Morphological and molecular changes in aging rat prelimbic prefrontal cortical synapses

Age-related impairments of executive functions appear to be related to reductions of the number and plasticity of dendritic spine synapses in the prefrontal cortex (PFC). Experimental evidence suggests that synaptic plasticity is mediated by the spine actin cytoskeleton, and a major pathway regulating actin-based plasticity is controlled by phosphorylated LIM kinase (pLIMK). We asked whether aging resulted in altered synaptic density, morphology, and pLIMK expression in the rat prelimbic region of the PFC. Using unbiased electron microscopy, we found an approximate 50% decrease in the density of small synapses with aging, while the density of large synapses remained unchanged. Postembedding immunogold revealed that pLIMK localized predominantly to the postsynaptic density where it was increased in aging synapses by approximately 50%. Furthermore, the age-related increase in pLIMK occurred selectively within the largest subset of prelimbic PFC synapses. Because pLIMK is known to inhibit actin filament plasticity, these data support the hypothesis that age-related increases in pLIMK may explain the stability of large synapses at the expense of their plasticity.     

alpha-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus

N-methyl-d-aspartate receptors (NMDARs) are critical determinants of bidirectional synaptic plasticity, however, studies of NMDAR function have been based primarily on pharmacological and electrophysiological manipulations, and it is still debated whether there are subunit-selective forms of long-term potentiation (LTP) and long-term depression (LTD). Here we provide ultrastructural analyses of axospinous synapses in cornu ammonis field 1 of hippocampus (CA1) stratum radiatum of transgenic mice with mutations to two key underlying postsynaptic density (PSD) proteins, postsynaptic density protein 95 (PSD-95) and the alpha-isoform of calcium-calmodulin-dependent protein kinase II (alphaCaMKII). Distribution profiles of synaptic proteins in these mice reveal very different patterns of subunit-specific NMDAR localization, which may be related to the divergent phenotypes of the two mutants. In PSD-95, Dlg, ZO-1/Dlg-homologous region (PDZ) 3-truncated mutant mice in which LTD could not be induced but LTP was found to be enhanced, we found a subtle, yet preferential displacement of synaptic N-methyl-d-aspartate receptor subunit 2B (NR2B) subunits in lateral regions of the synapse without affecting changes in the localization of N-methyl-d-aspartate receptor subunit 2A (NR2A) subunits. In persistent inhibitory alphaCaMKII Thr305 substituted with Asp in alpha-isoform of calcium-calmodulin kinase II (T305D) mutant mice with severely impaired LTP but stable LTD expression, we found a selective reduction of NR2A subunits at both the synapse and throughout the cytoplasm of the spine without any effect on the NR2B subunit. In an experiment of mutual exclusivity, neither PSD-95 nor alphaCaMKII localization was found to be affected by mutations to the corresponding PSD protein suggesting that they are functionally independent of the other in the regulation of NR2A- and NR2B-containing NMDARs preceding synaptic activity. Consequently, there may exist at least two distinct PSD-95 and alphaCaMKII-specific NMDAR complexes involved in mediating LTP and LTD through opposing signal transduction pathways in synapses of the hippocampus. The contrasting phenotypes of the PSD-95 and alphaCaMKII mutant mice further establish the prospect of an independent and, possibly, competing mechanism for the regulation of NMDAR-dependent bidirectional synaptic plasticity.     

双氢睾酮对快速老化小鼠海马CA1区突触可塑性和N-甲基-D-天冬氨酸受体1的影响

目的 探讨双氢睾酮(DHT)对快速老化小鼠(SAMP8)海马CA1区突触可塑性和N-甲基-D-天冬氨酸受体1(NMDAR1)的影响.方法 6月龄雄性SAMP8小鼠21只随机分为假手术组、去势组及去势+DHT补充治疗组,每组7只.DHT剂量为1mg/(kg·d),皮下注射21d后,通过Golgi染色观察海马CA1区顶树突树突棘的变化;免疫组织化学及图像分析检测突触素和NMDAR1表达的改变.结果 1.Golgi染色,去势组海马CA1区顶树突树突棘个数明显减少;DHT补充治疗后,树突棘个数明显增多.2.去势组海马CA1区突触素和NMDAR1的表达明显减少,平均吸光度值明显低于其他组(P<0.05).DHT补充治疗能明显增加突触素和NMDAR1的表达.结论 DHT可调节海马CA1区突触可塑性,使树突棘密度增多.DHT对突触可塑性的影响可能与其调节锥体细胞的NMDAR1有关.     

