRNA in the hippocampi and dentate gyri were elevated by AMP to 110–151% of controls in the rats of 3 ages. Reversible ³H-glutamate binding assay showed an increase of 25% and a 12% decrease in the binding levels in the cortices of AMP-treated P4 and P21 rats. The AMP administration also produced a 27% reduction and 62% elevation in the binding of the hippocampi of P4 and P60 rats. The results reveal age- and region-dependent changes in the expression of the glutamate receptors induced by AMP and may indicate differential plastic capability of the neurons to the drug perturbation.     

Developmental expression offlt3 mRNA in the mouse brain

The expression and localization offlt3 mRNA were investigated using Northern blotting andin situ hybridization during the developmental process of the mouse brain. By Northern blotting, the expression of theflt3 gene was not detected in embryonic, neonatal, or early postnatal brains, but was markedly increased with age. In adult mice,flt3 was abundantly expressed in the cerebellum, moderately in the pons, and faintly in the thalamus and the cerebral cortex. Byin situ hybridization, some neurons that expressedflt3 formed synaptic connections with each other. The present findings suggest that theflt3 gene expression is related to the cell type and the developmental process.     

Altered vasoactive intestinal polypeptide gene expression in the fetal rat suprachiasmatic nucleus following prenatal serotonin deficiency

This study has evaluated the possible role of serotonin, a potential morphogen, in the regulation of vasoactive intestinal polypeptide (VIP) gene expression in the target neurons of the suprachiasmatic nucleus (SCN) before and after the onset of the serotonin neurotransmitter function. VIP gene expression was quantified by in situ hybridization of the corresponding mRNA on cryostat sections with subsequent film autoradiography and densitometry. The content of VIP mRNA was measured in the SCN in fetuses at the 21st embryonic day (E21) and in postnatal rats at day 11 (P11) following chronic depletion of serotonin by p-chlorophenylalanine, an inhibitor of serotonin synthesis. This inhibitor was daily injected to pregnant rats for E13–20 or to postnatal animals for P2–10. Results of this study indicate that prenatal serotonin depletion caused a significant increase in VIP mRNA content in the SCN compared to control fetuses. On the contrary, the same treatment performed postnatally did not change VIP mRNA levels in the SCN. These data suggest that the VIP gene expression in differentiating target neurons of the SCN might be under serotonin inhibitory control during prenatal neurogenesis, prior to the onset of the serotoninergic neurotransmission.     

Constitutive expression of c-myb mRNA in the adult rat brain

In this study, we demonstrated the c-myb mRNA expression in the adult rat brain using an in situ hybridization technique. We found c-myb mRNA signals in the various regions of the forebrain and midbrain including the cerebral cortex, thalamus, hippocampus, hypothalamus, superior and inferior colliculi and central gray. In the cerebellum, a diffuse signal was found in the granular layer while some positive cells were detected in the molecular layer as well. In addition, a number of cells showed intense signals in many nuclei of the medulla oblongata. The constitutive expression of c-myb mRNA in the different kinds of neural cells suggests that this gene might be involved in the normal function of these neurons.     

褪黑素加强大鼠脑内前强啡肽原mRNA的表达

采用原位杂交技术结合计算机图像处理技术,观察褪黑素对大鼠脑内某些核团内神经细胞的前强啡肽原（ＰＰＤ）ｍＲＮＡ表达的影响。实验大鼠分２组,给药药组大鼠间隔１２ｈ胜利腔注射ＭＥＩ２次,每次剂量９０ｍｇ／ｋｇ,对照组注射配药液。     

Biphasic expression of TGF-β1 mRNA in the rat brain following permanent occlusion of the middle cerebral artery

Two patterns of transforming growth factor-β1 (TGF-β1) expression were identified in brains of normotensive rats following permanent occlusion of the middle cerebral artery (MCAO). First, a relative increase of TGF-β1 mRNA by 37% was found at 12 h after MCAO in the ipsilateral cingulate cortex as compared to the homotopic contralateral region. The cingulate cortex is located distant from the ischemic territory. Treatment with the glutamate receptor antagonists MK-801 and NBQX did not reduce this expression (34% and 26% increase, respectively). Therefore, peri-infarct depolarization waves were probably not responsible for induction. Secondly, an increase of TGF-β1 mRNA by 116% was found at 7 days after MCAO within infarcted tissue. This expression was not reduced by the glutamate receptor antagonists MK-801 (increase 140%) and NBQX (increase 137%), either. TGF-β1 mRNA expression in the cingulate cortex at 12 h after MCAO is possibly mediated by neurons and astroglia and may support cell survival. Expression in the infarcted tissue at 7 days after MCAO is most likely related to the invasion of monocytes and may be involved in the downregulation of inflammatory events, in neoangiogenesis, and in formation of a glial scar around the infarct.     

