Altered hippocampal synaptic transmission in transgenic mice with astrocyte-targeted enhanced CCL2 expression

Elevated expression of neuroinflammatory factors in the central nervous system (CNS) contributes to the cognitive impairment in CNS disorders such as injury, disease and neurodegenerative disorders. However, information on the role of specific neuroimmune factors in normal and abnormal CNS function is limited. In this study, we investigated the effects of chronic exposure to the chemokine CCL2 on hippocampal synaptic function at the Schaffer collateral-CA1 synapse, a synapse that is known to play an important role in cognitive functions such as memory and learning. Synaptic function was measured in vitro using hippocampal slices obtained from transgenic mice that express elevated levels of CCL2 in the CNS through astrocyte expression and their non-transgenic littermate controls. Extracellular field potential electrophysiological recordings showed a significant reduction in the magnitude of synaptic responses in hippocampal slices from the CCL2 transgenic mice compared with slices from non-transgenic littermate controls. Two forms of short-term synaptic plasticity (post-tetanic potentiation and short-term potentiation) thought to be important cellular mechanisms of short-term memory were enhanced in hippocampal slices from CCL2 transgenic mice compared to non-transgenic hippocampal slices, whereas long-term synaptic plasticity (LTP), which is critical to long-term memory formation, was not altered. Western blot analysis of hippocampus from the CCL2 transgenic mice and non-transgenic mice showed no change in level of neuronal specific enolase, a neuronal specific protein, GFAP, an astrocyte specific protein, and several synaptic proteins compared with non-transgenic littermate controls. These results show that CCL2, which is known to be chronically produced at elevated levels within the CNS in a number of CNS disorders, can significantly alter hippocampal function and implicate a role for CCL2 in the cognitive dysfunction associated with these CNS disorders.     

Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production

Production of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), in the brain is increased in various diseases. To investigate the relationships between the effect of overproduction of IL-6 in the brain on central and peripheral production of TNF, IL-1β and IL-6 itself, we used transgenic mice (NSE-hlL-6) where neuronal human IL-6 expression under the control of the neuronal specific enolase promoter results in astrocytosis and gliosis. These mice had higher cerebral endogenous IL-6 (12-fold). 11.-I β (12-fold) and TNF (4-fold) production measured in brain homogenates after intracerebroventricular (i.c.v.) injection of 2.5 μg LPS, lipopolysaccharide (LPS) than wild-type mice (no TNF or IL- I were detectable in saline-injected NSE or control mice). Cerebral cytokines production was also increased in NSE-hIL-6 mice treated i.p. with LPS doses that do not normally induce cytokines in the brain. The induction of peripheral (serum or spleen) TNF, IL-1β or IL-6 was the same in all these experiments in NSE-hIL-6 and wild-type mice. Furthermore, using microglial cell clone pretreated in vitro with IL-6 we noted an increase in LPS-induced TNF and IL-6 production and proliferation of pretreated cells than control. This study indicates that overproduction of IL-6 in the central nervous system (CNS) may ultimately result in increased central production of inflammatory cytokines, probably due to increased proliferation and activation of the cells which produce cytokine in the CNS.     

Altered short-term hippocampal synaptic plasticity in mutant alpha-synuclein transgenic mice

Hippocampal synaptic plasticity was studied in transgenic mice over-expressing human alpha-synuclein containing the A30P Parkinson's disease mutation. Medial perforant path-dentate granule cell synapses showed enhanced paired-pulse depression (PPD) for short interpulse intervals (< 200 ms), without differences in basal transmission. Extracellular calcium reduction failed to rescue the enhanced PPD. Paired-pulse facilitation in the CA1 region was normal in slices from transgenic mice, but enhanced synaptic depression was revealed upon repetitive stimulation of the Schaffer collaterals. Long-term potentiation in the CA1 field was not impaired in slices from transgenic mice. These results suggest that mutant alpha-synuclein accumulation impairs short-term changes in synaptic strength when neurotransmitter availability is limited due to enhanced release probability or repetitive synaptic activity.     

