星形胶质细胞条件培养基对培养海马神经元GluR2和PICK1 mRNA表达的调控

目的 探讨癫痫发病过程中,星形胶质细胞对神经元AMPA受体亚单位表达的调节机制.方法 收集谷氨酸刺激的星形胶质细胞条件培养基,作用于培养的海马神经元,用RT-PCR方法检测神经元GluR2和PICK1 mRNA表达的变化.结果 在星形胶质细胞条件培养基作用2h、8h、12h后,培养的海马神经元GluR2mRNA表达明显下降,而PICK1 mRNA表达则升高,与对照组相比,差异有显著意义(P＜0.05).离子型谷氨酸受体拮抗剂D-AP5和CNQX不能完全阻断条件培养基的作用.结论 在癫痫发病过程中,星形胶质细胞激活能通过上调PICK1的表达,实现下调神经元AMPA受体GluR2亚单位的表达.     

衰老大鼠海马结构内生长抑素样神经元的变化——免疫金银染色法及图像定量分析研究

本研究结合免疫金银染色法和图像定量分析,观察了幼年(2个月)、中年(10个月)和老年(24个月)Wistar 雄性大鼠海马结构内生长抑素(SOM)样神经元的衰老变化,结果如下:1.海马内的 SOM 样神经元从中年即开始锐减(P<0.05),而齿状回和下托内的阳性神经元到老年才开始呈显著性减少(P<0.05)。2.中年组 SOM 样神经元的胞体面积显著增大(P<0.01);而老年时的胞体有轻度萎缩现象,且轮廓不规整,突起伸长增粗,但边缘粗糙,似有皱折。3.SOM样神经元的灰度值随增龄而升高.提示神经元的 SOM 含量和活性在衰老进程中是逐渐降低的。     

Subunit interaction with PICK and GRIP controls Ca2+ permeability of AMPARs at cerebellar synapses

At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. Here we examine how glutamate receptor interacting protein (GRIP, also known as AMPAR binding protein or ABP) and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. We find that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors.     

Aging and cholinergic deafferentation alter GluR1 expression in rat frontal cortex

Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of aged rats compared to young adults. Cholinergic afferents from the NBM modulate glutamatergic transmission in neocortex, and glutamate is known to be involved in dendritic plasticity. To begin to identify possible mechanisms underlying age-related differences in plasticity after NBM lesion, we assessed the effect of cholinergic deafferentation on expression of the AMPA receptor subunit GluR1 in frontal cortex of young adult and aging rats. Young adult, middle-aged, and aged rats received sham or 192 IgG-saporin lesions of the NBM, and an unbiased stereological technique was used to estimate the total number of intensely GluR1-immunopositive neurons in layer II-III of frontal cortex. While the number of GluR1-positive neurons was increased in both middle-aged and aged rats, lesions markedly increased the number of intensely GluR1-immunopositive neurons in frontal cortex of young adult rats only. This age-related difference in lesion-induced expression of AMPA receptor subunit protein could underlie the age-related differences in dendritic plasticity after NBM lesions.     

Brainstem and spinal projections of augmenting expiratory neurons in the rat

To examine the role of Ca(2+) entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca(2+)-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca(2+)-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca(2+)-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.     

Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation

Postnatal glutamatergic principal neuron synapses are typically presumed to express only calcium-impermeable (CI), GluR2-containing AMPARs under physiological conditions. Here, however, we demonstrate that long-term potentiation (LTP) in CA1 hippocampal pyramidal neurons causes rapid incorporation of GluR2-lacking calcium-permeable (CP)-AMPARs: CP-AMPARs are present transiently, being replaced by GluR2-containing AMPARs approximately 25 min after LTP induction. Thus, CP-AMPARs are physiologically expressed at CA1 pyramidal cell synapses during LTP, and may be required for LTP consolidation.     

