Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.     

Axon development in mouse cerebellum: embryonic axon forms and expression of synapsin I

A fundamental question in central nervous system development is the timing of synaptogenesis in relation to invasion of targets by afferent axons. A related question is how growth cones transform into synaptic terminals. These two aspects of axon maturation were examined in developing mouse cerebellum, by labeling single axons with horseradish peroxidase, to study their form and cytology, and by immunocytochemical staining of a synaptic vesicle antigen, synapsin I, a phosphoprotein found on synaptic vesicles in all mature CNS synapses. From embryonic day 16 to postnatal day 3, horseradish peroxidase-labeled afferent axons extend well into the cerebellum and have simple forms. At embryonic day 16, axon growing tips are synapsin I-negative. Synapsin I is first expressed at embryonic day 17, and by embryonic day 18, fibers are stained throughout the cerebellum. Synapsin I expression coincides with a general increase in synaptic specializations, although growing tips continue to have the cytology of growth cones. During the period that axons have primitive shapes, synapsin I is distributed throughout the terminal arbor, corresponding to the presence of small vesicles along neurite lengths, even at non-synaptic sites. After postnatal day 3, when synaptic terminals develop into stereotypic shapes and engage in characteristic synaptic relations, synapsin I is restricted to boutons. Thus, the synapse-specific protein synapsin I is expressed in fetal mouse brain, long before nerve endings have the structure and connections of adult brain. In cerebellar axons, the expression of this protein follows axon arrival, coincides with the appearance of elementary synapses, and accompanies the transformation of growing tips into stereotypic synaptic boutons. The time course of expression of synapsin I, a phosphoprotein that may be involved in synaptic efficacy, suggests that transmitter release may influence early axon-target cell interactions.     

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     

Expression of brain-derived neurotrophic factor in the rat forebrain and upper brain stem during postnatal development: an immunohistochemical study

The present study was undertaken to characterize the regional and temporal patterns of brain-derived neurotrophic factor (BDNF) in the rat forebrain and upper brain stem during postnatal development using an immunohistochemical approach. Results indicated that BDNF-immunoreactive (IR) cells could be divided into three groups based on their postnatal developmental patterns: (group 1) BDNF-IR cells were first detected between postnatal days (PND) 1 and 7, and thereafter they increased in number and remained stable during later stages of ontogeny; (group 2) BDNF-IR cells progressively increased in number with age, and then decreased in adults; (group 3) numerous BDNF-IR cells detected between PND 1 and 7 showed a dramatic reductions in number with few IR cells in adults. In contrast, the developmental pattern of most BDNF-IR fibers differed from that of IR neurons, i.e. they appeared between PND 1-28 and thereafter continued to increase in number showing a maximum level in adults. Additionally, BDNF-IR cells in the superficial layer of the neocortex and IR fibers in the stratum oriens of CA2 first appeared as late as PND 28 and in adults, respectively. After colchicine treatment, reexpression or a marked increase in the number of BDNF-IR neurons was observed in many areas of the adult brain where a progressive decrease in BDNF-IR cell numbers during development and scant or some IR neurons in adults were shown. These results showed both transient and persistent expression of BDNF in various regions of the developing rat brain.     

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。     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND?10, decreased until PND 30, and thereafter remained at the same level as on PND?0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND?10 and PND?15, after which the immunoreactivity gradually decreased and reached a plateau at PND?30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED?20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND?0. Immunoreactivity in the neurons of the subiculum was found after PND?5 and that of the polymorphic cell layers was found after PND?15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions.     

Increased synapsin I expression in cerebral malaria

Synapsin I is a neuronal phosphoprotein contained in the synaptic vesicles of mammalian central and peripheral nervous systems. It regulates both neurotransmitter release and synaptic formation. Variations in synapsin I expression in the brain have been reported to cause brain malfunction. In severe malaria, neurological complications, such as convulsion, delirium and coma, suggest abnormalities in the release of neurotransmitters. This study evaluated synapsin I expression in cerebral malaria (CM). An immunohistochemical method was used to study the semi-quantitative and qualitative expression of synapsin I in the brain of CM patients (10 cases) who died with Plasmodium falciparum, compared with non-cerebral malaria (NCM) (4 cases), and control brain tissues (5). Synapsin I was expressed in the gray matter of the cerebral cortex and the molecular layer of the cerebellum, as a diffusely dense precipitate pattern in the neuropil, with no immunoreactivity in the neurons, neuronal dendrites, glial cells, endothelial cells, and Purkinje cells. The findings were similarly demonstrated in CM, NCM, and control brain tissues. However, in the granular layer of the cerebellum, a significant increase in synapsin I expression was observed in the granule cells, and the glomerular synaptic complex, from the CM group, compared with the NCM, and control brain tissues (all P < 0.05). Parasitemia showed a positive correlation with synapsin I expression in the granule cells (on admission: Spearman’s ρ = 0.600, P = 0.023) (before death: Spearman’s ρ = 0.678, P = 0.008), and glomerular synaptic complex (before death: Spearman’s ρ = 0.571, P = 0.033). It was hypothesized that CM causes pre-synaptic excitation and eventually activation of synapsin I, leading to increased neurotransmitter release. Synapsin I inhibitor should be investigated further as a target for a therapeutic intervention to alleviate neurological symptoms in severe malaria.     

