Changes in the mRNAs encoding subtypes I, II and III sodium channel alpha subunits following kainate-induced seizures in rat brain

Several lines of evidence underscore a possible role of voltage-gated Na+ channels (NaCH) in epilepsy. We compared the regional distribution of mRNAs coding for Na+ channel subunit I, II and III in brains from control and kainate-treated rats using non-radioactive in situ hybridization with subtype-specific digoxigenin-labelled cRNA probes. Labelling intensity was evaluated by a densitometric analysis of digitized images. Heterogeneous distribution of the three Na+ channel mRNAs was demonstrated in brain from adult control rats, which confirmed previous studies. Subtype II mRNAs were shown to be abundant in cerebellum and hippocampus. Subtype I mRNAs were also detected in these areas. Subtype III mRNAs were absent in cerebellar cortex, but significantly expressed in neurons of the medulla oblongata and hippocampus. The three subtypes were differentially distributed in neocortical layers. Subtype II mRNAs were present in all of the layers, but mRNAs for subtypes I and III were concentrated in pyramidal cells of neocortex layers IV–V. During kainate-induced seizures, we observed an increase in Na+ channel II and III mRNA levels in hippocampus. In dentate gyrus, subtype III mRNAs increased 3 h after K A administration to a maximum at 6 h. At this latter time, a lower increase in NaCh III mRNAs was also recorded in areas CA1 and CA3. NaCh III overexpression in dentate gyrus persisted for at least 24 h. In the same area, NaCh II mRNAs were also increased with a peak 3 h after K A injection and a return to control levels by 24 h. No changes in NaCh I mR NAs were seen. The K A-induced up-regulation in NaCh mR NAs probably resulted in an increase in hippocampal neuronal excitability.     

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.     

Decline in age-dependent, MK801-induced injury coincides with developmental switch in parvalbumin expression: somatosensory and motor cortex

MK801-induced activation of caspase-3 is developmentally regulated, peaking at postnatal day (P) 7 and decreasing with increasing postnatal age thereafter. Further, at P7, cells displaying activation of caspase-3 lack expression of calcium binding proteins (CaBPs). To further explore this relationship, we investigated postnatal expression of calbindin (CB), calretinin (CR) and parvalbumin (PV) in two brain regions susceptible to MK801-induced injury, the somatosensory cortex (S1) and layer II/III of motor cortex (M1/M2). Expression of CB and especially PV was low to absent prior to P7 but substantially increased from P7 through to P21 and adulthood. In contrast, CR expression was more variable at early developmental ages, stabilized to lower levels after P7 and showed a marked decline by P21. The results suggest that not only does calcium buffering capacity increase developmentally but also acquisition of enhanced buffering may be one mechanism by which neurons survive agent-induced alterations in calcium homeostasis.     

Expression of sodium channel α- and β-subunits in the nervous system of themyelin-deficient rat

Summary Using subtype-specific riboprobes and a non-isotopein situ hybridization technique, the pattern of expression of the mRNAs for voltage dependent sodium channel -subunits I, II, III and NaG, and the ₁-subumt were compared inmyelin-deficient rats and unaffected male littermates. Tissues examined included the hippocampus, cerebellum, spinal cord and dorsal root ganglia. Previous studies have demonstrated that the expression of sodium channel - and ₁-subunits follows a distinct temporal and spatial pattern during development, characterized in part by greater expression of -subunit III and its mRNA during development than in the adult. We examined animals of 20–22 days of age, a time when, according to earlier reports, the unaffected animals should nearly have reached an adult expression pattern. Normal male littermates were indeed found to express a sodium channel subunit mRNA pattern generally consistent with previous reports on adult rats.Myelin-deficient animals exhibited an expression pattern identical to the unaffected littermates, indicating that myelination is not required for the progression from the embryonic to the adult expression pattern of sodium channel subunits.     

Expression of mKirre, a mammalian homolog of Drosophila kirre, in the developing and adult mouse brain

mKirre, a mammalian homolog of the Drosophila kirre, is expressed in bone marrow stromal cells and the brain. Although mKirre has been shown to support the hematopoietic stem cells, little is known about the function of mKirre in the brain. In the present study, to gain insights into the function of mKirre, we investigated the expression pattern of mKirre gene in the developing and adult mouse brain using in situ hybridization. In the adult brain, mKirre mRNA was highly expressed in the olfactory bulb, the piriform cortex, the cochlear nucleus, and the cerebellum. At embryonic day (E) 11.5, we could observe mKirre mRNA in the differentiating zones of various regions, such as the caudate-putamen, the geniculate body, the thalamus, the amygdala, and the brainstem. Its gene expression in these regions at E11.5 also persisted to the adult, in which its expression levels were much less prominent. After birth, we could first observe high expression of mKirre mRNA in the glomerular and mitral layers of the olfactory bulb, the cortical plate of the neocortex, the cochlear nucleus, and the molecular and granule cell layers of the cerebellum. In the hippocampus, its gene expression was first observed in the dentate gyrus at postnatal day 7. The spatiotemporal expression pattern of mKirre mRNA suggests important roles of mKirre in later developmental processes, especially the synapse formation.     

