mRNA EXPRESSION OF THE LIPID AND MECHANO-GATED 2P DOMAIN K + CHANNELS DURING RAT BRAIN DEVELOPMENT

mRNAs encoded by genes for the lipid-sensitive mechano-gated K + channels TREK-1, TREK-2, and TRAAK were detected in rat brain at different life-cycle stages: 18-day embryos, postnatal days 1, 7, 28, and 60 (adulthood). mRNA expression of TREK-1 or TREK-2 showed no appreciable changes during the development of cortex and hippocampus. TRAAK mRNA expression increased with development and reached an apparent maximum at postnatal day 28 in hippocampus and day 60 in cortex. These data suggest that TRAAK might be important in the development of rat brain.     

Immunohistochemical colocalization of TREK-1, TREK-2 and TRAAK with TRP channels in the trigeminal ganglion cells

TREK belongs to a subfamily of tandem pore domain K⁺ channels, and consists of three subunits, TREK-1, TREK-2 and TRAAK. We examined the distribution of TREK-1, TREK-2 and TRAAK immunoreactive neurons in rat trigeminal sensory neurons. In the trigeminal ganglia, 31%, 43% and 60% of neurons were immunoreactive for TREK-1, TREK-2 and TRAAK, respectively. Mean sizes of TREK-1, TREK-2 and TRAAK immunoreactive trigeminal ganglion neurons were 447 ± 185, 445 ± 23 and 492 ± 12 mm², respectively. Furthermore, TREK channels were colocalized with cationic TRP channels, TRPV1, TRPV2 and TRPM8. TREK-1 immunoreactive neurons were colocalized with TRPV1 (57%), TRPV2 (11%) and TRPM8 (33%). TREK-2-immunoreactive neurons were colocalized with TRPV1 (33%), TRPV2 (9%) and TRPM8 (19%). TRAAK immunoreactive neurons were colocalized with TRPV1 (47%), TRPV2 (10%) and TRPM8 (22%). The present results revealed that TREK-1, TREK-2 and TRAAK channels colocalized with thermosensitive TRP channels in some small trigeminal ganglion neurons.     

Changes of synapsin I messenger RNA expression during rat brain development

Synapsin I is a synaptic phosphoprotein that is involved in the short-term regulation of neurotransmitter release. In this report we present the first extensive study of the developmental expression of its corresponding messenger ribonucleic acid (mRNA) by in situ hybridization and northern blot analysis in rat brain. Synapsin I mRNA showed pronounced differences in expression in different brain regions during postnatal development. The early expression of synapsin I mRNA in ontogenetically older regions such as the thalamus, the piriform cortex and the hippocampus coincides with the earlier maturation of these regions, in contrast to its later expression in ontogenetically younger areas such as the cerebellum and the neocortex. An intriguing expression pattern was found in the hippocampus. In all hippocampal subregions synapsin I mRNA expression increased from postnatal day (PND) 1 to 17. After PND 17, however, there was a marked dissociation between persisting high expression levels in CA3 and the dentate gyrus and a strong decline in synapsin I mRNA expression in CA1. The persistence of synapsin I in some adult rat brain regions indicates that it plays a part in synapse formation during plastic adaption in neuronal connectivities.     

Noggin在大鼠中枢神经系统发育过程中的表达

目的 研究Noggin基因在大鼠中枢神经系统(CNS)发育过程中的表达。方法 地高辛标记的cRNA探针原位杂交组织化学技术。结果 ISHH结果显示，在胚胎期(E16)大鼠，noggin mRNA阳性细胞主要位于大脑皮质、海马、丘脑与下丘脑的部分核团。新生期(P1-P2)大鼠，noggin在大脑皮质与海马的表达均降低，而在丘脑与延脑的表达增强；生后1周(P1W)noggin在脑内的表达明显降低，生后2周noggin在脑内的表达开始升高，在大脑皮质与海马升高尤为明显。生后1个月，noggin表达继续升高，在额叶皮质、顶叶皮质、扣带皮质、梨状皮质及海马的齿状回可检测到强阳性信号；而在丘脑的侧核、网状核、腹内侧核与腹外侧核可见中等强度的阳性信号。此外，在下丘脑的室旁核和视上核亦可见密集深染的阳性神经元。生后3个月，noggin阳性细胞在脑内的表达开始降低；生后18个月，noggin表达降至最低，仅见散在的阳性神经元。此外，在不同发育期大鼠的脊髓亦未观察到noggin mRNA阳性细胞。结论 提示noggin基因参与大鼠生后CNS的发育。     