Effects of aging and caloric restriction on dentate gyrus synapses and glutamate receptor subunits

Caloric restriction (CR) attenuates aging-related degenerative processes throughout the body. It is less clear, however, whether CR has a similar effect in the brain, particularly in the hippocampus, an area important for learning and memory processes that often are compromised in aging. In order to evaluate the effect of CR on synapses across lifespan, we quantified synapses stereologically in the middle molecular layer of the dentate gyrus (DG) of young, middle aged and old Fischer 344 × Brown Norway rats fed ad libitum (AL) or a CR diet from 4 months of age. The results indicate that synapses are maintained across lifespan in both AL and CR rats. In light of this stability, we addressed whether aging and CR influence neurotransmitter receptor levels by measuring subunits of NMDA (NR1, NR2A and NR2B) and AMPA (GluR1, GluR2) receptors in the DG of a second cohort of AL and CR rats across lifespan. The results reveal that the NR1 and GluR1 subunits decline with age in AL, but not CR rats. The absence of an aging-related decline in these subunits in CR rats, however, does not arise from increased levels in old CR rats. Instead, it is due to subunit decreases in young CR rats to levels that are sustained in CR rats throughout lifespan, but that are reached in AL rats only in old age.     

Activation of postsynaptic Ca(2+) stores modulates glutamate receptor cycling in hippocampal neurons

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP(3)Rs) with the IP(3)R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal synaptic transmission. The regulation of postsynaptic AMPAR trafficking involves synaptotagmin-SNARE-mediated vesicle exocytosis and interactions between AMPARs and the PDZ domains in GRIP/PICK1. We show that inhibitors of synaptotagmin-SNARE-mediated exocytosis, or interactions between AMPARs and GRIP/PICK1, attenuated AdA-enhanced increases in EPSC amplitudes. These results suggest that IP(3)R-mediated Ca(2+) release can enhance AMPAR EPSC amplitudes through mechanisms that involve AMPAR-PDZ interactions and/or synaptotagmin-SNARE-mediated receptor trafficking.     

Multiple changes in noradrenergic mechanisms in the coeruleo-hippocampal pathway during aging. Structural and functional correlates in intraocular double grafts

Age-related changes of the coeruleo-hippocampal noradrenergic system were investigated using intraocular double transplants. Pieces of fetal hippocampus were grafted into the anterior chamber of the eye and placed into contact with previously inserted locus coeruleus grafts. Ages of both transplants and hosts were varied to enable studies of intrinsic versus extrinsic determinants of aging in an isolated neuronal circuit. Four different experimental groups, with the approximate age in months of grafts/hosts at the time of recording given in parentheses, were studied; young grafts in the eyes of young hosts (3/7), young grafts in the eyes of old hosts (3/23), mature transplants in adult host rats (8/12) and aged transplants in the eyes of aged rats (21/25). Extracellular recordings from the hippocampal part of the double grafts were performed. Superfusion with alpha-adrenergic antagonists and the alpha 2-agonist clonidine elicited significant increases in the discharge rate of the grafted hippocampal neurons in all groups except the aged transplants in the aged hosts (21/25), where a small excitation was elicited with clonidine and no effect at all was seen with alpha-adrenergic antagonists. The host age did not seem to be important since young transplants in the old hosts (3/23) showed a similar increase in discharge rate as transplants in the young and adult hosts. Tyrosine hydroxylase immunohistochemistry and high-performance liquid chromatography revealed that hippocampal transplants remaining in oculo for a minimum of 6-10 months became permanently hyperinnervated by noradrenergic fibers from the locus coeruleus grafts. The density of noradrenergic fibers was significantly lower in young transplants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dominant role of the GluR2 subunit in regulation of AMPA receptors by CaMKII

GluR1 and GluR2 subunits compose AMPA receptors (AMPARs) in the mature hippocampus, and both the GluR1 subunit and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are required for synaptic plasticity, memory and learning. Although GluR1 phosphorylationby CaMKII is preserved, the functional regulation of AMPARs by phosphorylation is lost in the presence of the GIuR2 subunit. Our findings define a previously unknown, dominant role of the GluR2 subunit in signaling mediated by CaMKII at AMPARs.     