Testosterone-driven seasonal regulation of vasopressin and galanin in the bed nucleus of the stria terminalis of the Djungarian hamster (Phodopus sungorus)

The sexually dimorphic vasopressin system of the bed nucleus of the stria terminalis (BNST) is the most sensitive neurotransmitter system regulated by sex steroids in rats and mice. In addition to vasopressin, the BNST neurons also express a second neuropeptide, galanin, whose expression also appears to be regulated by testosterone in laboratory rodents. Seasonal fluctuations of sex steroids in photoperiodic rodents feed back on the brain to regulate the expression of sex steroid sensitive genes. The seasonal rhythm of circulating sex steroids is generated by photoperiod-controlled melatonin secretion, resulting in a seasonal stimulation and involution of the gonads. We have studied the seasonal expression of vasopressin and galanin in BNST neurons and their target areas in the Djungarian hamster (Phodopus sungorus). Furthermore, we analyzed the effect of testosterone on vasopressin and galanin by testosterone supplementation in animals where reproduction was inhibited by exposure to a short photoperiod. Exposure to short photoperiod induced a major reduction in the expression of vasopressin in BNST neurons, as well as in their target areas, the lateral septum (LS) and the lateral habenula (LHb). Galanin expression in the BNST and its target areas was also strongly reduced, although this reduction did not result in an almost complete disappearance of the neuropeptide as observed for vasopressin. Testosterone was able to reverse this reduction for both vasopressin and galanin. However, while the mRNA expression in BNST neurons recovered within 2-4 days, recovery of the neuropeptide immunoreactivity in the target areas, LS and LHb, required more than 3 weeks. The photoperiod-driven testosterone rhythm thus appears to be a major regulator of extra-hypothalamic vasopressin and galanin in the Djungarian hamster. The long delay between mRNA recovery in the cell body and the neuropeptide recovery in the target areas may be due to progressive filling up of the axon terminals. Alternatively, this delay might be indicative of a seasonal structural plasticity.     

小鼠蛛网膜下腔出血后海马CA1区mGluR1和ERK1/2的表达变化

目的:探讨小鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后代谢型谷氨酸受体1(metabotropicglutamate receptor 1,mGluR1)及细胞外信号调节激酶1/2(ERK1/2)调控神经细胞凋亡的分子机制。方法:采用非开颅血管内穿线法制备小鼠SAH模型,随机分为3组:假手术组、SAH+生理盐水(SAH+NS)组、SAH+LY367385(mGluR1抑制剂,SAH+LY367385)组,于SAH后10 min侧脑室注射生理盐水或LY367385(500 nmol/L)5μl,术后行神经功能评分。分别在SAH后6、24、48 h 3个时间点取右侧脑组织标本,逆转录-聚合酶链式反应(RT-PCR)检测各组mGluR1的表达变化,免疫印迹法(Western Blot)检测p-ERK1/2蛋白的表达,TUNEL法检测右侧海马CA1区神经细胞的凋亡。结果:与假手术组比较,SAH+NS组小鼠神经功能评分均显著降低(P<0.05),随SAH时间延长,各组小鼠mGluR1、p-ERK1/2蛋白均有不同程度增强,凋亡细胞增多(P<0.05)。与SAH+NS组比较,SAH+LY367385组小鼠神经功能评分增加,mGluR1、p-ERK1/2蛋白表达均有不同程度下调,神经细胞凋亡数目有所减少。SAH后6～48 h,mGluR1的表达与p-ERK1/2呈正相关。结论:mGluR1和ERK在SAH的发病机制中发挥了重要作用,SAH后海马内mGluR1的表达增强可通过激活ERK信号途径诱导神经细胞的凋亡。     