Caspase-3在出生后大鼠海马中的表达

目的 探讨Wistar大鼠海马生后发育过程中,活化的Caspase-3与凋亡之间的关系. 方法 应用免疫荧光方法观测活化的Caspase-3和赫斯特荧光染料33342(Hoechst 33342)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 在CA1区,活化的Caspase-3的表达在生后7d(P7)达到高峰;在CA3区,P2达到高峰,然后逐渐减弱.在DG,P7后又有所增强,到P14达到高峰,并在所观测的其余时段维持此水平.观测的3个区的凋亡细胞数目都在P7达到高峰,然后逐渐减少. 结论 在大鼠海马生后发育过程中,活化的Caspase-3的表达存在特定的时空格局.活化的Caspase-3在CA1区与CA3区有丝分裂后期细胞和DG神经前体细胞中的作用和机制不同.     

Age-related and regional differences in secretin and secretin receptor mRNA levels in the rat brain

In the present study expression levels of secretin and secretin receptor mRNAs in several brain regions of rats ranging in age from postnatal days 7 to 60 were investigated by quantitative real-time PCR. Expression of secretin and secretin receptor was detected in the central amygdala, hippocampus, area postrema, nucleus of the tractus solitary and cerebellum. The cerebellum expressed secretin receptor at significantly higher levels than that found in other brain regions within all the ages examined. In contrast, secretin mRNA was significantly higher in the nucleus of the tractus solitary than in the other four brain regions examined in postnatal day-21, -30 and -60 rats. Within most brain regions, both secretin and secretin receptor mRNAs were more abundant in postnatal day-7 and -14 rats as compared to postnatal day-21, -30 and -60 rats. Thus, secretin and its receptor are widely expressed in rat brain and the expression of both genes is developmentally regulated during the first few weeks following birth.     

Pregnenolone and dehydroepiandrosterone administration in neonatal rats alters the immunoreactivity of hippocampal synapsin I, neuropeptide Y and glial fibrillary acidic protein at post-puberty

It is well documented that neurosteroids administered during the neonatal period influence the development of several brain systems. In our previous study, pregnenolone administered to rats during the neonatal period altered adenosinergic and dopaminergic functions in the striatum and cerebral cortex. The present study examined the effects of the treatment with pregnenolone and dehydroepiandrosterone (DHEA) from the postnatal day (P) 3-P7 on synapsin I (a marker for presynaptic terminals) and glial fibrillary acidic protein (GFAP: a marker for astroglia) levels in the hippocampus of Sprague-Dawley rats at 3 and 7 weeks of age. In addition, neuropeptide Y and dynorphin A immunoreactivity was measured. The administration of pregnenolone and DHEA to neonatal rats significantly altered the expression of synapsin I in the dentate gyrus and CA3 region at post-puberty but not at pre-puberty. A significantly greater expression of GFAP-immunoreactive astrocytes or processes was demonstrated in the pregnenolone- and DHEA-treated groups at both pre-puberty and post-puberty. A significant increase in the number and size of GFAP-immunoreactive astrocytes and in the extension of arborization was seen in the overall hippocampus. The number of neuropeptide Y-positive cells in the hilus region was also significantly increased in the neurosteroid-treated group at post-puberty. No differences were detected in dynorphin A immunoreactivity among the experimental groups. These results of this study suggest that pregnenolone and DHEA play an important role in the development of hippocampus.     

Prox1 expression patterns in the developing and adult murine brain.

Prox1, a homeobox gene related to the Drosophila gene prospero, is necessary for retina, lens, liver, pancreas, and lymphatics development. However, not much is yet known about Prox1 expression during central nervous system development. Here we provide a detailed analysis of Prox1 mRNA and protein expression during prenatal and postnatal murine brain development. Prenatally, Prox1 is expressed in the subventricular zone or in early differentiating regions of the brain. At these stages, Prox1 mRNA, but not Prox1 protein, was also detected in several regions of the prethalamus and hypothalamus. At an early postnatal stage, Prox1 expression is mainly detected in several nuclei of the thalamus, the cerebellum, and the hippocampus. In adulthood, Prox1 expression remains only in the hippocampus and cerebellum. These complex patterns of expression suggest that Prox1 activity is differentially required during brain development and adulthood.     