Chronic complement C3 gene expression in the CNS of transgenic mice with astrocyte-targeted interleukin-6 expression

Both cytokines and complement are thought to play significant, but poorly understood roles, in the pathogenesis of a variety of neurodegenerative diseases. In this study, we examined the expression of C3, the central component of complement, in the brains of transgenic mice with constitutive astrocyte expression of the proinflammatory cytokine, interleukin-6 (IL-6). Immunohistochemistry studies demonstrated elevated deposition of C3 in the brains of transgenic animals compared with normal animals. Northern blot analysis of mRNA from brain and other tissues demonstrated an age-related increase in C3 gene expression only in the brains of transgenic animals, indicative of local synthesis. In situ hybridization studies revealed coincidence between C3 and IL-6 transgene expression, as well as areas of neuronal and white matter damage observed in cerebellum and hind brain. Furthermore, C3 mRNA expression was observed in ependymal cells, perivascular mononuclear cells, endothelial cells, and scattered cells throughout the white matter and the brain stem. The overlap in C3 mRNA expression with areas of pathology suggests that complement may contribute to the inflammation and cellular injury observed in this model. The transgenic mice used in these studies provide a novel and valuable tool for examining the role of complement in central nervous system pathobiology. © 1996 Wiley-Liss, Inc.     

Metallothionein-I overexpression decreases brain pathology in transgenic mice with astrocyte-targeted expression of interleukin-6

Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) causes significant damage and alters the expression of many genes, including a dramatic upregulation of metallothionein-I (MT-I). The findings in this report support the idea that the upregulation of MT-I observed in GFAP-IL6 mice is an important mechanism for coping with brain damage. Thus, GFAP-IL6 mice that were crossed with TgMTI transgenic mice (GFAP-IL6xTgMTI) and overexpressed MT-I in the brain showed a decreased upregulation of cytokines such as IL-6 and a diminished recruitment and activation of macrophages and T cells throughout the CNS but mainly in the cerebellum. The GFAP-IL6 mice showed clear evidence of increased oxidative stress, which was significantly decreased by MT-I overexpression. Interestingly, MT-I overexpression increased angiogenesis in GFAP-IL6 mice but not in control littermates. Overall, the results strongly suggest that MT-I+II proteins are valuable factors that protect against cytokine-induced CNS injury.     

Increased tumor necrosis factor and interleukin-6 production in the central nervous system of interleukin-10-deficient mice

Interleukin-10 (IL-10) inhibits tumor necrosis factor (TNF) production. We investigated the role of endogenous IL-10 in brain TNF production. We injected IL-10-knockout mice with lipopolysaccharide (LPS,2.5 microg/mouse i.c.v.). Brain TNF and IL-6 levels were more elevated and persisted longer in IL-10-deficient mice compared with wild type mice, suggesting that IL-10 is an important negative feedback inhibitor of TNF and IL-6 production in the CNS.     

Astrocyte-targeted expression of interleukin-6 protects the central nervous system during neuroglial degeneration induced by 6-aminonicotinamide

6-aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes mainly in the brainstem. We have examined the role of interleukin-6 (IL-6) in this degenerative process by using transgenic mice with astrocyte-targeted IL-6 expression (GFAP-IL6 mice). This study demonstrates that transgenic IL-6 expression significantly increases the 6-AN-induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL-6 induced significant increases in proinflammatory cytokines IL-1, IL-12, and tumor necrosis factor-alpha as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor-beta, neurotrophin-3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP-IL6 mice relative to controls after 6-AN. Moreover, oxidative stress and apoptotic cell death were significantly reduced by transgenic IL-6 expression. IL-6 is also a major inducer in the CNS of metallothionein I and II (MT-I+II), which were significantly increased in the GFAP-IL6 mice. MT-I+II are antioxidants and neuroregenerative factors in the CNS, so increased MT-I+II levels in GFAP-IL6 mice could contribute to the reduction of oxidative stress and cell death in these mice.     

Ontogeny of synaptic transmission in the rat hippocampus

Electrophysiological characteristics of the hippocampal slices of juvenile (14-27 days) or young (36-40 days) Wistar rats have been compared. In the juvenile rats measurements were taken daily, from postnatal day (PN) 14 to PN27. Input-output curves were used to quantify the ontogeny of excitatory processes. The dynamic of population spike (PS) maturation was not even during the investigated postnatal period. After day 19 transient decrease of PS amplitude was observed until day 22. There were also some differences between the shape of input-output curves from the slices of rats of different ages. In general, PS was saturated at lower intensities in younger animals. The slices from 19-day-old rats did not display saturated input-output curve with 2-20 V stimuli intensities. But input-output curves on PN20-22 were rather similar to that obtained before PN19. The periods of gradual increase and subsequent decrease of PS amplitudes during early ontogeny correlate with the appearance of certain forms of behaviour. This fact suggests that hippocampal PS amplitude depression may be relevant functionally.     