Thermosensitivity of the two-pore domain K+ channels TREK-2 and TRAAK

TREK-1, TREK-2 and TRAAK are members of the two-pore domain K⁺ (K_2P) channel family and are activated by membrane stretch and free fatty acids. TREK-1 has been shown to be sensitive to temperature in expression systems. We studied the temperature-sensitivity of TREK-2 and TRAAK in COS-7 cells and in neuronal cells. In transfected COS-7 cells, TREK-2 and TRAAK whole-cell currents increased ∼20-fold as the bath temperature was raised from 24°C to 42°C. Similarly, in cell-attached patches of COS-7 cells, channel activity was very low, but increased progressively as the bath temperature was raised from 24°C to 42°C. The thresholds for activation of TREK-2 and TRAAK were ∼25°C and ∼31°C, respectively. Other K_2P channels such as TASK-3 and TRESK-2 were not significantly affected by an increase in temperature from 24°C to 37°C. When the C-terminus of TREK-2 was replaced with that of TASK-3, its sensitivity to free fatty acids and protons was abolished, but the mutant could still be activated by heat. At 37°C, TREK-1, TREK-2 and TRAAK were sensitive to arachidonic acid, pH and membrane stretch in both cell-attached and inside-out patches. In cerebellar granule and dorsal root ganglion neurones, TREK-1, TREK-2 and TRAAK were generally inactive in the cell-attached state at 24°C, but became very active at 37°C. In cell-attached patches of ventricular myocytes, TREK-1 was also normally closed at 24°C, but was active at 37°C. These results show that TREK-2 and TRAAK are also temperature-sensitive channels, are active at physiological body temperature, and therefore would contribute to the background K⁺ conductance and regulate cell excitability in response to various physical and chemical stimuli.     

Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS

TREK-1 is a member of the two-pore-domain potassium channel family which is expressed predominantly in the CNS. Using an anti-peptide polyclonal antiserum, we have determined the distribution of TREK-1 in the brain and spinal cord of adult rats. Specificity of the antiserum was tested using a TREK-1-transfected cell line and confirmed with c-myc-tagged TREK-1. In thin tissue sections, immunoreactivity was widespread throughout the rat brain and spinal cord. TREK-1-like signals were observed in the cerebral cortex, basal ganglia, hippocampus, and various other subcortical nuclei in the hypothalamus, thalamus, mesencephalon and rhombencephalon. TREK-1 labelling appeared to be over the entire cell membrane, including the cell body and processes. Cells that morphologically resembled projection neurones and interneurones but not glial cells were labelled. As interneurones and known GABAergic projection neurones were the predominant population labelled, we investigated the possibility that TREK-1 is expressed in GABA-containing neurones using a specific anti-GABA antiserum. Expression of TREK-1 in GABA-containing neurones was observed in a number of areas, including the isocortex, hippocampus and thalamus. Thus, TREK-1 expression defines a unique and specific subset of interneurones and principal cells.These studies indicate a widespread distribution of TREK-1 potassium channels throughout the rat brain and spinal cord, with expression in a number of areas being demonstrated to be present on GABA-containing neurones.     

Hetrin基因在大鼠脑发育过程中表达的初步研究

本文目的是 :观察 hetrin基因在大鼠脑发育过程中的表达变化规律 ,藉以探讨 hetrin在神经系统发育过程中的作用。以地高辛 (dig)标记 hetrin基因 3 ' -末端 c RNA为探针 ,采用原位杂交法研究 hetrin m RNA在生后第 10 d(P10 )、P3 0和 P60大鼠脑中的分布状况 ;同时采用半定量 RT-PCR法分析出生前后大鼠脑内 hetrin m RNA表达水平的变化。结果发现 ,hetrin m RNA原位杂交阳性信号定位于神经元胞质内 ,在 P10、P3 0和 P60大鼠的脑组织中均可见明显的杂交信号。 RT-PCR结果显示 ,在胚胎第 9d(E9)大鼠脑内 hetrin m RNA开始表达 ,但表达量很低 ,以后其表达量逐渐升高。出生后大鼠不同脑区 hetrin m RNA的表达量变化趋势不同。本实验结果表明 ,在胚胎期及出生后大鼠脑的多个部位都有 hetrin m RNA表达 ,而且 hetrin m RNA表达量与神经细胞突起生长及新突触连接的建立在时间上有一定的同步性 ,提示 hetrin m RNA的表达与神经突起生长存在着一定的关系 ,进一步支持存在 hetrin基因的神经细胞突起具有诱向生长功能     