大鼠小脑和下橄揽核中含Calbindin－D28KmRNA的神经元的生后发育

本实验应用原位杂交组织化学方法观察了大鼠小脑皮质和下橄榄核中含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ＫｍＲＮＡ的神经元的生后发育过程。发现在刚出生时，小脑浦肯野氏细胞已含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ＫｍＲＮＡ，其表达水平在生后第３周时达高峰并持续至成年期。但在下橄榄核中，含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ＫｍＲＮＡ的神经元在生后第７天时才出现，其数量及标记强度在生后第３、４周时迅速增加，并达成年水平。结合以往的资料分析，在小脑中，Ｃａｌｂｉｎｄｉｎ－Ｄ２８Ｋ可能与浦肯野氏细胞的成熟（突起的形成及伸长、突触的形成）过程有关。而在下橄榄核中，Ｃａｌｂｉｎｄｉｎ－Ｄ２８Ｋ主要参与成年期神经元的正常生理功能。     

Quantitation and localization of ENaC subunit expression in fetal, newborn, and adult mouse lung

The newborn lung is cleared of fetal liquid by active Na+ transport. The heterotrimeric (alpha, beta, gamma) epithelial Na+ channel, ENaC, mediates this process. To understand the role of individual ENaC subunits in Na+ transport during development, we quantified murine ENaC (mENaC) subunit messenger RNA (mRNA) expression levels of fetal, neonatal, and adult mouse lung by Northern blot analysis and studied regional expression by in situ hybridization. alphamENaC and gammamENaC mRNA expression increased sharply in late fetal gestation and reached near-adult levels by Day 1 of postnatal life. betamENaC expression increased more gradually through late fetal and early postnatal life and increased progressively until adulthood. In situ hybridization studies showed similar localization patterns of alphamENaC and gammamENaC subunit expression in fetal and postnatal lung. gammamENaC and alphamENaC subunits were initially localized to fetal lung bud tubules and by late gestation both subunits were expressed in all regions (acinar and bronchiolar) of the distal lung epithelium. betamENaC was detected from 16 d gestation onward and was expressed most intensely in small airways. There was little expression of betamENaC in the alveolar region. In postnatal lung all three subunits were expressed intensely in small airways. In adult lung, alphamENaC and gammamENaC were expressed in a pattern consistent with an alveolar type II (ATII) cell distribution. The timing of quantitative changes in mENaC subunit expression is consistent with a role of Na+ transport in liquid clearance of the perinatal lung. Intense expression of mENaC subunits in medium and small airway epithelium and in ATII cells suggests that these regions are a primary location for liquid absorption in the perinatal and postnatal murine lung.     

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     

Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression

Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.     

Differing expression of insulin-like growth factor I in the developing and in the adult rat cerebellum

Insulin-like growth factor I (IGF-I; somatomedin C) is a trophic peptide of importance for the development of several tissues and organs. In the present study we have mapped the cellular distribution and dynamic changes of IGF-I immunoreactivity in the rat cerebellum from its postnatal development to maturity. In vitro hybridization of IGF-I mRNA was used to demonstrate that the IGF-I immunoreactive material was synthesized in the cerebellum during a limited time period of cerebellar differentiation. IGF-I immunoreactivity was absent in primordial nerve cells but was present in neuroglial cells during the first two days after birth and then rapidly increased in intensity in the latter during the next few days. Proliferative nerve cells in the external granular layer did not express IGF-I immunoreactivity, while migrating and differentiating nerve cells as well as neuroglial cells showed intense labelling. Starting about 2 weeks postnatally, the IGF-I immunoreactivity declined, first in the neuroglial cells and eventually in the nerve cells. No IGF-I immunoreactivity could be demonstrated in the normal adult cerebellum. Colchicine pretreatment did, however, enable demonstration of IGF-I immunoreactivity in adult cerebellar nerve cells but not in neuroglial cells. In vitro hybridization revealed IGF-I mRNA in the developing cerebellum but only at very low levels in the adult cerebellum. It is concluded that IGF-I is likely to be a factor of importance for the development and maturation of nerve cells and neuroglial cells in the brain. The neuroglial cells in normal adult cerebellum as well as in other parts of the central nervous system do not show any IGF-I immunoreactivity, in contrast to neuroglial cells in the automatic and peripheral nervous systems.     