Place cells of aged rats in two visually identical compartments

Aged rats perform poorly on spatial learning tasks, a cognitive impairment which has been linked to the failure of hippocampal networks to fully encode changes in the external environment [Barnes CA, Suster MS, Shen J, McNaughton BL. Multistability of cognitive maps in the hippocampus of old rats. Nature 1997;388(6639):272-5; Wilson IA, Ikonen S, Gureviciene I, McMahan RW, Gallagher M, Eichenbaum H, et al. Cognitive aging and the hippocampus: how old rats represent new environments. J Neurosci 2004;24(15):3870-8]. To examine whether the impairment in hippocampal processing extends to conditions in which self-motion provides the cues for environmental change, we have analyzed spatial firing patterns of hippocampal pyramidal neurons in young and aged rats, as well as in young rats with selective cholinergic lesions, another model of cognitive aging. The rats walked between two visually identical environments, pitting self-motion cues that indicated environmental change against visual inputs that indicated no differences between environments. Our results indicated that place cells in both aged and cholinergic-lesioned rats were equally likely as those of young rats to create new spatial representations in the second compartment. These findings suggest that the hippocampal network of aged rats is able to process changes in internally generated cues without rigidity, but that incomplete processing of external landmark cues may lead to impaired spatial learning.     

Modification of ionotropic glutamate receptor–mediated processes in the rat hippocampus following repeated,brief seizures

The seizure-induced molecular and functional alterations of glutamatergic transmission in the hippocampus have been investigated. Daily repeated epileptic seizures were induced for 12 days by intraperitoneal administration of 4-aminopyridine (4-AP; 4.5 mg/kg) in adult Wistar rats. The seizure symptoms were evaluated on the Racine's scale. One day after the last injection, the brains were removed for in vitro electrophysiological experiments and immunohistochemical analysis. The glutamate receptor subunits NR1, NR2A, NR2B, GluR1, GluR1_flop, GluR2, and KA-2 were studied using the histoblotting method. The semi-quantitative analysis of subunit immunoreactivities in hippocampal layers was performed with densitometry. In the hippocampus, increase of GluR1, GluR1_flop and NR2B immunostaining was observed in most of the areas and layers. The significant decrease of GluR2 staining intensity was observed in the CA1 and dentate gyrus. Calcium permeability of hippocampal neurons was tested by a cobalt uptake assay in hippocampal slices. The uptake of cobalt increased in the CA1 area and dentate gyrus, but not in the CA3 region following 4-AP treatment. Effects of AMPA and NMDA (N-methyl-d-aspartate) glutamate receptor antagonists (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) and D-APV respectively) were measured in hippocampal slices using extracellular recording. Analysis of the population spikes revealed the reduced effectiveness of the AMPA receptor antagonist GYKI 52466, while the effect of the NMDA receptor antagonist d-(2R)-amino-5-phosphonovaleric acid was similar to controls. The results demonstrated that repeated convulsions induced structural and functional changes in AMPA receptor–mediated transmission, while NMDA and kainate receptor systems displayed only alterations in receptor subunit composition.     

GluR2/3 label expression of the AMPA-type glutamate receptor in the hippocampal formation of the homing pigeon stabilizes just after birth

The compositions of the glutamate AMPA-type receptors influence the neural response and the subunits GluR2/3 has been referred to as essential for receptor trafficking and synapse consolidation. We investigate the GluR2/3 occurrence and expression in the hippocampal formation of newly born homing pigeons by a semi-quantitative approach, the Western-blotting technique and by immunohistochemistry. Immunoreactivity for GluR2/3 occurs before hatching has been evident in neuropil that was fully dispersed over the hippocampus proper (HP) and the area parahippocampalis (APH). Although many HP cells are NeuN-positives, a specific neuronal protein indicating that they are already differentiated as neurons while not one contains GluR2/3 at the hatching day (P0). Few neurons at the APH seem to express GluR2/3 at P0, but 3 days later (P3) the GluR2/3 labeling can be recognized in many HP neurons, showing a distribution pattern that resembles the adult, gradually increasing in intensity until P10. Also, the Western-blot shows an augment between P0 and P3, remaining stable after that. The enhancement of the neuronal label at P3 coincides with the retraction of the GluR2/3 label in neuropil, reducing their occurrence during the maturational period to become restricted to the dorsomedial portion as reported for adults. As the HP GluR2/3-containing cells are supposedly projecting neurons, taking together, the results signalize the relevance of the GluR2/3 in post-hatch formation of avian hippocampal circuitry in which the third day seems to be the critical period.     