氯胺酮对新生大鼠海马NMDA受体亚型mRNA表达的影响

本研究利用反转录聚合酶链式反应(RT-PCR)观察了将氯胺酮给予生后早期大鼠后,海马组织中NMDA受体亚型mR-NA的表达变化。实验用生后7d的SD大鼠40只,随机分为2大组:给药后即刻组(PND7组)和给药后3周组(PND28组)。PND7组为腹腔注射不同剂量氯胺酮后24h内处死,PND28组用相同给药方法给药并在相同环境中饲养3周(即至生后28d)后处死。RT-PCR结果显示在PND7组,腹腔注射氯胺酮引起海马组织内NR2A,NR2B和NR2C亚型mRNA的表达上调,NR1受体亚型mRNA的表达无明显变化。在PND28组,氯胺酮注射引起NR1和NR2A亚型mRNA的表达水平增高,而NR2B和NR2C亚型mRNA的表达没有变化。氯胺酮的给药量和给药方式对上述受体亚型的表达变化没有明显影响。各受体亚型在不同发育阶段的海马组织中的表达水平也有一定的差异。本研究的结果提示氯胺酮对发育阶段大鼠脑内NMDA受体的表达具有上调作用,从而对大鼠神经系统的发育可能产生影响。     

Chronological changes in prosaposin in the developing rat brain

Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain.     

细胞外信号调节激酶系统 (ERKs)在正常发育大鼠视皮层基因表达的研究

细胞外信号调节蛋白激酶 (ERKs)是皮层神经元生长、发育和分化的关键因子。本研究目的在于研究 ERKs(ERK1、ERK2和 ERK3 ) m RNA在视皮层各层的分布、表达量以及发育过程变化。实验用健康雄性 SD大鼠 ,于生后 (P) 14、2 1、2 8、45和 90 d(成年 )灌注固定 ,取全脑 ,切取视皮层。用 4%多聚甲醛固定 ,石蜡包埋 ,4μm厚切片。地高辛标记特异性寡核苷酸探针 (ERK1、ERK2 )和 c DNA探针 (ERK3 )。用原位杂交方法检测三种 ERKs亚型的 m RNA在各年龄组大鼠视皮层的表达。结果证明 :ERKs m RNA在出生后大鼠正常发育视皮层的表达 ,ERK1和 ERK2 m RNA的分布具有明显的层的特异性 ,表达于除 I层 (分子层 )之外的 II-VI层 ,ERK2较 ERK1m RNA的表达更广泛、信号密度更强。ERK1和 ERK2 m RNA的转录在发育敏感期增高 ,从 P2 1～P2 8逐渐增加 ,P45时达到高峰 ,到成年时降低为相当于 P2 1的水平。 ERK3 m RNA在大鼠出生后视皮层的信号表达强 ,比较恒定 ,无明显的层分布特异性。本研究结果提示 ,出生后正常大鼠发育期视皮层 ERK1和 ERK2的 m RNA表达呈上调趋势 ,而 ERK3 m RNA在大鼠出生后视皮层的表达量中等 ,比较恒定 ,缺乏发育性变化特点。表明 ERK1和 ERK2可能是参与出生后在视觉环境刺激下视皮层发育可塑性调节的重要     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.     

Postnatal expression of α2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of α and β subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of α3, α5, α7, and β4 subunit mRNAs and α7 binding sites during hippocampal and cortical development. Here, we examined the expression of α2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. α2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of α2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of α2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, α2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, α2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing α2 subunits could regulate GABAergic activity during a critical period of network formation.     