The cyclin-dependent kinase 5 activator,p39, is expressed in stripes in the mouse cerebellum

Cyclin-dependent kinase 5 (Cdk5) activity is required for CNS development. The Cdk5 activator, p35, is well characterized but its isoform, p39, has been less studied. Previously, p39 mRNA expression in rat brain was shown to peak at 3 weeks postnatal, and the level remains high in the adult cerebellum [Neurosci Res 28 (1997) 355]. However, p39 protein expression and specific localization in the cerebellum, where p39 mRNA level significantly exceeds that of p35, have not been examined. Here, we explored the specific cerebellar localization of the p39 protein in the developing and adult mice. Adult cerebellar Purkinje cell somata and dendritic arbors were strongly positive for p39 but only rare and barely detectable p39 was observed in Purkinje cell axons. Cdk5 also localized in Purkinje cell somata and dendrites of the adult cerebellum, but p35 localized only in Purkinje cell somata, further suggesting a functional difference between p35 and p39. During development, cerebellar p39 was first noted at P10. Primary cultures of a developing cerebellum also showed strong p39 immunoreactivity in Purkinje cell somata and dendrites, but weak p39 immunoreactivity in Purkinje cell axons. Starting from P10, p39 was observed in a subset of Purkinje cells that form parasagittal bands throughout the vermis and hemispheres. These bands were bilaterally symmetrical and continuous from one lobule to another. Conversely, Cdk5 and p35 showed a uniform staining pattern. The pattern of p39 closely resembled that of zebrin II/aldolase C, suggesting that p39 may play a role in the adult cerebellum rather than in pattern development. This premise is consistent with the normal pattern of zebrin II/aldolase C zones and stripes in mutant p39-/- mice. The alternating p39 parasagittal band pattern may reflect a role for p39 or Cdk5/p39 in the functional compartmentation of the cerebellum.     

含GABA_A受体α_6亚单位mRNA神经元在大鼠脑内的生后发育

用原位杂交组织化学方法观察了大鼠小脑皮质和耳蜗核中含ＧＡＢＡＡ受体α６亚单位ｍＲＮＡ神经元的生后发育过程。结果发现小脑在生后发育中，杂交信号在生后第５ｄ最早出现于内颗粒层，在生后第２１ｄ达到高峰并持续至成年期。但在外生发展始终未见到阳性信号。在耳蜗核中，α６亚单位ｍＲＮＡ到生后第７ｄ方出现，其表达水平在此后的阶段内迅速增加，从生后第１４ｄ开始，杂交信号的增强趋于缓慢，至生后第３ｗ达到成年水平。α６亚单位ｍＲＮＡ在小脑及耳蜗核的生后发育早期即有较强表达，提示其与上述两个系统的成熟过程有关。     

Developmental regulation of phosphoprotein gene expression in the caudate-putamen of rat: an in situ hybridization study.

The regional and cellular ontogeny of the mRNA encoding the dopamine- and cAMP-regulated phosphoprotein, DARPP-32, has been studied in rat striatum by quantitative in situ hybridization histochemistry. The mRNA for DARPP-32 exhibited a characteristic developmental profile. The hybridization signal was first visible on the day of birth, at which time DARPP-32 mRNA was concentrated in patches in the caudate-putamen. By the end of the first postnatal week, the majority of neurons in the caudate-putamen expressed the DARPP-32 message. Levels of mRNA per cell increased markedly during the second postnatal week, and peaked around the beginning of the third week. The adult level of DARPP-32 mRNA was lower than that observed at the apex of mRNA expression, on a per cell basis, while the proportion of neurons expressing detectable levels of message remained relatively constant. In the nucleus accumbens and olfactory tubercle, DARPP-32 mRNA development lagged somewhat behind that observed in the caudate-putamen, but was similar in other respects. A non-quantitative study employing an oligonucleotide probe complementary to the mRNA encoding another cAMP-regulated phosphoprotein, ARPP-21, revealed a similar developmental sequence to DARPP-32. The present results suggest that for DARPP-32 mRNA, genetic and, possibly, environmental factors play a role in determining the developmental patterns observed.     

The temporal and spatial development of CRF binding sites in the postnatal mouse cerebellum.

This study describes the distribution and relative level of labeling of binding sites for corticotrophin releasing factor (CRF) in the postnatal mouse cerebellum. At birth low levels of labeling are present throughout the cerebellum. However, this labeling is most densely distributed in the caudal and lateral aspects of the cerebellum. By P3 CRF binding sites are present throughout the cerebellum, although the greatest level of labeling is in the posterior lobe of the vermis, especially lobules IX and X; this correlates with the early differential pattern of CRF distribution in cerebellar afferents within these same lobules. At P10, the adult pattern of distribution and level of labeling begins to emerge. The presence of CRF and CRF binding sites at birth, and during postnatal growth, suggests that this peptide could play a role in the regulation of developmental events within the cerebellum.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer