Biomedical subjects

N G Cooper

Publications and source records attributed to N G Cooper.

At least 19 recordsLinked to original sources

PubMed2001

Visual-mediated regulation of retinal CaMKII and its GluR1 substrate is age-dependent.

Previous studies have shown that multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) and one of its substrates, the glutamate receptor, are key players in experience-driven synaptic plasticity in several areas of the central nervous system (CNS). To determine if CaMKII and the glutamate receptor are regulated by visual activity in the retina, we compared dark-reared (DR; 1 week) rats with control rats raised in a diurnal light-dark cycle (LD), at the following ages: postnatal day 12 (P12d), 2-month (2m) and 6-month (6m) old. The mRNA levels of CaMKIIalpha and beta were determined by a competitive reverse transcription polymerase chain reaction (competitive RT-PCR) method. The protein levels of these two subunits were evaluated by immunoblots. The data show that the mRNAs for CaMKIIalpha and beta were increased about 8-fold and 10-fold, respectively, in the retinae of DR P12d rats. As for the proteins, 2- and 2.6-fold elevations for CaMKIIalpha and beta, respectively, were evident. The GluR1 subunit of the AMPAR (AMPAR-GluR1) was also evaluated in antibody-treated blots and found to be increased about 2-fold after 1 week of dark rearing in the retinae of P12d rats. This type of experience-driven molecular change was age-dependent, showing less increase in 2m old rats and not present in 6m old rats. Returning DR 2m old rats to the LD environment for 1 week was sufficient to restore the dark-induced changes to the levels of the age-matched LD controls. Based on the data, a theoretical model for activity-dependent modulation of the developing retinal synapses is proposed.

Age Factors↗

PubMed2001

The mRNA level of the potassium-chloride cotransporter KCC2 covaries with seizure susceptibility in inferior colliculus of the post-ischemic audiogenic seizure-prone rat.

Cardiac arrest and resuscitation were used to induce brain damage and susceptibility to sound-triggered seizures in Sprague-Dawley rats. Glucose preloading was used to vary seizure susceptibility. Because loop diuretics can block these seizures, we investigated changes in KCC2, a potassium-chloride cotransporter, in the inferior colliculus - the origin of the seizures. Using polymerase chain reaction (PCR), we found that collicular KCC2 mRNA levels covaried with seizure susceptibility in these animals. Using quantitative PCR, we found that a fivefold increase in collicular KCC2 mRNA levels was associated with a doubling of seizure incidence. A hypothesis linking KCC2 activity to seizure susceptibility is presented.

Animals↗

PubMed2001

Characterization of apoptosis-genes associated with NMDA mediated cell death in the adult rat retina.

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal (CaMKII-alphaB) is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). AIP (myristoylated autocamtide-2-related inhibitory peptide), a specific inhibitor of CaMKII provides neuroprotection against NMDA-mediated neurotoxicity. In this study, gene-arrays were used to investigate which apoptosis-associated genes are altered after exposure to NMDA. The data indicate an increased expression (2-7-fold) of five such genes encoding proteins that could be involved in NMDA induced cell death. The up-regulated genes are: FasL; GADD45; GADD153; Nur77 and TNF-R1. Treatment with AIP blocked their altered expression. The results suggest that multiples genes are involved in NMDA-induced excitotoxicity and that AIP, a specific inhibitor for CaMKII, regulates the expression of these apoptosis-associated genes in the retina.

Animals↗

PubMed2001

The modification of NMDA receptors by visual experience in the rat retina is age dependent.

Extensive studies have shown that the activation of N-methyl-D-aspartate receptors (NMDARs) and the subsequent rise in the levels of postsynaptic calcium are critical events in the initiation of synaptic plasticity. Modification of the amount, or of the subunit composition of NMDARs, alters receptor function thereby affecting the development and/or efficacy of synaptic transmission. In the present study, a Western blot analysis was employed to investigate the effects of visual experience and age on the differential expression of NMDARs in the rat retina. A crude synaptic membrane fraction (SPM) was prepared and assayed with antibodies specific for either the NR1, NR2A or NR2B subunits. Relative to control animals raised in a diurnal light-dark cycle, a period of 1 week of dark-rearing caused an increase in the relative amount of NR1, a decrease in the level of NR2A, and no change in the level of NR2B subunit expression in postnatal day 12 rats. At 2 months of age, 1 week of dark-rearing had less effect, and at 6 months of age there was no difference between dark-reared and control animals. The effect of light exposure on dark-reared animals was tested for the 2-month-old animals. Light exposure for long periods (days), but not short periods (h), could reverse the dark-rearing effects. These data provide evidence for a developmentally regulated plasticity of NMDAR subunits in the retina.