Deafness associated changes in two-pore domain potassium channels in the rat inferior colliculus

Two-pore potassium channels can influence neuronal excitability by regulating background leakage of potassium ions and resting membrane potential. The present study used quantitative real time PCR and in situ hybridization to determine if the decreased activity from deafness would induce changes in two-pore potassium channel subunit expression in the rat inferior colliculus (IC). Ten subunits were assessed with quantitative real-time PCR at 3 days, 3 weeks and 3 months following bilateral cochlear ablation. TASK-1, TASK-5 and THIK-2 showed significant decreases in expression at all three times assessed. TASK-5, relatively specific to auditory neurons, had the greatest decrease. TWIK-1 was significantly decreased at 3 weeks and 3 months following deafness and TREK-2 was only significantly decreased at 3 days. TASK-3, TWIK-2, THIK-1, TRAAK and TREK-1 did not show any significant changes in gene expression. In situ hybridization was used to examine TASK-1, TASK-5, TWIK-1 and THIK-2 in the central nucleus, dorsal cortex and lateral (external) cortex of the IC in normal hearing animals and at 3 weeks following deafening. All four subunits showed expression in neurons throughout IC subdivisions in normal hearing rats, with TASK-5 having the greatest overall number of labeled neurons. There was no co-localization of subunit expression with glial fibrillary acidic protein immunostaining, indicating no expression in glia. Three weeks following deafening there was a significant decrease in the number of neurons expressing TASK-1 and THIK-2 in the IC, while TASK-5 had significant decreases in the central nucleus and dorsal cortex and TWIK-1 in the lateral and dorsal cortices.     

细胞外信号调节激酶系统(ERKs)在正常发育和单眼剥夺大鼠视皮层蛋白表达的研究

为了研究正常发育和单眼视觉剥夺大鼠视皮层 ERK1/ ERK2基因蛋白质表达的变化 ,本研究建立了 SD大鼠单眼视觉剥夺模型 ,以多克隆抗 ERK1/ ERK2抗体和单克隆抗双磷酸化 ERK抗体检测出生后第 1、14、2 1、2 8、45 d和成年 (90 d)正常发育和单眼视觉剥夺 (14～ 45 d)视皮层 ERK1/ ERK2蛋白表达和活性改变。结果表明 ,出生后大鼠视皮层 ERK1/ ERK2蛋白表达逐渐增加 ,至 45 d达到高峰 ,此后下降至成年达稳定水平。磷酸化 ERK蛋白表达仅见于成年大鼠视皮层。单眼视觉剥夺导致ERK1/ ERK2蛋白表达减少。提示 ERK的蛋白表达依赖于正常的视觉输入信号的刺激 ,异常的视觉经验造成表达的明显下调 ,阻断正常的发育进程。说明 ERK1和 ERK2可能参与发育敏感期视皮层神经元可塑性的调节     

大鼠海马γ-氨基丁酸-A受体在生后发育中的表达

目的:研究大鼠生后发育过程中海马组织γ-氨基丁酸-A(GABA-A)受体的表达规律。方法:用免疫组织化学和PCR技术,检测不同年龄大鼠海马组织GABA-A受体及编码该受体的mRNA表达,并用图像分析方法进行定量研究。结果:大鼠海马组织在生后3 d已经出现GABA-A受体免疫反应,以后逐渐增强,到生后30 d达到最高值,海马各区GABA-A受体免疫反应强度没有显著的差别;编码GABA-A受体的mRNA表达也有类似的增龄性增多,但其表达高峰值提前到14 d。结论:生后大鼠海马GABA-A受体表达在一定时期内呈增龄性表达增强的趋势。     

Gerbil angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT1 Angiotensin II receptors. The recently cloned gerbil AT1 receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT1 receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region. We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life. The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT1 receptors in the development and function of the corticohippocampal system.     

NR1、NR2A与PSD-95在生后大鼠海马发育中的表达

目的:探讨N-甲基-D-天冬氨酸受体亚单位1(N-methy1-D-aspartate receptor subunit1,NR1)、N-甲基-D-天冬氨酸受体亚单位2A(N-methy1-D-aspartate receptor subunit2A,NR2A)与突触后密度蛋白-95(Postsynapticdensity protein 95,PSD-95)在Wistar大鼠海马生后发育过程中的表达。方法:应用免疫荧光染色方法检测NR1、NR2A与PSD-95在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况。结果:NR1于生后各期海马CA1、CA3区和DG的表达均增强,P14~P21达高峰期后减弱。NR2A于生后在海马CA1和CA3区的表达略有减弱,P4后增强至高峰期P14,然后轻微减弱,在DG中NR2A的表达生后略有减弱,P4后在增强的趋势中于P7、P14和P28后均有不同程度的减弱,高峰期在P28。PSD-95于生后各期海马CA1、CA3区和DG的表达均增强,P21~P28达高峰期后轻微减弱。结论:NR1、NR2A和PSD-95在CA1、CA3区和DG中的表达具有特异的时空分布模式,此模式可能与其在生后发育中发挥的不同生理功能相关。