全脑缺血-再灌注降低成年大鼠海马CA1区抑制性突触功能

目的观察全脑缺血-再灌注对成年大鼠海马CA1区抑制性突触功能的影响。方法成年雄性大鼠,随机分为:1)假手术组(SH)、2)缺血-再灌注3 d组(IR-3)、3)缺血-再灌注7 d组(IR-7)。用四动脉阻断法制作全脑缺血模型,使用全细胞电压钳技术记录海马脑片CA1区锥体细胞诱发的抑制性突触后电流(eIPSCs)。结果与SH组相比:低刺激强度时,IR-3及IR-7组eIPSCs的幅值明显降低(P<0.05);IR-7组eIPSCs的上升时间明显变短(P<0.05);IR-3组eIPSCs的配对抑制明显变大(P<0.05)。结论全脑缺血-再灌注降低了大鼠海马CA1区抑制性突触功能。     

磷酸化的细胞周期相关蛋白在大鼠海马神经元发育和老化过程中的表达

目的： 观察正常Wistar大鼠发育及老化过程中海马神经元磷酸化的细胞周期相关蛋白的表达,探讨这一过程中海马神经元的细胞周期的变化以及磷酸化细胞周期相关蛋白在其中的调控作用。方法：采用免疫荧光方法观察不同发育时期(1天、11天、1月、3月、15月) 磷酸化周期素依赖激酶2（CDK2）、磷酸化细胞分裂周期激酶2（CDC2）、磷酸化视网膜母细胞瘤（Rb）蛋白表达的规律,并应用Western blotting方法测定不同阶段大鼠海马内磷酸化CDK2、磷酸化CDC2、磷酸化Rb的含量。结果：在各年龄组中神经元特异性核蛋白（NeuN）阳性细胞数量随着年龄增加而逐渐减少,提示神经元数量随年龄的增加而逐渐减少。磷酸化CDK2、磷酸化Rb阳性细胞的数量随着年龄增加而逐渐增多,老年组增加明显,与其它各组间有显著差异,磷酸化CDC2阳性细胞在各年龄组神经元中表达量均较低;蛋白定量亦提示老年组磷酸化CDK2、磷酸化Rb的含量较其它组明显增高。结论：海马神经元数量随年龄增加逐渐减少,而其中磷酸化CDK2和磷酸化Rb却随年龄的增长逐渐增多,提示老化过程中部分神经元再次进入细胞周期,说明磷酸化细胞周期相关蛋白可能参与了这一过程中海马神经元的凋亡。     

Gap junctions on GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus (CA1 region)

Summary Gap junctions were identified for the first time on chemically defined neurons in the central nervous system. Gap junctions were thus demonstrated on GABAergic neurons containing the calcium-binding protein parvalbumin (PV) in the rat hippocampus. Thin and semithin (0.5 m thick) sections were cut alternately and consecutively from osmium-fixed tissue which was embedded in epoxy resin and usable for conventional electron microscopic studies. The semithin sections were processed for postembedding immunocytochemistry using an anti-PV serum. Structures corresponding to the PV-immunoreactive (PV-I) profiles on the semithin sections were easily identified on electron micrographs from the adjacent thin sections. Using this technique gap junctions were found (1) between PV-I dendrites, (2) between PV-I dendrites and PV-I somata and (3) between PV-I dendrites and small processes whose origin could not be identified. Despite a systematic search, we did not find gap junction between PV-negative processes.     