Age Factors↗

PubMed2001

Expression of ionotropic glutamate receptors in the retina of the rdta transgenic mouse.

BACKGROUND: The expression of retinal CaMKII is up-regulated in the retina of the rdta mouse in which rod photoreceptors are genetically ablated. As ionotropic glutamate receptors are known substrates of CAMKII, this study set out to determine if the protein levels of ionotropic glutamate receptors in the rdta mouse retina are also affected. RESULTS: The NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots. The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls. The GluR1 receptor subunit and its phosphorylation are simultaneously increased in retinae of the rdta mice. CONCLUSIONS: These data indicate that the NMDA receptors and AMPA (GluR1) receptors are altered in the retinae of rdta mice that lack rod photoreceptors. Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

Actins↗

PubMed2001

Molecular cloning and sequence analyses of calcium/calmodulin-dependent protein kinase II from fetal and adult human brain. Sequence analyses of human brain calciuum/calmodulin-dependent protein kinase II.

The aims of this study were to characterize specific mRNAs and the expression pattern for isoforms of calcium/calmodulin-dependent protein kinase II (CaMKII) in the human brain. We cloned and sequenced the CaMKII alpha and beta subunit cDNAs, and used them to study the CaMKII expression in human brain. Four distinct isoforms of CAMKII were isolated. Two of them were characterized as CaMKII alpha and beta subunits. The other two showed similar nucleotide sequences, but one had a 33-bp insertion relative to the alpha subunit, and the other had a 75-bp deletion relative to the beta subunit. These alterations are located within the variable regions. These two isoforms were characterized as CaMKII alphaB and beta(e). Northern blot analysis showed that a 4.4-kb messenger RNA for the alpha isoform and a 3.9-kb messenger RNA for the beta isoform were expressed in both human fetal and adult brain to different degrees. The results indicate that CaMKII expression is developmentally regulated. The CaMKII isoform expression was confirmed in human fetal and adult brain using RT-PCR with specific primers, which flanked the CaMKII variable regions. The CaMKII alpha, alphaB, beta, beta' and beta(e) isoforms were characterized in both human fetal and adult brain.

Alternative Splicing↗

PubMed2000

Neuroprotective effect of AIP on N-methyl-D-aspartate-induced cell death in retinal neurons.

Excessive activation of glutamate receptors mediates neuronal death, but the intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Previously, we have demonstrated that calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) containing a nuclear localizing signal but not CaMKII-alpha is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). The present study describes a prospective function of CaMKII-alpha(B) in signal transduction leading to apoptosis. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that cell death occurs in the inner nuclear and ganglion cell layers following injection of 4 mM NMDA. A specific AIP (myristoylated autocamtide-2-related inhibitory peptide) with proven cell permeability inhibits CaMKII activity in vivo. Neuroprotection achieved by 500 microM AIP was complete when administered 2 h before and coincident with the NMDA application. Additionally, 100 microM of AIP protects only partially against the NMDA-induced excitotoxicity. The conformationally active fragment of caspase-3 (17 kDa), known to be involved in neuronal apoptosis was apparent within 30 min and at 2 h postinjection with NMDA. This activation was inhibited by 500 microM AIP when administered 2 h before and coincident with the NMDA application. The results suggest that CaMKII-alpha(B) isoform plays a role in excitotoxicity-induced neuronal apoptosis.

Animals↗

PubMed2000

Photoreceptor regulated expression of Ca(2+)/calmodulin-dependent protein kinase II in the mouse retina.

The objective of this investigation is to determine mechanisms for regulation of retinal calmodulin kinase II (CaMKII). To this end, the expression and activity of CaMKII are examined in the retina of the rdta mouse, in which rod photoreceptors have been genetically ablated [47]. CaMKII levels are compared between rdta mice and the normal, littermate control mice. It is demonstrated that retinal CaMKII protein, enzyme activity and mRNA are significantly increased in response to the genetic ablation of rod photoreceptors. The data indicate that CaMKII expression/activity in amacrine and ganglion cells is negatively regulated by the rod photoreceptor-mediated visual input. The regulation appears to occur primarily at the transcriptional level. It is shown that the cytoplasmic polyadenylation element binding protein (CPEB), a regulatory factor for translation that is known to promote CaMKIIalpha translation in dendrites [83], is also present in the mouse retina. However, the polyadenylation-mediated translational control mechanism is not activated in this experimental paradigm.