The role of interleukin-6 in central nervous system demyelination

正Demyelination of the central nervous system(CNS)is a hallmark of multiple sclerosis(MS),chronic inflammatory and neurodegenerative disease.Chronic demyelination favors neurodegeneration of denuded axons,which is a major cause of irreversible neuronal deficits and disability in MS patients(Lucchinetti et al.,2000).MS remains an incurable disease,despite formida-     

Lack of interleukin-6 expression is not protective against focal central nervous system ischemia

BACKGROUND AND PURPOSE: Interleukin-6 (IL-6) appears to be involved in the inflammatory response associated with central nervous system (CNS) ischemia. Although IL-6 levels increase after stroke, it is not known whether IL-6 directly influences CNS ischemic injury. In this study, we used a focal reversible stroke model to investigate whether mice lacking IL-6 were protected against acute ischemic injury. METHODS: We bred IL-6-deficient C57 black mice (I-129 IL-6 KO back-crossed with C57), including homozygous knockouts (IL-6 -/-), heterozygous littermates (IL-6 +/-), and normal littermates (IL-6 +/+). The status of all animals was confirmed by DNA sampling and polymerase chain reaction analysis. Reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 2 hours (experiment 1) or 45 minutes (experiment 2). At 24 hours, animals were evaluated on a 28-point clinical scale, blood and cerebrospinal fluid were obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. RESULTS: In experiment 1 (severe ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=15), 57+/-13 mm(3); IL-6 +/- (n=15), 58+/-23 mm(3); and IL-6 +/+ (n=15), 58+/-18 mm(3) (P=NS). ELISA testing confirmed very low to absent levels of IL-6 in the serum and cerebrospinal fluid of knockout animals. Brain mRNA levels of the other proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist, were 50% lower in IL-6-deficient ischemic animals than in normal animals. In experiment 2 (mild ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=10), 16+/-8 mm(3); IL-6 +/- (n=10), 14+/-4 mm(3); and IL-6 +/+ (n=10), 19+/-12 mm(3) (P=NS). CONCLUSIONS: In this study, infarct size and neurological function at 24 hours were not different in animals deficient in IL-6 after transient CNS ischemia. This suggests that IL-6 does not have a direct influence on acute ischemic injury. Further study investigating the role of IL-6 on long-term recovery after stroke is in progress.     

Metallothionein-I overexpression alters brain inflammation and stimulates brain repair in transgenic mice with astrocyte-targeted interleukin-6 expression

Transgenic expression of IL-6 in the CNS under the control of the GFAP gene promoter, glial fibrillary acidic protein-interleukin-6 (GFAP-IL-6) mice, raises an inflammatory response and causes significant brain damage. However, the results obtained in the GFAP-IL-6 mice after a traumatic brain injury, such as a cryolesion, demonstrate a neuroprotective role of IL-6. Thus, the GFAP-IL-6 mice showed faster tissue repair and decreased oxidative stress and apoptosis compared with control litter-mate mice. The neuroprotective factors metallothionein-I+II (MT-I+II) were upregulated by the cryolesion to a higher extent in the GFAP-IL-6 mice, suggesting that they could be related to the neuroprotection afforded by the transgenic expression of IL-6. To examine this possibility, we have crossed GFAP-IL-6 mice with transgenic mice overexpressing MT-I (TgMT), producing double transgenic GFAP-IL-6 TgMT mice. The results obtained after cryolesion in GFAP-IL-6 TgMT mice, as well as in TgMT mice, consistently supported the idea that the increased MT-I+II levels observed in GFAP-IL-6 mice are a fundamental and important mechanism for coping with brain damage. Accordingly, MT-I overexpression regulated the inflammatory response, decreased oxidative stress and apoptosis significantly, and increased brain tissue repair in comparison with either GFAP-IL-6 or control litter-mate mice. Overall, the results demonstrate that brain MT-I+II proteins are fundamental neuroprotective factors.     

Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice

Neurons are generated from neural progenitor cells not only during development but also in the mature brain. To develop an in vivo system for analyzing neurogenesis, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of regulatory regions of the nestin gene. GFP fluorescence was observed in areas and during periods connected with neurogenesis, including embryonic neuroepithelium, neonatal cerebellum, and hippocampal dentate gyrus and rostral migratory pathway from the subventricular zone to the olfactory bulb in the adult. GFP-positive cells in the adult brain included immature neuronal cells expressing polysialylated NCAM. BrdU labeling experiments revealed that newly generated interneurons which migrated rostrally from the subventricular zone expressed GFP until they reached the olfactory bulb. These results indicate that nestin promoter-GFP transgenic mice can be utilized to visualize the regions of neurogenesis throughout the life of the animals and to follow the migration and differentiation of newly generated neurons.     

