Biomedical subjects

G Tocco

Publications and source records attributed to G Tocco.

At least 37 records · Page 2Linked to original sources

PubMed1997

Induction of tumor suppressor p53 and DNA fragmentation in organotypic hippocampal cultures following excitotoxin treatment.

The p53 tumor suppressor gene encodes a cell cycle regulatory protein that is induced by DNA damage and has been implicated in apoptosis. To investigate whether excitotoxic cell death due to kainic acid (KA) and cell death due to N-methyl-D-aspartate (NMDA) share similar molecular mechanisms, we studied p53 expression and DNA fragmentation in organotypic hippocampal slice cultures following excitotoxin treatment. Cellular analyses showed that both p53 induction and DNA fragmentation occurred only in injured neurons following exposure to either excitotoxin. The temporal profiles of these changes demonstrated that p53 induction preceded DNA fragmentation. The extent of regional alterations in p53 expression and DNA fragmentation correlated with drug-related toxicity (i.e., NMDA > KA). These results support the hypothesis that p53 is a marker of neuronal death in the CNS and suggest the possibility that excitotoxin-mediated neuronal death may occur through a p53-dependent pathway.

Animals↗

PubMed1997

Complement and glutamate neurotoxicity. Genotypic influences of C5 in a mouse model of hippocampal neurodegeneration.

Using mice genetically deficient in the complement (C)-system component C5, this study explored a potential novel role of the C-system in Ca(2+)-mediated control of glutamate AMPA receptor functions. We found that Ca2+ preincubation of frozen brain tissue sections enhances AMPA binding capacity more dynamically in C5 deficient (C5-) than congenic C5 sufficient (C5+) mice. The Ca(2+)-mediated response was mostly localized to the CA3 and CA1 subdivisions of the pyramidal layers of the hippocampal formation. In C5- mice, kainic acid (KA) excitotoxicity that models hippocampal neurodegeneration abolished the Ca(2+)-mediated induction of hippocampal AMPA binding. The changes in AMPA binding preceded temporally and overlapped anatomically the appearance of apoptotic features in the same hippocampal neuron layers. C5- mice showed greater hippocampal neurodegeneration then C5+ mice. NMDA binding controlled for specificity of glutamate-mediated changes and found no C5 genotypic influences. The study gives further credence to the role of the C-system in modifying the intensity and outcome during response to conditions leading to hippocampal neurodegeneration.

Animals↗

PubMed1997

Characterization of calpain-mediated proteolysis of GluR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in rat brain.

Previous results have indicated that GluR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C-terminal (C-Ab) and the N-terminal (N-Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen-thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C-Ab and in the formation of a doublet band labeled with N-Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain-induced changes in GluR1 immunological properties were not associated with modifications of [3H]AMPA or 6-cyano-7-[3H]nitroquinoxaline-2,3-dione ([3H]CNOX) binding. Treatment of frozen-thawed brain sections with concentrations of calcium as low as 0.2 mM resulted in a large decrease in the 105-kDa GluR1 band and in the concurrent appearance of the 98-kDa band. This treatment was associated with increased [3H]-AMPA and [3H]CNOX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium-dependent processes regulating the characteristics of receptors in intact tissues.

Amino Acid Sequence↗

PubMed1996

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats.

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [3H]AMPA, [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]glutamate binding on rat brain tissue sections. Preincubation of brain sections with PS produced an increase in [3H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [3H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [3H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [3H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties, which could be involved in producing long-lasting changes in synaptic operation.

Animals↗

PubMed1996

Increased expression of cyclin D1 in the adult rat brain following kainic acid treatment.

Recent evidence has implicated aberrant cell cycle regulation as a possible mechanism of apoptosis in non-dividing cells. We previously demonstrated increased expression of the p53 tumor suppressor gene, a prominent cell cycle regulator, in apoptotic neurons. Here we investigated the potential involvement of cyclin D1, a G1 phase cell cycle protein under p53 regulation, in kainic acid-mediated neuronal degeneration. Adult male Sprague-Dawley rats were treated systemically with kainic acid and sacrificed between 1 h and 5 days following seizure onset. Cyclin D1 expression was studied by Western blot analysis and immunohistochemistry using a rabbit polyclonal anti-cyclin D1 antibody. In untreated control rats low levels of cyclin D1 expression were detected in multiple brain regions. Between 8 and 16 h after the onset of kainic acid-induced seizures, increased cyclin D1 immunoreactivity was observed in vulnerable hippocampal pyramidal cells. Five days after seizure onset increased cyclin D1 expression was evident in reactive astrocytes. These results support a role for cyclin D1 in certain neuronal death pathways, and suggest that cyclin D1 has multiple and cell type-specific functions in the central nervous system.