Animals↗

PubMed2000

Developmental changes in the expression of NMDA receptor subunits (NR1, NR2A, NR2B) in the cat visual cortex and the effects of dark rearing.

The present study used Western blots to determine changes in the level of expression of the three major NMDA receptor subunits, NR1, NR2A, and NR2B, in relation to the 'critical period' in cat visual cortex. NR2A rose dramatically (10-fold) from very low levels at 1 week to a peak at 5 weeks and gradually declined into adulthood (twofold). NR2B showed a similar time course to NR2A, but the rise from 1 to 5 weeks was of lesser magnitude (twofold). NR1 was expressed at comparable levels at 1, 5, 10 weeks and declined markedly (fivefold) in older animals. No significant effects of dark rearing on the levels of NR2B and NR1 were found. However, NR2A expression was significantly elevated in normal compared to dark reared visual cortex (twofold) at 5 weeks and significantly elevated in dark reared compared to normal visual cortex at 20 weeks (twofold). The close agreement between NR2A expression and both the time course of the critical period and the effects of dark rearing on that time course further indicates a role of this subunit in visual cortical critical period plasticity.

Age Factors↗

PubMed2000

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal is altered in retinal neurons exposed to N-methyl-D-aspartate.

This study investigated N-methyl-D-aspartate (NMDA) mediated cell death and its possible regulation by calcium/calmodulin-dependent protein kinase II (CaMKII) in the adult rat retina. To investigate cell death, the terminal deoxyribonucleotidyltransferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that apoptosis occurs in the inner nuclear layer (INL) and ganglion cell layer (GCL) following NMDA injection. The level of antibody binding to CaMKII-alpha, the activity of CaMKII, and the mRNA level for the alpha(B) subunit of CaMKII were found to be elevated for short time periods (30 min, 2 h) after a single intravitreal injection of NMDA. In contrast to this, there was a decrease in CaMKII activity and in the CaMKII-alpha(B) mRNA levels at longer time periods (24 h) following injection of NMDA. These effects were specific for the mRNA for the alpha(B) subunit, an alternatively spliced product of the CaMKII-alpha gene, that contains a nuclear localizing signal (NLS) known to target this protein to the nucleus. It is suggested that regulated expression of CaMKII-alpha(B) could be involved in the NMDA-mediated cell death in retinal neurons.

Animals↗

PubMed2000

Selective photoreceptor damage in albino rats using continuous blue light. A protocol useful for retinal degeneration and transplantation research.

PURPOSE: To develop a retinal degeneration model with selective photoreceptor loss and RPE sparing, to be used as recipient for evaluating retinal transplants. METHODS: Albino rats were exposed to blue light, continuously, for 1-7 days (24-168 h) in a specially designed cage. Eyes were histologically analyzed at periods between 2 h and 8 months after the light exposure. Electroretinograms (ERGs) were recorded from some rats at 12-216 days after exposure. Using behavioral methods, visual thresholds of some rats were determined before exposure and re-measured between 18 and 52 days following exposure. RESULTS: Apoptotic nuclei appeared exclusively in the photoreceptor layer after 1-5 days exposure to blue light. Light microscopy revealed that 2-4 days of light exposure reduced the outer nuclear layer (normally eight to ten rows) to 1 row of cells in the central retina and to two to three rows in the periphery, both in the superior and the inferior retina. Average ERG a- and b-wave amplitudes of light-damaged rats were both reduced by about 98%. Visual performance in the behavioral test was substantially impaired. CONCLUSIONS: Continuous exposure of albino rats to moderate blue light for 2-5 days selectively eliminates most of the photoreceptors while leaving the RPE initially intact.

Animals↗

PubMed1999

Regulation of calcium/calmodulin-dependent protein kinase II in the adult rat retina is mediated by ionotropic glutamate receptors.