Cannabinoid administration increases 5HT1A receptor binding and mRNA expression in the hippocampus of adult but not adolescent rats

The endocannabinoid and serotonin systems share a high level of overlap in terms of the physiological processes that they regulate, however, little is known about their functional interactions particularly during adolescence, a vulnerable period for both the development of psychosis and for initiation to substance use. In the present study, the effects of cannabinoid treatment on serotonin 5HT1A receptor density and mRNA expression were investigated in two age groups: Adolescent (postnatal day 35) and adult (postnatal day 70) rats were injected with the synthetic cannabinoid HU210 (25, 50 or 100 μg/kg) or vehicle for 1, 4 or 14 days and sacrificed 24 h after the last injection. 5HT1A receptor density was measured in different brain regions using [³H]8-OH-DPAT quantitative autoradiography whereas mRNA expression was measured in adjacent brain sections. Higher levels of both serotonin 5HT1A receptor binding and mRNA expression were observed in limbic regions in adolescent control animals compared to adults. 5HT1A receptor density was increased by 23% in the CA1 region of the hippocampus of adult rats treated with 100 μg/kg HU210 for 4 days compared to vehicle treated controls. The same treatment increased mRNA expression by 27% and by 14% in the CA1 region and dentate gyrus of the hippocampus respectively. 5HT1A receptor density was increased by 22% in the CA1 of adult animals treated with 50 μg HU210, by 26% in the dentate gurus of adult rats treated with 100 μg for 14 days. By contrast, 5HT1A receptor density or mRNA expression was not affected in the brain of adolescent animals in any of the brain regions examined. These results suggest that cannabinoid treatment has differential effects on serotonin-related neurochemistry in adolescent compared to adult rats. The effects in the adult brain may compromise hippocampal function and could account for the cognitive deficits seen in habitual heavy cannabis users.     

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.     

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.     

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.     

钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠生后海马发育中的表达

目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）的表达。 方法 应用免疫荧光方法检测CaMKⅡ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（n =48）。结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强，生后第10天（P10）达高峰期，此后逐渐减弱；于CA3区的表达在P4和P10时均较高。其中，CaMKⅡ在CA3区的表达高于在CA1区和DG的表达，在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达。结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式，这可能与其在生后发育过程中的突触发生，树突、轴突形成，海马的成熟以及学习记忆功能相关。     

Transient expression of juvenile-type neurocan by reactive astrocytes in adult rat brains injured by kainate-induced seizures as well as surgical incision

Neurocan is one of the major chondroitin sulfate proteoglycans expressed in nervous tissues. The expression of neurocan is developmentally regulated, and full-length neurocan is detected in juvenile brains but not in adult brains. In the present study, we demonstrated by western blot analysis that full-length neurocan transiently appeared in adult rat hippocampus when it was lesioned by kainate-induced seizures. Immunohistochemical studies showed that neurocan was detected mainly around the CA1 region although the seizure resulted in neuronal cell degeneration in both the CA1 and CA3 regions of the hippocampus. Double-labeling for neurocan mRNA and glial fibrillary acidic protein demonstrated that many reactive astrocytes expressed neurocan mRNA. The re-expression of full-length neurocan was also observed in the surgically injured adult rat brain. In contrast, the expression of other nervous tissue chondroitin sulfate proteoglycans, such as phosphacan and neuroglycan C, was not intensified but rather was either reduced in the kainate-lesioned hippocampus or in the surgically injured cerebral cortex. These observations indicate that induction of neurocan expression by reactive astrocytes is a common phenomenon in the repair process of adult brain injury, and therefore, it can be postulated that juvenile-type neurocan plays some roles in brain repair.     

Differential effects of neonatal norepinephrine lesions on immediate early gene expression in developing and adult rat brain

Activity regulated cytoskeletal protein (Arc), c-fos and zif268 are immediate early genes (IEGs) important for adult brain plasticity. We examined developmental expression of these IEGs and the effect of neonatal noradrenergic lesion on their expression in developing and mature brain. N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), a specific noradrenergic neurotoxin, was administered to rats on postnatal day (PND) 3 and in situ hybridization was used to assay Arc, c-fos and zif268 mRNA on PND 13, 25 and 60. In contrast to decreases in Arc, c-fos and zif268 expression produced by noradrenergic lesions of mature brain, lesions on PND 3 yield a strikingly different effect. Neonatal lesions produce increases in c-fos and zif268 expression in specific frontal cortical layers on PND 13, while Arc shows no change. These lesions lead to increases in zif268 expression in frontal cortical layers on PND 25, with no changes in c-fos or Arc expression, and on PND 60 they produce a significant increase in c-fos expression in hippocampus with no significant changes in Arc or zif268 expression. 2-[2-(2-Methoxy-1,4-benzodioxanyl)]imidazoline hydrochloride (RX821002), an alpha-2 adrenergic receptor (A2AR) antagonist, administered to control PND 60 animals produces elevations of Arc, zif268 and c-fos mRNAs. This response was eliminated in animals lesioned with DSP-4 on PND 3. These data indicate that norepinephrine regulation of IEG expression differs in developing and mature brain and that loss of developmental norepinephrine leads to abnormally high postnatal IEG expression. Previous studies have shown an important role for norepinephrine in brain development. Our data support the idea that norepinephrine plays an important role during CNS development and that changes in noradrenergic signaling during development may have long lasting effects, potentially on learning and memory.