NMDA receptor mediated changes in IGF-II gene expression in the rat brain after injury and the possible role of nitric oxide

This study was undertaken in order to investigate the role of insulin-like growth factor (IGF)-II, c-fos, N-methyl-D-aspartate (NMDA) receptors, and nNOS in the cellular processes following a penetrating brain injury. IGF-II mRNA levels, as determined by Northern analysis, were decreased at 4, 8, and 24 h after brain injury, in the lesioned, compared to the contralateral intact hemisphere. Forty-eight and 72 h after the injury, there was no difference between the lesioned and the contralateral intact hemisphere in IGF-II mRNA levels. c-fos mRNA levels followed a parallel, but opposite course: They were increased at 4, 8 and 24 h after the injury, while at 48 and 72 h c-fos mRNA levels in the lesioned hemisphere did not differ from those in the intact. Administration of MK-801 reversed the injury-induced decrease in IGF-II mRNA levels. Administration of MK-801 resulted in an increase in IGF-II mRNA in both the intact and the lesioned hemispheres. Brain injury resulted in an increase in nNOS immunopositive cells in the hippocampal formation, which was detectable at 4 and 12, but not 48 h after the injury. These results suggest that IGF-II, c-fos, NMDA receptors and nNOS are involved in the cellular responses to brain injury.     

Effects of dizocipine maleate on mitochondrial ultramicrostructure in neurons following traumatic brain injury in neonatal rats*★ A quantitative time-course analysis

BACKGROUND： The effects of N-methyl-D-aspartic acid （NMDA） receptor antagonist on neurodegeneration in the immature brain following traumatic brain injury （TBI） are still widely unknown. OBJECTIVE： To study the effects of dizocipine maleate （MK-801）, a non-competitive NMDA receptor antagonist, on mitochondrial ultramicrostructure of neurons in the ipsilateral cingulate cortex and hippocampus after TBI in neonatal rats, and to analyze the optimal time interval of MK-801 administration （1 mg/kg）. DESIGN： Completely randomized controlled study.
SETTING： Shanghai Jiao Tong University.
MATERIALS： Eight 7-day-old neonatal SD rats, irrespective of gender, were provided by Experimental Animal Center, Medical College of Fudan University. The experiment was approved by a local ethics committee. MK-801 was provided by Sigma. A CM-120 transmission electron microscope （Philips, Holland） was used for tissue analysis.
METHODS： This study was performed at the Departments of Anatomy, Neuromorphology, and Biophysics, Medical College of Shanghai, Jiaotong University, between October 2006 and January 2007. Focal models of contusion and laceration of brain were established by the free-falling impact method. Eight rats were randomly divided into a normal control group （n = 2 ） and a MK-801 group （n = 6）. Rats in the normal control group did not receive model establishment and administration, and they were only analyzed by an electron microscope. In the MK-801 group, the cingulate cortex was damaged using a contusion device. MK-801 （1 mg/kg） was intraperitoneally injected 30 minutes before lesion, immediately after lesion, and 30 minutes after lesion （n = 2 for each time point）.
MAIN OUTCOME MEASURES： The cingulate cortex and hippocampal tissues from the injured side were removed 24 hours after lesion and routinely processed for analysis of neuronal ultramicrostructure using transmission electron microscopy. RESULTS： Differential therapeutic effects of MK-801 （1 mg/kg） at d     

Striatal cannabinoid CB1 receptor mRNA expression is decreased in the reserpine-treated rat model of Parkinson's disease

High levels of both endocannabinoids and endocannabinoid receptors are present in the basal ganglia. Attention has recently focused on the role of endocannabinoids in the control of movement and in movement disorders of basal ganglia origin such as Parkinson's disease. We investigated CB1 cannabinoid receptor mRNA expression in the reserpine-treated rat model of Parkinson's disease using in situ hybridization. Reserpine treatment caused a topographically organized reduction in CB1 receptor mRNA expression in the striatum (ranging from 11.6% medially to 53.6% laterally and dorsally). No change in CB1 receptor mRNA expression was observed in the cerebral cortex or septum. This reduction in CB1 receptor mRNA expression may be secondary to increased endocannabinoid stimulation of the receptor as increased basal ganglia endocannabinoid levels have been shown to occur in this model of Parkinson's disease. The data support the idea that cannabinoid receptor antagonists may provide a useful treatment for the symptoms of Parkinson's disease.     

Ontogenic expression of two α-1 adrenergic receptor subtypes in the rat brain

α-1A/D and α-1B adrenergic receptor subtype mRNA expression was studied during pre-and postnatal rat brain development. Oligonucleotide probes were generated to distinguish these two homologous subtypes byin situ histochemical analysis in E14, E16, E19, P0, P8, P14, P21, and adult animals. α-1B adrenergic receptor mRNA expression was noted as early as the E14 animal and demonstrated specific regional and temporal expression. α-1A/D adrenergic receptor mRNA expression was limited in the E19 and P0 animal but increased in intensity with aging. Specific regional and temporal expression differed between the two subtypes. The regional localization for both subtypes appeared to be stable after P21 but the intensity of expression for both subtypes decreased between P21 and adulthood, which is a finding that correlates with previous ligand-binding data. Different subtypes of homologous receptors have very different ontogenic patterns of distribution and may account for previous discrepancies in ligand-binding data and differential tissue responses to various ligands during development.     

Postinjury Magnesium Treatment Attenuates Traumatic Brain Injury-Induced Cortical Induction of p53 mRNA in Rats

Administration of magnesium has been shown to be neuroprotective in experimental models of traumatic brain injury (TBI). The present study examined the effect of magnesium on posttraumatic regional induction of p53, a gene associated with induction of cell death. Male Sprague-Dawley rats (350-400 g, n = 26) were anesthetized with sodium pentobarbital and subjected to either lateral fluid percussion brain injury of moderate severity (2.4-2.6 atm; n = 22) or sham surgery (n = 4). At 15 min postinjury, animals randomly received an intravenous bolus of either 125 micromol magnesium chloride (n = 12) or saline vehicle (n = 10). Expression of p53 mRNA was not observed in any uninjured animal. By 6 h postinjury in vehicle-treated, brain-injured animals, p53 mRNA was induced in the cortex, dentate hilus, and CA3 regions of the hippocampus and geniculate nuclei of the thalamus, ipsilateral to the impact site. Posttraumatic magnesium treatment significantly reduced the number of labeled cells in the injured cortex (P < 0.05), but not in the hippocampus or thalamus. p53 mRNA expression returned to near baseline in all animals by 24 h postinjury. These data suggest that the neuroprotective effects of magnesium treatment may be related, in part, to a downregulation in expression of a gene associated with induction of cell death and further support the utility of magnesium as a pharmacotherapy for TBI.     

[H]MK-801 binding sites in neonate rat brain

[³H]MK-801 binding sites are present in neonate rat brain as early as 3 days after birth. Immature hippocampus and cortex contain approximately one sixth the concentration of binding sites of the adult, while brainstem concentration is twice as high as that of adult. [³H]MK-801 binding is stimulated by glutamate and glycine and blocked by phencyclidine and Mg²⁺ both in 7-day-old neonate and adult, indicating that as early as 7 days postnatally, theN-methyl-d-asparatate-type glutamate receptor and MK-801 binding site are functionally coupled.     

大鼠脑缺血区皮层XIAP表达的动态变化及其意义

目的观察大鼠脑缺血区皮层XIAP表达的动态变化,探讨其在缺血后抗神经元凋亡过程中的可能机制。方法采用RT-PCR,免疫组化和western blot等方法分别从mRNA和蛋白水平测定脑缺血区皮层不同时间点XIAP表达的变化。结果XIAP转录水平和蛋白表达的改变呈时间依赖性;RT-PCR显示模型组大鼠在脑缺血后3h就有XIAP mRNA转录水平的明显上调,持续至24h后逐渐下降;免疫组化和western blot显示在脑缺血后6h有XIAP蛋白表达的增加,12h时XIAP蛋白表达至高峰（P〈0．01）,48h基本恢复至缺血前水平。结论XIAP在脑缺血过程中可能具有抗凋亡作用。     

Distribution of serotonin 2A and 2C receptor mRNA expression in the cervical ventral horn and phrenic motoneurons following spinal cord hemisection

Cervical spinal cord injury leads to a disruption of bulbospinal innervation from medullary respiratory centers to phrenic motoneurons. Animal models utilizing cervical hemisection result in inhibition of ipsilateral phrenic nerve activity, leading to paralysis of the hemidiaphragm. We have previously demonstrated a role for serotonin (5-HT) as one potential modulator of respiratory recovery following cervical hemisection, a mechanism that likely occurs via 5-HT2A and/or 5-HT2C receptors. The present study was designed to specifically examine if 5-HT2A and/or 5-HT2C receptors are colocalized with phrenic motoneurons in both intact and spinal-hemisected rats. Adult female rats (250-350 g; n = 6 per group) received a left cervical (C2) hemisection and were injected with the fluorescent retrograde neuronal tracer Fluorogold into the left hemidiaphragm. Twenty-four hours later, animals were killed and spinal cords processed for in situ hybridization and immunohistochemistry. Using (35)S-labeled cRNA probes, cervical spinal cords were probed for 5-HT2A and 5-HT2C receptor mRNA expression and double-labeled using an antibody to Fluorogold to detect phrenic motoneurons. Expression of both 5-HT2A and 5-HT2C receptor mRNA was detected in motoneurons of the cervical ventral horn. Despite positive expression of both 5-HT2A and 5-HT2C receptor mRNA-hybridization signal over phrenic motoneurons, only 5-HT2A silver grains achieved a signal-to-noise ratio representative of colocalization. 5-HT2A mRNA levels in identified phrenic motoneurons were not significantly altered following cervical hemisection compared to sham-operated controls. Selective colocalization of 5-HT2A receptor mRNA with phrenic motoneurons may have implications for recently observed 5-HT2A receptor-mediated regulation of respiratory activity and/or recovery in both intact and injury-compromised states.     

Xenon is an inhibitor of tissue-plasminogen activator: adverse and beneficial effects in a rat model of thromboembolic stroke

Preclinical evidence in rodents has proven that xenon may be a very promising neuroprotective agent for treating acute ischemic stroke. This has led to the general thinking that clinical trials with xenon could be initiated in acute stroke patients in a next future. However, an unappreciated physicochemical property of xenon has been that this gas also binds to the active site of a series of serine proteases. Because the active site of serine proteases is structurally conserved, we have hypothesized and investigated whether xenon may alter the catalytic efficiency of tissue-type plasminogen activator (tPA), a serine protease that is the only approved therapy for acute ischemic stroke today. Here, using molecular modeling and in vitro and in vivo studies, we show (1) xenon is a tPA inhibitor; (2) intraischemic xenon dose dependently inhibits tPA-induced thrombolysis and subsequent reduction of ischemic brain damage; (3) postischemic xenon virtually suppresses ischemic brain damage and tPA-induced brain hemorrhages and disruption of the blood–brain barrier. Taken together, these data indicate (1) xenon should not be administered before or together with tPA therapy; (2) xenon could be a golden standard for treating acute ischemic stroke if given after tPA-induced reperfusion, with both unique neuroprotective and antiproteolytic (anti-hemorrhaging) properties.     

Distribution of angiotensin II type-2 receptor (AT2) mRNA expression in the adult rat brain

Radioactively labeled cRNA probes were used for in situ hybridization histochemistry to establish a detailed map of the sites of expression of the recently cloned angiotensin II, type 2 (AT₂) receptor mRNA in the adult rat brain. The distribution of the AT₂ receptor mRNA was consistent with that of the AT₂ binding sites, which were previously established by autoradiographic binding studies. Thus, high AT₂ receptor mRNA expression was observed in the lateral septum, in several thalamic nuclei, in the subthalamic nucleus, in the locus coeruleus, and in the inferior olive. Due to the superior resolution and sensitivity of in situ hybridization, AT₂ receptor expression was localized at the cellular level, and some additional brain nuclei expressing AT₂ receptor mRNA have been identified. These include the red nucleus, the pedunculopontine tegmental nucleus, the bed nucleus of the supraoptic decussation, the paragenual nucleus, and numerous brainstem nuclei. Several brain nuclei, such as the motor hypoglossal nucleus and the cerebellar nuclei, where AT₂ receptor binding had previously been identified in young animals only, showed a high expression of the AT₂ receptor mRNA in the adult rat. No correlation was found between the expression of the AT₂ and the type 1 (AT₁) receptor mRNAs. A combination of the in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemistry shows that the AT₂ receptor in the lateral septum showed that the AT₂ receptor was not detected in GFAP immunoreactive astroglial cells, therefore indicating that AT₂ is neuronal rather than glial in this brain region. © 1996 Wiley-Liss, Inc.     

颅脑损伤大鼠肠道防御素-5 mRNA表达

目的探讨弥漫性脑损伤后肠道防御素-5（RD-5）变化。方法大鼠32只，分为对照组8只；弥漫性脑损伤组24只，再分成3个亚组（24h，3d，7d）。观察回肠RD-5mRNA的表达，回肠肠粘膜的病理改变以及血液内毒素的变化。结果弥漫性脑损伤后24hRD-5 mRNA的表达显著升高（P〈0．01），伤后3d降低至正常水平以下；回肠肠粘膜损伤，血液内毒素伤后24h最高（P〈0．01），3至7d仍显著高于正常水平（P〈0．01）。结论弥漫性脑损伤早期RD-5 mRNA的表达增强可能是机体的一种保护性反应。