This study is concerned with the transmitter-mediated regulation of the alpha(50 kDa) and beta(60 kDa) subunits of calcium calmodulin dependent protein kinase II (CamKII) in the adult rat retina. The level of antibody binding to the CamKII and the activity of CamKII were found to be increased after intravitreal injection of glutamate. Changes in the levels of the antibody-binding to the subunits of CamKII were observed in different subcellular fractions of the retina with a maximum response observed in crude synaptic membrane fractions. The glutamate mediated increases in CamKII were specific and blocked by 3,5-Dimethyl-1 adamantanamine; 3,5-Dimethylamantadine (Memantine), (+/-) 2-Amino-5-Phosphopentonic (AP-5) and 6-Cyano-7-Nitroquinoxaline-2,3-Dione (CNQX) but not with dl -2-Amino-3-Phosphono-Propionic (AP-3). The results indicate that the retinal neurotransmitter, glutamate, can regulate retinal CamKII activity through ionotropic but not metabotropic glutamate receptors. NMDA-receptors were found to be necessary but insufficient to stimulate CamKII. A model in which cooperative interaction between NMDA and non-NMDA glutamate receptors/ion channels is presented to explain the glutamate stimulated increases in CamKII activity in the retina.

Animals↗

PubMed1998

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of rat somatosensory barrel cortex.

The postnatal development of glutamic acid decarboxylase (GAD; GAD67 and GAD65) expression was studied in the rat somatosensory cortex. Delineation of barrels in layer IV by GAD67 immunoreactivity occurred between postnatal days P3 and P6 and remained evident into adulthood. At birth, a band of GAD67-positive elements was already present in superficial layer V. This band was prominent until P6 and gradually disappeared after P9. In parallel, there was a gradual appearance of GAD67-immunoreactive cells neuropil and puncta, which began in layer VI/subplate at P1 and achieved the adult laminar pattern by about P13. This later GAD67 immunoreactivity was responsible for the demarcation of barrels in layer IV. Development of GAD65 immunoreactivity was delayed relative to GAD67. GAD65 immunoreactivity, which was in little evidence before P6, increased markedly in density and in delineation of cell bodies over the next several weeks. During this prolonged developmental process, GAD65 first showed a negative image of the barrels compared with the septae and the surrounding cortex. Subsequently, there was a filling in of the barrels resulting in rather uniform GAD65 immunoreactivity across the barrel field and surrounding cortex. These results suggest that the development of the gamma-aminobutyric acid (GABA) synthetic system in the barrel cortex involves several processes: the disappearance of a precocious GAD67 system in layer V, the temporally overlapping maturation of the mature GAD67 system in an inside-outside manner, and the delayed and prolonged development of the GAD65 system.

Age Factors↗

PubMed1998

Temperature-sensitive interactions between RPE and rod outer segment surface proteins.

Phagocytosis of rod outer segments by the retinal pigment epithelium is distinguished by the two distinct temperature-dependent steps of binding and ingestion. This study was designed to see if retinal pigment epithelial (RPE) plasma membrane proteins interact with ROS plasma membrane proteins at temperatures favoring either binding or ingestion. A modified blot overlay assay was used whereby Western blots of RPE plasma membrane proteins were overlaid with biotinylated ROS plasma membrane proteins. RPE/ROS interactions were detected by streptavidin-HRP and the ECL method at 25 degrees C (ingestion), 15 degrees C (binding), and 4 degrees C (little or no binding or ingestion). Unlabeled ROS proteins served as the negative control. Competition with excess unlabeled ROS proteins were used to test the specificity of the protein interactions. Some protein interactions were somewhat temperature dependent. For example, two RPE plasma membrane proteins (200 kDa and 173 kDa) interacted with ROS plasma membrane proteins at both 25 degrees C and 15 degrees C, but not at 4 degrees C. A strongly labeled protein at 50 kDA protein was present at 25 degrees C but weakly labeled at 15 degrees C and at 4 degrees C. Other protein interaction were more clearly temperature dependent. For example, a 110 kDa RPE protein interacted with ROS proteins only at 25 degrees C. Another RPE protein (55 kDa) interacted only at 15 degrees C. These latter data provide correlations between binding events in the assay and previously described stages of phagocytosis.

Animals↗

PubMed1998

Molecular cloning and analysis of Ca2+/calmodulin-dependent protein kinase II from the chicken brain.