Animals↗

PubMed1996

Polysynaptic potentiation in the lateral septum following stimulation of the fimbria in anesthetized rats.

In anesthetized rats, electrical stimulation of fimbria fibers evoked, in the ipsilateral lateral septum (LS), a field potential consisting of two negative components: an initial negativity (N2-3 complex wave) of high amplitude at 6.7 ms (+/- 0.8 ms; peak latency) and a slow negative wave (N4 wave) of small amplitude at 14.4 ms (+/- 2.4 ms). The N2-3 complex wave represents the monosynaptic activation of LS neurons while the N4 wave corresponds to polysynaptic activation of neurons in the mediolateral part of the LS. In this study, we investigated the effects of high-frequency stimulation of fimbria fibers on LS field potentials and compared them with those observed in the CA3 area. Tetanic stimulation of the fimbria did not change the characteristics of the N2-3 wave but induced a long-lasting increase in amplitude and slope of the N4 wave. A positive correlation was found between the magnitude of CA3 LTP and lateral septal polysynaptic potentiation of the N4 component. These results indicate that patterns of stimulation delivered to the same input fibers (fimbria fibers) produce similar changes in a polysynaptic input to the LS and in a monosynaptic input to the CA3 and emphasize the complexity of signal processing in serial networks.

Anesthesia↗

PubMed1996

Two synaptotagmin genes, Syt1 and Syt4, are differentially regulated in adult brain and during postnatal development following kainic acid-induced seizures.

The synaptotagmins together with other vesicle proteins are thought to be essential for the docking and/or fusion of synaptic vesicles with the plasma membrane that occurs following depolarization and calcium influx in presynatic terminals. Syt4, the fourth identified member of the synaptotagmin family, is inducible in PC12 cells by depolarization and secretagogues, and in limbic regions of the adult rat brain by kainic acid-induced seizures. In the present study, we examined the time course of the seizure-induced changes in the expression of Syt4 and Syt1, both in adult animals and during the postnatal period. Syt4 was transiently induced in several structures of the adult rat brain following seizure activity with peak inductions between 4 and 8 h and overal return to control values by 30 h. No induction was observed following seizure activity in 7-day-old animals. The brain regions most sensitive to increased induction were, in decreasing order of sensitivity, hippocampal pyramidal cells dentate granule cells and piriform cortex pyramidal cells. The brain areas showing the greatest Syt4 stimulation in adults were also the areas in which Syt4 was induced by seizures earlier in development. In contrast, Syt1 mRNA was depressed in adult brains following seizure activity, particularly in the dentate granule cells. Our results suggest that the differential regulation of different synaptotagmin genes following excessive neuronal activity might participate in rapid adaptation of subsequent transmitter release.

Animals↗

PubMed1996

Regional distribution and time-course of calpain activation following kainate-induced seizure activity in adult rat brain.

Systemic injection of kainic acid (KA) in adult rat elicits a pattern of neuronal pathology which exhibits several features of human temporal lobe epilepsy. KA-induced seizure activity is accompanied by the activation of the calcium-dependent protease calpain in limbic structures. In the present study, we evaluated the spatio-temporal activation of calpain after the onset of seizure activity by immunohistochemistry using an antibody for the spectrin breakdown product (sbdp) generated by calpain-mediated spectrin proteolysis. In addition, we compared the changes in sbdp immunoreactivity with those in immunoreactivity to subunits of the Glu/AMPA receptors (GluR1 and GluR2/3). One hour after seizure onset, sbdp accumulation was observed in selected interneurons in stratum oriens and in the hilus of the dentate gyrus. By 4 h, sbdp immunoreactivity was prominent in dendritic fields of the hippocampus as well as in neurons in thalamus and piriform cortex. By 8 h, sbdp immunoreactivity had disappeared from interneurons but was localized in pyramidal cell bodies in hippocampus. Intense labeling of cell bodies and dendritic fields persisted until 5 days following KA treatment. Changes in GluR subunit immunoreactivity were mirror images of those seen for sbdp. In general, increased sbdp immunoreactivity in dendritic fields was associated with decreased GluR1 immunoreactivity. However, increased sbdp immunoreactivity in neuronal perikarya was also associated with increased GluR immunoreactivity. These results indicate that calpain activation following seizure onset exhibits a specific spatio-temporal pattern, with activation in restricted interneurons preceding widespread activation in pyramidal neurons. Calpain activation also precedes neuronal pathology and could thus represent an initial trigger for neuronal pathology. Finally, the results suggest that calpain activation produces rapid alterations in GluR subunit properties which could be involved in the hyperexcitability observed following seizure activity.