Delayed neuronal cell death and microglial cell reactivity in the CA1 region of the rat hippocampus in the cardiac arrest model

Morphological changes in the CA1 region of the hippocampus in the rat cardiac arrest model were studied with the in situ nick-end labeling (TUNEL) method and light and electron microscopy. The TUNEL-positive pyramidal cells first appeared on day 1, increased in number with time, and reached a peak at 7 days after recirculation. At the ultrastructural level, cell shrinkage, nuclear fragmentation, and an increased number of atuophagic vacuoles of the pyramidal cells were observed in the CA1 region. The brief ischemia activates the microglial cells in the CA1 region, and these cells were found to increase in number with time. The microglial cells were seen to adhere to degenerating pyramidal cells and to phagocytose the apoptotic neurons selectively.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs) is involved in BMP signaling through phosphorylation of SMADS and TAK1 in early mouse embryo

Hepatocyte growth factor-regulated tyrosine kinase substrate that is encoded by Hgs promotes degradation of ubiquitinated signaling molecule in the early endosome. We previously reported that a targeted mutation in Hgs results in embryonic lethality soon after gastrulation in the mouse. Here, we report that downstream target genes for BMP signaling were highly down-regulated in the Hgs mutant embryos. We also showed that Hgs is required for phosphorylation of SMAD1/5/8 and TAK1/p38 to transduce BMP signaling. Furthermore, we found that HGS functions to localize TAK1 in early endosome for its activation. These results suggest that HGS is critical to localize TAK1 to early endosome for transducing BMP signaling for proper development. Our data revealed a new mechanism to modify BMP signaling by Hgs during early mouse development.     

Comparative Ultrastructural Analysis of Mitochondria in the CA1 and CA3 Hippocampal Pyramidal Cells Following Global Ischemia in Mongolian Gerbils

Post‐ischemic injury of the hippocampus unrolls at different levels and has both functional and structural implications. The deficiency in neuron energy metabolism is an initiating factor. We performed transmission electron microscopic (TEM) comparative analysis of mitochondria in excitatory spine synapses in CA1 stratum radiatum and CA3 hippocampal areas after 5 min of global cerebral ischemia in Mongolian gerbils, 4 and 7 days after reperfusion. Electron microscopy and unbiased morphometric methods were used to evaluate synaptic plasticity, and the number and size of mitochondria in synaptic terminals. We compared the morphological organization of mitochondria in presynaptic terminals between CA1 and CA3 areas in control and post‐ischemic condition according to the following morphometric parameters: mitochondrial volume fraction, mitochondrial frequency in CA1 and CA3 terminals, mean number of mitochondria per presynaptic terminal, frequency of damaged mitochondria in terminals, and density of presynaptic terminals. Our ultrastructural study revealed statistically significant differences in morphometric parameters between CA1 and CA3 areas in control conditions, as well as in post‐ischemic conditions. Also, we found temporal differences in measured parameters obtained 4 and 7 days after reperfusion. This study showed significant morphological differences in the organization of mitochondria in excitatory spine synapses between CA1 and CA3 areas, which corresponded with already known differences in functionality and sensitivity to the ischemic insult. Our conclusion is that revealed post‐ischemic changes in mitochondrial distribution in presynaptic CA1 and CA3 terminals could be an indicator of hippocampal metabolic dysfunction and synaptic plasticity. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

gamma-Hydroxybutyrate induces cyclic AMP-responsive element-binding protein phosphorylation in mouse hippocampus: an involvement of GABA(B) receptors and cAMP-dependent protein kinase activation

gamma-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate gamma-hydroxybutyrate's action. Acute administration of gamma-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP-responsive element-binding protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABA(B) receptor antagonist [3-[1-(R)-[(3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of gamma-hydroxybutyrate, confirming a GABA(B) receptor-mediated mechanism. In addition, acute gamma-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent protein kinase activity in the hippocampus, and pretreatment with the cAMP-dependent protein kinase inhibitor H-89 could prevent the effect of gamma-hydroxybutyrate on cAMP-responsive element-binding protein phosphorylation, indicating a direct involvement of cAMP-dependent protein kinase in gamma-hydroxybutyrate-induced cAMP-responsive element-binding protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding protein was not observed in the hippocampus of mice subjected to repeated gamma-hydroxybutyrate exposure, suggesting the development of a gamma-hydroxybutyrate-induced desensitization of the signaling pathway leading to cAMP-responsive element-binding protein activation. Since cAMP-responsive element-binding protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying gamma-hydroxybutyrate-induced neuroadaptations.     