Altered central nervous system cytokine-growth factor expression profiles and angiogenesis in metallothionein-I+II deficient mice.

To study the importance of metallothionein-I and -II (MT-I+II) for brain inflammation and regeneration, the authors examined normal and MT-I+II knock-out (MT-KO) mice subjected to a cortical freeze injury. Normal mice showed profound neurodegeneration, inflammation, and gliosis around the injury, which was repaired by 20 days postlesion (dpl). However, in MT-KO mice the lesion-associated inflammation was still present as late as 90 dpl. Scanning electron microscopy demonstrated that the number of capillaries was lower, and ultrastructural preservation of the lesioned parenchyma was poorer in MT-KO mice, suggesting an altered angiogenesis. To gain insight into the mechanisms involved, a number of cytokines and growth factors were evaluated. The number of cells expressing the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha was higher in MT-KO mice than in normal mice, which was confirmed by RNase protection analysis, whereas the number of cells expressing the growth factors bFGF, TGFbeta1, VEGF, and NT-3 was lower. Increased expression of proinflammatory cytokines could be involved in the sustained recruitment of CD-14+ and CD-34+ inflammatory cells and their altered functions observed in MT-KO mice. Decreases in trophic factors bFGF, TGFbeta1, and VEGF could mediate the decreased angiogenesis and regeneration observed in MT-KO mice after the freeze lesion. A role for MT-I+II in angiogenesis was also observed in transgenic mice expressing IL-6 under the control of the promoter of glial fibrillary acidic protein gene (GFAP-IL6 mice) because MT-I+II deficiency dramatically decreased the IL-6-induced angiogenesis of the GFAP-IL6 mice. In situ hybridization analysis indicated that the MT-III expression was not altered by MT-I+II deficiency. These results suggest that the MT-I+II isoforms have major regulatory functions in the brain inflammatory response to injury, especially in the angiogenesis process.     

Chronic interleukin-6 alters the level of synaptic proteins in hippocampus in culture and in vivo

There is now considerable evidence that the level of expression of the proinflammatory cytokine, interleukin-6 (IL-6), is increased in the central nervous system (CNS) during neuroinflammatory conditions such as occurs in neurological disorders and in disease and injury. However, our understanding of the consequences of increased expression of IL-6 on the CNS is still limited, especially with respect to the developing nervous system, which is known to be particularly vulnerable to environmental factors. To address this issue, we investigated the properties of cultured hippocampal neurons exposed chronically to IL-6 during the main period of morphological and physiological development, which occurs during the first 2 weeks of culture. IL-6 was tested at 500 U/mL, considered to reflect a pathophysiologic concentration. The morphological features of neuronal development in the control and IL-6-treated cultures appeared similar. However, Western blot analysis showed a significant reduction in the level of Group-II metabotropic receptors (mGluR2/3) and L-type Ca(2+) channels in the IL-6-treated cultures. A similar reduction in mGluR2/3 and L-type Ca(2+) channel protein was observed in transgenic mice that over-express IL-6 in the CNS through astrocyte production starting early in development. Analysis of Ca(2+) signals produced by spontaneous synaptic network activity in the hippocampal cultures and effects of a mGluR2/3 agonist and antagonist showed that the reduced levels of mGluR2/3 impact on the functional properties of hippocampal synaptic network activity. These results have important implications relative to the mechanisms responsible for altered CNS function during conditions associated with increased levels of IL-6 in the CNS.     

Action of phencyclidine on synaptic transmission in the hippocampus

A neurophysiological investigation of the effects of phencyclidine (PCP) and ketamine on synaptic transmission was carried out at the level of two excitatory connections of the hippocampal formation: the interhippocampal projections from contralateral CA3 (cCA3) to CA1 and the entorhino-dentate pathway.In urethane-anesthetized rats PCP i.v. produced a moderate depression of the population EPSP elicited in the stratum radiatum of CA1 by cCA3 stimulations (16–40%) and a large decrease (up to 97%) of the amplitude of the corresponding population spike recorded at the level of the CA1 pyramidal cell bodies (ED₅₀:1.83 mg/kg).Single-unit analysis of CA1 pyramidal cell activation triggered by cCA3 stimulations indicated that i.v. PCP did not decrease the amplitude of individual action potentials suggesting that the decrease in the size of the population spike was due to a decrease in the number of CA1 pyramidal cells activated by the stimulus. Moreover, PCP administered i.v. in the same dose range (0.6-4.0 mg/kg) reduced the maintained activity of CA1 pyramidal cells and their excitation by iontophoretically applied glutamate or ACh.Similar effects on both field potentials and single-unit activity in the CA1 area were also observed following the administration of larger i.v. doses of ketamine (ED₅₀: 7.2 mg/kg), but the effects of the latter drug were of considerably shorter duration than those of PCP.     