Animals↗

PubMed1996

Hereditary deficiencies in complement C5 are associated with intensified neurodegenerative responses that implicate new roles for the C-system in neuronal and astrocytic functions.

Possible roles of the complement (C) system in the normal and injured brain were explored with inbred mice that carried a frameshift mutation in the C5 gene. A congenic pair was used: the C5-sufficient (C5+) B10.D2/nSnJ strain with the functional allele (Hc1) from the C57BL/10J donor strain was compared with the C5-deficient (C5-) B10.D2/oSnJ with the Hc0 allele from the C5-deficient DBA/2J donor strain. In response to the excitotoxin kainic acid (KA), C5- mice had more hippocampal pyramidal neuron death and greater induction of astrocyte mRNAs (GFAP, apoE, apoJ). In primary astrocyte cultures from unlesioned mice, an inflammatory stimulus (LPS) caused greater production of IL-6 and TNF production in C5- mice. These enhanced responses to KA and LPS suggest that hereditary C5 deficits modify responses to neurodegenerative stimuli of neurons and astrocytes. Moreover, unlesioned C5- mice had smaller input-output slopes for the NMDA component of the EPSP amplitude, but enhanced the Ca(+2)-dependent AMPA binding. Thus, C5 deficits also modify basal properties of glutamatergic neurotransmission that pertain to synaptic plasticity. These findings are also discussed in relation to roles of the C-system in Alzheimer disease (AD). C5 deficiencies may also be considered in the choice of strains as transgene hosts and for genetic analysis of normal and pathological brain functions. In recent transgenic studies for AD, C5- hosts showed greater neurodegeneration, consistent with the present data. These pleiotropic associations of C5 deficiency indicate roles for the C-system in neurodegeneration, but also in normal neural functions.

Animals↗

PubMed1996

Induction of glucose-regulated protein (glucose-regulated protein 78/BiP and glucose-regulated protein 94) and heat shock protein 70 transcripts in the immature rat brain following status epilepticus.

Prior to 21 days of age, the immature rat brain is relatively resistant to excitotoxicity caused by the glutamate analogue, kainate. As stress-inducible proteins (GRP78, GRP94 and HSP70) have been proposed to possess molecular chaperone activity and protect cells from the deleterious effects of damaged proteins, we examined the pattern of expression of their respective messenger RNAs following systemic kainate at different postnatal ages. In untreated rats between seven and 21 days old, there was a higher basal level of grp78 and grp94 expression compared to hsp70. Unlike hsp70, which was inducible only in 21-day-old rats, kainate-mediated grp94 induction occurred in several regions of the brain as early as postnatal day 7. Grp78 messenger RNA expression was also increased by kainate treatment in 14-day-old rats, and the induction was most pronounced in the kainate-resistant dentate gyrus. With increasing age, longer lasting expression of both grp78 and grp94 messenger RNAs was observed in kainate-vulnerable regions, similar to observations in the adult rat brain. These results demonstrate non-overlapping expression patterns of glucose-regulated proteins and HSP70 in the immature central nervous system, suggesting that they serve different functions. While hsp70 induction could be a marker for potential cell injury and death, increased expression of grp78 and grp94 could play a neuroprotective role in the developing rat brain.

Animals↗

PubMed1995

Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain.

Subtractive library construction and differential screening were used to identify a cDNA for a cell type-specific immediate early gene induced in rat PC12 pheochromocytoma cells. Sequencing identified the protein product of this gene as rat synaptotagmin IV (SytIV). Synaptotagmins are synaptic vesicle proteins thought to play a role in depolarization-induced, calcium-mediated exocytosis and neurotransmitter release. SytIV mRNA accumulation is transiently induced in PC12 cells by potassium depolarization, calcium ionophore, ATP, and forskolin. In contrast, growth factors and phorbol 12-myristate 13-acetate induce little or no SytIV mRNA accumulation. Kainic acid-induced seizures in rats are followed by accumulation of SytIV message in the hippocampus and piriform cortex. The SytIV gene may provide a direct link between depolarization-induced neuronal gene expression and subsequent modulation of synaptic structure and function.