Sprouting and synaptic reorganization in the subiculum and CA1 region of the hippocampus in acute and chronic models of partial-onset epilepsy

Repeated seizures induce permanent alterations in the hippocampal circuitry in experimental models and patients with intractable temporal lobe epilepsy (TLE). Most studies have concentrated their attention on seizure-induced reorganization of the mossy fiber pathway. The present study examined the projection pathway of the CA1 pyramidal neurons to the subiculum, which is the output of the hippocampal formation in five models of TLE. We examined the laminar pattern of Timm's histochemistry in the stratum lacunosum-moleculare of CA1 in three acute and two chronic models of TLE: intraventricular kainic acid (KA), systemic KA, systemic pilocarpine, chronic electric kindling and chronic i.p. pentylenetetrazol. The laminar pattern of Timm histochemistry in the stratum moleculare of CA1 was permanently remodeled in epileptic models suggesting sprouting of Timm containing terminals from the adjacent stratum lacunosum. Ultrastructural examination confirmed that Timm granules were localized in synaptic terminals. As the source of Timm-labeled terminals in this region was not known, sodium selenite, a selective retrograde tracer for zinc-containing terminals, was iontophoretically injected in vivo in rats exposed to systemic pilocarpine, systemic KA or chronic pentylenetetrazol. The normal projection of CA1 pyramidal neurons to the subiculum is topographically organized in a lamellar fashion. In normal rats, the extent of the injection site (terminals) and the retrogradely labeled pyramidal neurons (cell soma) corresponded to the same number of lamellas. In epileptic rats, the retrograde labeling extended 42-67% farther than the normal dorso-ventral extent including lamellas above and below the expected. This is direct evidence for sprouting of CA1 pyramidal axons into the subiculum and stratum lacunosum-moleculare of the CA1 region confirming the alterations of the laminar pattern of Timm's histochemistry. Sprouting of the CA1 projection to subiculum across hippocampal lamellas might lead to translamellar hyperexcitability, and to amplification and synchronization of epileptic discharges in the output gate of the hippocampal formation.     

Platelet-activating factor-induced synaptic facilitation is associated with increased calcium/calmodulin-dependent protein kinase II,protein kinase C and extracellular signal-regulated kinase activities in the rat hippocampal CA1 region

Platelet-activating factor (PAF) is an important inflammatory lipid mediator affecting neural plasticity. In the present study, we demonstrated how PAF affects synaptic efficacy through activation of protein kinases in the rat hippocampal CA1 region. In cultured hippocampal neurons, 10 to 1000 nM PAF stimulated autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of synapsin I and myristoylated alanine-rich protein kinase C substrate (MARCKS). In hippocampal CA1 slices, field excitatory postsynaptic potentials (fEPSPs) induced by stimulation of the Schaffer collateral/commissural pathways were significantly increased 10–50 min after exposure to 100 to 1000 nM PAF. Immunoblotting analysis showed that 100 nM PAF treatment for 10 or 50 min significantly and persistently increased CaMKII autophosphorylation in the hippocampal CA1 region. Increased protein kinase Cα (PKCα) autophosphorylation was also seen at the same time point after PAF exposure. By contrast, extracellular signal-regulated kinase (ERK) phosphorylation was slightly but significantly increased at 10 min after PAF exposure. Consistent with increased CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (GluR1) (Ser-831) phosphorylation as a CaMKII postsynaptic substrate significantly increased after 10 or 50 min of treatment, whereas synapsin I (Ser-603) phosphorylation as a presynaptic substrate increased at 10 min in the hippocampal CA1 region. Phosphorylation of MARCKS (Ser-152/156) and NMDA receptor subunit 1 (NR1) (Ser-896) as PKCα substrates also significantly increased after 10 min but had not further increased by 50 min in the CA1 region. Increased of fEPSPs induced by PAF treatment completely and/or partly inhibited by KN93 and/or U0126 treatment. These results suggest that PAF induces synaptic facilitation through activation of CaMKII, PKC and ERK in the hippocampal CA1 region.     