Preserved memory capacities in aged Lou/C/Jall rats

Although memory impairments are a hallmark of aging, the degree of deficit varies across animal models, and is likely to reflect different states of deterioration in metabolic and endocrinological properties. This study investigated memory-related processes in young (3-4 months) and old (24 months) Sprague-Dawley rats (SD), which develop age-linked pathologies such as obesity or insulin-resistance and Lou/C/Jall rats, which do not develop such impairments. In short- and long-term memory recognition tasks, old Lou/C/Jall rats were never impaired whereas old SD rats were deficient at 1 and 24h latencies. The expression of N-methyl-d-aspartate receptors (NMDAR)-mediated synaptic plasticity in CA1 hippocampal networks shifted towards lower activity values in old Lou/C/Jall rats whereas long-term potentiation was impaired in age-matched SD rats. Age-related decrease in NR2A subunits occurred in both strains, extended to NR2B, NR1 and GluR1 subunits in older animals (28 months) but only in SD rats. Therefore, the Lou/C/Jall rats can be considered as a model of healthy aging, not only in terms of its preserved metabolism, but also in terms of cognition and synaptic plasticity.     

Age-related toxicity to lactate, glutamate, and beta-amyloid in cultured adult neurons

The age-related susceptibility of the brain to neurodegenerative disease may be inherent in the susceptibility of individual neurons to various stressors. Neurons were isolated from embryonic, young- and old-aged rat hippocampus, cultured in serum-free medium and exposed to lactic acid, glutamate or beta-amyloid. Yields of isolated adult cells were 1 million cells/hippocampus, 12,000 cells/mg tissue, independent of age. For lactic acidosis, there was a non-significant 10% increment in killing of neuron-like cells from old rats compared to young. For glutamate, there was a 5-10% increment in killing of neuron-like cells from old rats compared to young rats and embryonic neurons. For cells exposed to the toxic fragment of beta-amyloid, A beta (25-35), toxicity was age, dose and time-dependent. Maximum toxicity in cells treated for 1 day with 25 microM A beta (25-35) was 16%, 24%, and 33% for embryonic, young and old cells. Similar results were found for A beta (1-40) (LD50 = 2 microM). These results suggest that aging imparts to individual cells an increased susceptibility to toxic substances relevant to neurodegenerative diseases.     

Age-related toxicity to lactate, glutamate, and β-amyloid in cultured adult neurons

The age-related susceptibility of the brain to neurodegenerative disease may be inherent in the susceptibility of individual neurons to various stressors. Neurons were isolated from embryonic, young- and old-aged rat hippocampus, cultured in serum-free medium and exposed to lactic acid, glutamate or β-amyloid. Yields of isolated adult cells were 1 million cells/hippocampus, 12,000 cells/mg tissue, independent of age. For lactic acidosis, there was a non-significant 10% increment in killing of neuron-like cells from old rats compared to young. For glutamate, there was a 5–10% increment in killing of neuron-like cells from old rats compared to young rats and embryonic neurons. For cells exposed to the toxic fragment of β-amyloid, Aβ (25–35), toxicity was age, dose and time-dependent. Maximum toxicity in cells treated for 1 day with 25 μM Aβ (25–35) was 16%, 24%, and 33% for embryonic, young and old cells. Similar results were found for Aβ (1–40) ( ₅₀ = 2 μM). These results suggest that aging imparts to individual cells an increased susceptibility to toxic substances relevant to neurodegenerative diseases.     