Adrenal Gland Neoplasms↗

PubMed1995

Developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor properties and expression in the rat hippocampal formation.

The developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor properties in rat hippocampus were evaluated with quantitative autoradiography of ligand binding and in situ hybridization performed in adjacent sections with antisense oligonucleotides for AMPA receptor subunits (GluR1-3, flip and flop splice variants). Specific 3H-AMPA binding in different hippocampal subfields increased between postnatal day 7 and 15 and was higher in CA3 during the postnatal period when compared to adult levels. This effect was mostly due to high levels of high affinity binding sites in cell body layers during the developmental period. By contrast, autoradiograms of 3H-AMPA binding predominantly to the low affinity binding sites indicated an absence of these sites in cell body layers and the overall levels of binding exhibited little overshoot compared to adult levels during the developmental period. The changes in binding of the antagonist of the AMPA receptor, 6-nitro-7-cyanoquinoxaline-2,3-dione were markedly different from those for the high affinity AMPA binding sites but quite similar to those for the low affinity sites. The binding was extremely low at postnatal day 7 and increased rapidly between postnatal day 7 and 15 and slowly between postnatal day 15 and adult. Low levels of binding were observed in the cell body layer at every postnatal age. The changes in expression of messenger RNAs for the different subunits of the AMPA receptors were well correlated with the modifications in high affinity AMPA binding sites measured in the cell body layers also exhibiting an increased expression of the receptors at the transcriptional level during the developmental period as compared to adult levels. The relative expression of the GluR2 subunits decreased during the postnatal period and the time course for this reduction paralleled that for the increased vulnerability of hippocampal pyramidal neurons to a variety of insults. The results indicate that both the messenger RNAs for the subunits and the AMPA receptors exhibit increased levels of expression during the postnatal period compared to adult levels. They also suggest that nascent receptors might bind AMPA with high affinity before their insertion in membranes into functional receptors that have low affinity for agonists and high affinity for antagonists. The changes in subunit composition of the receptors during the postnatal period may have important implications for mechanisms of plasticity as well as of neuropathology.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1994

Calpain-mediated regulation of AMPA receptors in adult rat brain.

Changes in AMPA receptors have been proposed to underlie changes in synaptic efficacy in hippocampus and other brain structures. Activation of calpain has also been discussed as a potential mechanism to produce lasting modifications of synaptic structure and function. We report here that preincubation of thin (10 microns) frozen rat brain sections with calcium at physiological temperature changes the immunological properties of AMPA receptors, an effect totally blocked by calpain inhibitors. Immunocytochemistry indicates that in situ calpain activation produces a decreased immunoreactivity for GluR1, and to a lesser extent for GluR2/3, in the neuropil throughout the brain and an increased immunoreactivity in cell bodies, particularly in hippocampus. Western blots of calcium-treated sections suggest that the decreased immunoreactivity for GluR subunits is due to partial proteolysis. These results strongly suggest the involvement of calpain in the regulation of glutamatergic synapses.

Animals↗

PubMed1994

p53 induction is associated with neuronal damage in the central nervous system.

The p53 tumor-suppressor gene encodes a growth-regulatory protein that has been implicated in programmed cell death. To investigate the possible role of p53 in neuronal death, we studied p53 expression associated with excitotoxicity in the adult rat brain. Within hours of systemic administration of the glutamate analogue kainic acid, p53 mRNA levels were increased in neurons exhibiting morphological features of damage within kainate-vulnerable brain regions. A similar distribution was found for neurons exhibiting DNA damage as evidenced by in situ end-labeling of fragmented DNA. Pretreatment with the protein synthesis inhibitor cycloheximide prevented both kainate-mediated p53 induction and neuronal damage. The distinctive pattern of excitotoxin-mediated p53 expression suggests that p53 induction is a marker of irreversible injury in postmitotic cells of the central nervous system and could have functional significance in determining selective neuronal vulnerability.

Animals↗

PubMed1994

Neural tropomodulin: developmental expression and effect of seizure activity.