不同声压级次声作用后小鼠海马内白细胞介素-6 mRNA的变化

目的研究不同声压级次声作用小鼠后海马内白细胞介素-6(interleuk in,IL-6)mRNA的改变和意义。方法BALB/C小鼠暴露于16 Hz、声压90 dB和130 dB次声。每天作用2 h,分别作用1、7、14、21和28 d后,采用原位杂交方法观察小鼠海马内IL-6 mRNA的改变。结果发现90 dB和130 dB次声作用后IL-6 mRNA的表达在海马区域明显增多;130 dB次声作用较90 dB次声作用强;在相同强度的次声作用下,作用次数的多少与海马内IL-6 mRNA的改变程度呈正相关。结论海马区域对次声敏感,次声的作用效应与声压级和作用时间有关;次声可以通过海马内IL-6 mRNA的改变造成脑损伤,这是次声导致脑损害的重要因素之一。     

Baclofen selectively inhibits excitatory synaptic transmission in the hippocampus

The effect of baclofen was investigated on mossy fiber, Schaffer collateral and perforant path synaptic transmission in hippocampal slices. Baclofen completely inhibits mossy fiber and Schaffer collateral synaptic transmission with an IC50 of 3.8 microM. The lateral perforant path is insensitive to baclofen, while the response in the medial zone was partially blocked. Baclofen does not appear to act in a GABA-like manner.     

Enhanced expression of erythropoietin in the central nervous system of SOD1(G93A) transgenic mice

In the present study, we investigated the changes of erythropoietin (Epo) expression in the central nervous system (CNS) of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). In wild-type SOD1 (wtSOD1) transgenic mice, little immunoreactivity was found in all cortical regions. In the cerebral cortex of symptomatic SOD1(G93A) transgenic mice, there was a significant increase in Epo immunoreactivity. In the hippocampal formation, layer-specific alterations in the staining intensity were observed in the CA1-3 areas and dentate gyrus. Epo immunoreactivity was significantly increased in the midbrain, cerebellar cortex and brainstem of SOD1(G93A) transgenic mice. On the contrary, Epo immunoreactivity was moderately stained in the spinal cord and was not different between wtSOD1 and SOD1(G93A) transgenic mice at the age of 8 weeks, 13 weeks and 18 weeks. In the staining of Epo receptor (EpoR), the changing pattern was similar with that of Epo in the spinal cord and hippocampal formation in wtSOD1 and SOD1(G93A) transgenic mice. Although further studies of functional features of Epo in ALS are needed, the first demonstration of increased immunoreactivity for Epo in the CNS of SOD1(G93A) transgenic mice may provide initial insights into the development of interventional strategies to alleviate motor neuron degeneration in human ALS.     

In vivo synaptic transmission in young and aged amyloid precursor protein transgenic mice

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cognitive impairment. We examined in vivo alterations in hippocampal neurotransmission in both young and aged PDAPP transgenic mice and nontransgenic littermates. We now report that in vivo abnormal neurotransmission in hippocampal circuits of PDAPP mice precedes beta deposition and neurodegeneration. These in vivo data provide the first evidence that dysfunction in hippocampal neuronal circuits may not be correlated with age-related extracellular beta plaque deposition.     

Genetically-targeted and conditionally-regulated ablation of astroglial cells in the central, enteric and peripheral nervous systems in adult transgenic mice

Ablation of tissue regions, specific genes, or specific cell types represent important means of studying function in the nervous system. Here we summarize recent experience using a strategy for the genetically-targeted and conditionally regulated ablation of astroglial cells in different parts of the nervous system. The strategy is based on the targeted expression of herpes simplex virus thymidine kinase to astroglial cells using the glial fibrillary acid protein promoter in transgenic mice, combined with treatment with the antiviral agent ganciclovir. Under different experimental conditions we find that transgene-expressing astroglial cells can be selectively ablated by ganciclovir in the enteric nervous system, or in the injured forebrain or sciatic nerve, providing models in which to study the functions of these cells.