The length of hippocampal cholinergic fibers is reduced in the aging brain

Cholinergic deficits occur in the aged hippocampus and they are significant in Alzheimer's disease. Using stereological and biochemical approaches, we characterized the cholinergic septohippocampal pathway in old (24 months) and young adult (3 months) rats. The total length of choline acetyltransferase (ChAT)-positive fibers in the dorsal hippocampus was significantly decreased by 32% with aging (F((1,9))=20.94, p=0.0014), along with the levels of synaptophysin, a presynaptic marker. No significant changes were detected in ChAT activity or in the amounts of ChAT protein, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin related kinase receptor (Trk) A, TrkB, or p75 neurotrophin receptor (p75(NTR)) in the aged dorsal hippocampus. The number and size of ChAT-positive neurons and the levels of ChAT activity, NGF and BDNF were not statistically different in the septum of aged and young adult rats. This study suggests that substantial synaptic loss and cholinergic axonal degeneration occurs during aging and reinforces the importance of therapies that can protect axons and promote their growth in order to restore cholinergic neurotransmission.     

PKC and polyamine modulation of GluR2-deficient AMPA receptors in immature neocortical pyramidal neurons of the rat

AMPA receptors (AMPARs) mediate the bulk of fast synaptic excitation in the CNS. We have recently shown that AMPAR-dependent synaptic transmission in immature neocortical pyramidal neurons is mediated by GluR2-deficient receptors that can be modulated by intra- or extracellular polyamines (PAs). Phosphorylation of AMPARs, e.g. by PKC, can lead to enhanced excitation, and PAs are known to modulate PKC activity. Therefore, PAs and PKC might interact to influence AMPAR function. To test this hypothesis, we made whole cell recordings from immature (P12–14) layer V pyramidal neurons and assayed two measures of PA influence on synaptic AMPAR function – inward rectification and use-dependent unblock (UDU), with the latter assayed by differences in rectification between a pair of EPSCs evoked at short (50 ms) latencies. We have previously shown that EPSCs in immature pyramidal neurons displayed inward rectification, which was enhanced by intracellular spermine, as was UDU. Staurosporin (ST), a PKC inhibitor, reversed the effect of PA on rectification and UDU, suggesting that PKC modulates postsynaptic activation of AMPARs. Similarly, polyamine-dependent rectification of spontaneous EPSCs was reversed by treatment with ST or GFX109203X, a specific PKC inhibitor. Chelating intracellular Ca²⁺ with BAPTA reproduced the effects of ST. In addition, PA immunoreactivity in layer V pyramidal neurons was reduced by PKC inhibition indicating that PKC activity influences PA metabolism. Taken together, these data support the involvement of postsynaptic PKC activation in both the inward rectification and UDU of EPSCs in immature rat cortex, and suggest an important mechanism by which excitatory synaptic transmission can be dynamically modulated by changes in either [Ca²⁺]_i or [PA]_i.     

Gene targeting reveals a role for the glutamate receptors mGluR5 and GluR2 in learning and memory

This work suggests that class I mGluRs are involved in long-term potentiation (LTP) at CA1 synapses within the hippocampus. Our data support a pathway linking class I-mGluRs with PKC and src to enhance the open probability of the NMDAR channel. This leads to LTP of the NMDAR, but not the AMPAR. We are currently analyzing double mGluR1 X mGluR5 knockouts with Collingridge for a loss of the LTP induction switch [Nature 368 (1994) 740.]. This induction of LTP of the NMDAR is necessary for "spatial" learning and memory to occur, since mice lacking the mGluR5 are deficient in the Morris water maze and context-dependent fear conditioning. We postulate that AMPARs may provide negative feedback inhibition to the NMDAR. Hence, in null mutants lacking the AMPAR subtype, GluR2, LTP in the CA1 region of hippocampal slices was markedly enhanced (twofold) and non-saturating, whereas neuronal excitability and paired-pulse facilitation were normal. The ninefold increase in Ca(2+) permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Enhanced LTP could result from enhanced AMPAR channel conductance or increased recruiting of previously silent synapses. Since the GluR2 null mutants showed reduced exploration and impaired motor coordination, we could make no conclusion about its role in learning and memory. Future work will be directed to inducible deletion of GluR2 only in CA1 after development is complete. These results support the correlation between LTP and learning and memory.