The goals of this study were to identify specific mRNA for isoforms of calmodulin-dependent protein kinase II in chicken forebrain, prepare a cDNA expression library, and perform a sequence analysis of the kinase cDNA. Specific mRNAs for alpha- and beta-subunits of the kinase were identified in Northern blots. The mRNA for the alpha-subunit is larger in the chicken that in the rat, and for the beta-subunit is smaller in the chicken than the rat. Nucleotide sequencing of selected clones demonstrated the presence of an alpha-subunit with a 33 nucleotide insert known as the alpha-B-isoform. Clones of the beta-subunit showed it to contain a deletion of six nucleotides relative to previously described sequences. Variability in the mRNAs of calmodulin kinase II, as shown here, reflect the presence of species-dependent variability in gene structure as well as the presence of different functional isoforms.

Animals↗

PubMed1997

Expression of two forms of glutamic acid decarboxylase (GAD67 and GAD65) during postnatal development of the cat visual cortex.

The postnatal development of GAD67 and GAD65 protein expression and of GAD67 positive neurons and GAD65 containing axon terminals in cat visual cortex was studied. Western blot analysis showed that the expression of both GAD67 and GAD65 increased to approximately two-thirds of the adult level during the first 5 postnatal weeks and gradually increased thereafter. In adult cats, immunohistochemistry showed that GABA and GAD67 containing neurons were found in all cortical layers. Faint cell body staining was seen with the antibody to GAD65, but it densely labeled puncta. In neonates, GABA and GAD67 immunoreactivity was most intense in two distinct bands, one superficial (Layer 1/Marginal zone), another deep (Layer VI/Subplate). Unlike in adults, GAD65 positive cell bodies were clearly evident in neonates and distributed similarly to, but less frequently than, GABA and GAD67. These GAD65 positive cells frequently had morphologies suggestive of embryonic cells and largely disappeared in older animals. During postnatal development, the neurochemical differentiation of GAD67 positive neurons and GAD65 positive axon terminals across visual cortical laminae followed an inside-outside developmental pattern, which reached adult levels after 10 weeks of age. These results suggest that postnatal development of the visual cortical GABA system involves three distinct processes: (A) a dying off of embryonic GABA cells which could play a role in formation of the cortical plate; (B) a period of relative quiescence of the VC GABA system in the first 5 postnatal weeks which could maximize excitatory NMDA effects during the rising phase of the critical period; (C) the prolonged postnatal maturation of the adult GABA system which could be involved in the crystallization of adult physiological properties and the disappearance of neural plasticity.

Aging↗

PubMed1996

The Ca2+/calmodulin-dependent protein kinase II-associated protein complex isolated from chicken retina.

Retinal cytosolic Ca2+/calmodulin-dependent protein kinase II (CaM KII) was isolated from hatched 6-wk chicken retinae by ultracentrifugation and affinity chromatography using calmodulin (CaM) and anti-CaM KII-alpha columns. Samples from different fractions were examined with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining or immunoblotting. Comparisons were made between the final antibody affinity eluates from retina and forebrain. Silver-stained gels showed that multiple proteins were present in the antibody affinity eluates from retina, including major proteins of 178, 56, and 45 kDa and several minor proteins. Immunoblots revealed that CaM KII-alpha was present in eluates from the retina and forebrain. CaM KII-beta was present in the antibody eluate from forebrain but not retina. The latter subunit was present in the crude homogenates of the retina. Regarding the antibody eluate from retina, the possibility that the major 56 kDa protein was tubulin was ruled out, but protein tau (tau) and synapsin I were present. The presence of multiple proteins in the antibody affinity eluate indicates that these proteins were coisolated in a CaM KII-alpha-associated protein complex. The finding that protein tau and synapsin I are associated with retinal CaM KII provides further insight into the mechanisms underlying the function of the kinase in this tissue. The lack of cytosolic CaM KII-beta subunit in the antibody affinity eluate from retina is indicative of a brain region-specificity in subunit composition of the kinase.

Animals↗

PubMed1995

A novel calcium-dependent activator of retinal rod outer segment membrane guanylate cyclase.

The membrane guanylate cyclase in retinal rod outer segments (ROS-GC) is known to be negatively regulated by calcium; when the calcium concentration is reduced below the dark-adapted level of about 500 nM, the enzyme is activated by a soluble protein. We now report that the enzyme is also positively regulated by calcium; a novel soluble protein is identified and purified from bovine retina which activates ROS-GC, with half-maximal activation occurring at 2-5 microM calcium. The activation is dose-dependent, and at its maximum, cyclase is stimulated up to 25-fold. The activator has a molecular mass of about 40 kDa and is a multimer of a 6-7 kDa peptide.

Animals↗