Tropomodulin is a 40.6 kDa tropomyosin-binding protein associated with actin filaments in muscle and the membrane cytoskeleton in erythrocytes. We have detected tropomodulin mRNA and protein in brains of rats by northern and western blot analyses. In situ hybridization of rat brain and spinal cord sections shows tropomodulin expression in the cerebellum, neocortex, hippocampus, and anterior horn of the spinal cord. Tropomodulin expression is first observed around day 15 after birth and increases through day 24. The temporal and spatial changes in tropomodulin expression during cerebellar development parallel those for brain tropomyosin. Tropomodulin mRNA increases in the dentate gyrus of the hippocampus following prolonged seizure activity induced by kainic acid administration; the increase is clearly evident 8 h after initiation of seizures and is still present 1 week later. However, Western blot analysis of tropomodulin protein level in the dentate gyrus before and after seizure induction show only slight increases in tropomodulin protein concentration, suggesting tight regulation of tropomodulin expression at the translational level. The developmental expression of tropomodulin, together with the induction of tropomodulin mRNA production in the dentate gyrus after kainic acid treatment, suggests a role for tropomodulin in neuronal organization and plasticity.

Animals↗

PubMed1994

Emergence neophobia correlates with hippocampal and cortical glutamate receptor binding in rats.

Previous work from our laboratory indicated that emergence neophobia is highly correlated with perforant path long-term potentiation (LTP) in rats. In the present study, we examined the relationship between hippocampal and cortical alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and emergence behavior in rats. Emergence neophobia was assessed in an exploratory task that provided a choice between a novel alley and a familiar nest box. Quantitative autoradiography using radiolabeled ligands specific for the AMPA subclass of glutamate receptors was performed on frozen brain sections. Both [3H]AMPA and [3H]CNQX (6-cyano-7-nitro-[3H]quinoxaline- 2,3-dione, an AMPA receptor antagonist) binding in the dentate gyrus (stratum moleculare), hippocampal area CA1 (stratum radiatum), and the parietal cortex overlying the hippocampus were significantly correlated with emergence behavior. The correlations indicated that neophobic rats, which had longer latencies to enter the novel alley, made fewer entries into the alley, and spent less time in the novel alley during a 10-min test than their neophilic counterparts, had higher levels of AMPA receptor binding. These results suggest that individual differences in specific hippocampal AMPA receptors reflect variability in a specific class of hippocampal-dependent behaviors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Co-expression of HSP72 and c-fos in rat brain following kainic acid treatment.

The relationship between heat shock protein 72 (HSP72) and c-fos gene expression following systemic administration of kainic acid was investigated by combining immunocytochemistry for HSP72 with in situ hybridization for c-fos. Increased HSP72 expression was detected in adult rat hippocampus 4 h after seizure-onset. Transient co-expression of c-fos and HSP72 occurred in neurons that are resistant to kainic acid, whereas prolonged co-expression was observed in vulnerable neurons. The spatial distribution and developmental time course of kainic acid-induced HSP72 expression were similar to those of kainic acid-induced neurodegeneration. The results demonstrate a relationship between c-fos and HSP72 gene expression and suggest that prolonged co-expression of these genes plays a role in kainic acid-induced neuronal death.

Animals↗

PubMed1993

Postsynaptic factors in the expression of long-term potentiation (LTP): increased glutamate receptor binding following LTP induction in vivo.

Several lines of evidence indicate that LTP in the hippocampus is associated with a change in the properties of postsynaptic glutamate receptors. In the present study, we used quantitative autoradiography to examine the binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate subclasses of glutamate receptors in frozen brain sections obtained from rats in which perforant-path LTP was induced in vivo. Induction of LTP resulted in a selective increase in [3H]AMPA binding in those hippocampal subfields receiving perforant-path axons. Increases in [3H]AMPA binding in dentate gyrus (stratum moleculare) were highly correlated with the magnitude of LTP recorded in this structure. Scatchard analyses of [3H]AMPA and 6-cyano-7-nitro-[3H]quinoxaline-2,3-dione (an AMPA receptor antagonist) binding in the dentate gyrus indicated that LTP induction resulted in an increase in the number of AMPA receptor binding sites. No changes in the binding of 3H-labeled N-[1-(thienyl)cyclohexyl]piperidine (an N-methyl-D-aspartate receptor antagonist) were observed in any hippocampal subfield. These results suggest that a modification in postsynaptic AMPA receptors plays a role in the expression of synaptic enhancement following LTP induction in the hippocampus.

Animals↗