The axon initial segment as a synaptic site: Ultrastructure and synaptology of the initial segment of the pyramidal cell in the rat hippocampus (CA3 region)

Summary The axon initial segments (ISs) of pyramidal cells in the rat hippocampus (CA3 region) were studied by means of light microscopy of Golgi-impregnated material and electron microscopy of random and serial thin sections.The ISs display three distinguishing characteristics; fascicles of microtubules, membrane undercoating and clusters of ribosomes. The ISs contain cisternal organelles which are often associated with synapses and are in continuity with smooth and rough endoplasmic reticulum.Small spines are recognized on the ISs both in the light and electron microscope. There are 10–25 on each IS and they are usually concentrated on the proximal 30 m of the IS. Axonic spines contain spine apparatuses, clusters of ribosomes, multivesicular bodies and other organelles. Several collaterals are also recognized to originate from the axon proximal to the start of a myelin sheath.The IS receives many synapses both on its shaft and spines. Almost all of them are of the symmetrical type with flattened vesicles but a few asymmetrical synapses with spherical vesicles occur. Pyramidal cell ISs are very rarely presynaptic at asymmetrical synapses with spherical vesicles. Based on serial sectioning studies, the number of synapses on one IS is estimated at 100–200. These abundant synaptic contacts on the IS suggest that it is an important synaptic site. The possibility that there are two different inhibitory systems controlling the output of the pyramidal cell is discussed.     

Axonal and dendritic arborization of an intracellularly labeled chandelier cell in the CA1 region of rat hippocampus

Summary During the course of an in vivo intracellular labeling study, a chandelier (axo-axonic) cell was completely filled with biocytin in the CA1 region of the hippocampus. Chandelier cells are known to provide GABAergic terminals exclusively to the axon initial segment of pyramidal cells. The lateral extent and laminar distribution of the dendritic arborization of the chandelier cell was very similar to that of pyramidal cells; the numerous basal and apical dendrites reached the ventricular surface and the hippocampal fissure, respectively. The dendrites, however, had very few spines. The neuron had an asymmetric axonal arbor occupying an elliptical area of 600 by 850 m in the pyramidal cell layer and stratum oriens, with over three-quarters of the axon projecting to the fimbrial side of the neuron. Counting all clusters of terminals, representing individually innervated axon initial segments, the chandelier cell was estimated to contact 1214 pyramidal cells, a number that exceeds previous estimations, based on Golgi studies, by several-fold. The findings support the view that chandelier cells may control the threshold and/or synchronize large populations of principal cells.     

Effect of age on blood vessels and neurovascular appositions in the CA1 region of the rat hippocampus

Rats aged 3, 9, 24 and 30 months were used in this study. Increased basal lamina thickening in capillaries, muscular large vessels and nonmuscular large vessels was shown with advancing age. There is also an age-related increase in the area of mitochondria in smooth muscle cells. These ultrastructural changes may underlie observed age-related functional changes in the vasculature. They may be a compensatory response of the vessel wall cells to a declining capacity to handle the continual and varying shear stress exerted by the blood. Ultrastructural differences between capillaries and the two types of large vessels are reported and discussed in terms of their functional significance. It was noted that there are more dendrites adjacent to capillaries than to large vessels, however, this was unaffected by increasing age. Since advancing age did not alter the number of neuronal processes adjacent to vessels, age-related compromises in vessel function may not be subjected to neuronal regulation.